Genetic Engineering

UNIT I: NUCLEIC ACID AND PROTEIN SYNTHESIS AND REGULATION
LESSON 1: NUCLEIC ACID - DNA

CONTENTS
1.0. AIMS AND OBJECTIVES
1.1. INTRODUCTION
1.2. FORMS OF DNA
1.3. LET US SUM UP
1.4 POINTS FOR DISCUSSION
1.5. LESSON - END ACTIVITIES
1.6. REFERENCE

The chapter in detail deals with the structure of DNA and its component nucleic acids.
1.1. INTRODUCTION
Deoxyribonucleic acid, or DNA, is a nucleic acid that contains the genetic instructions
used in the development and functioning of all known living organisms. The main role of
DNA molecules is the long-term storage of information and DNA is often compared to a set
of blueprints, since it contains the instructions needed to construct other components of cells,
such as proteins and RNA molecules. The DNA segments that carry this genetic information
are called genes, but other DNA sequences have structural purposes, or are involved in
regulating the use of this genetic information. Two scientists named, Rosalind Franklin and
Maurice Wilkins, decided to try to make a crystal of the DNA molecule. If they could get
DNA to crystallize, then they could make an x-ray pattern, thus resulting in understanding of
how DNA works. These two scientists were successful and obtained an X-ray pattern. The
pattern appeared to contain rungs, like those on a ladder between two strands that are side by
side. It also showed by an X shape that DNA had a helix shape.
In 1953 two scientists, James Watson and Francis Crick, were trying to put together a
model of DNA. When they saw Franklin and Wilkin's picture of the X-ray they had enough
information to make an accurate model. They created a model that has not been changed
much since then. Their model showed a double helix with little rungs connecting the two
strands. These rungs were the bases of a nucleotide.
DNA is a long polymer of simple units called nucleotides, with a backbone made of
sugars and phosphate atoms joined by ester bonds. Attached to each sugar is one of four types
of molecules called bases. It is the sequence of these four bases along the backbone that
encodes information. This information is read using the genetic code, which specifies the
sequence of the amino acids within proteins. The code is read by copying stretches of DNA
into the related nucleic acid RNA, in a process called transcription. Most of these RNA
molecules are used to synthesize proteins, but others are used directly in structures such as
ribosomes and spliceosomes.
Within cells, DNA is organized into structures called chromosomes and the set of
chromosomes within a cell make up a genome. These chromosomes are duplicated before
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cells divide, in a process called DNA replication. Eukaryotic organisms such as animals,
plants, and fungi store their DNA inside the cell nucleus, while in prokaryotes such as
bacteria it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such
as histones compact and organize DNA, which helps control its interactions with other
proteins and thereby control which genes are transcribed
Fig. 1 STRUCTURE OF DNA

1.2. FORMS OF DNA
Six different morphological forms of DNA double helix have been described. These
are A, B, C, D, E and Z forms. Only B and Z conformations occur as cellular DNA, other
forms are found rigidly in controlled, experimental conditions. All DNA molecules of any
source, natural 0r artificial, can take on A or B conformation. Some DNA forms are inter
convertible also.
The differences in these DNA forms are associated with:
The number of base pairs present in each turn of DNA helix.
The pitch or angle between each base pair.

The helical diameter of DNA molecule.
The handedness of coiling of double helix.
Character
A-DNA
Direction of helix
Appearance
Width or diameter of
Helix
Base pairs per turn
Length of each coil
of helix
Right handed
Short and wide
23A
Right handed
Left handed
Longer and thinner Long and thin
20A
18A
10.9 or 11
34A
10
34A
12
45A
2.7A
3.4A
3.75A
Distance between
adjacent nucleotides
B-DNA
Z.DNA
I
I
Major groove
Flattened out on helix

Extremely narrow and Wide and of intersurface.
very deep.
mediate depth.
Minor groove
Very
wide
shallow.
Location of axis of
Through
groove.
and
Narrow
inter
depth.
majorThrough
pairs.
and of
Extremely narrow and very
mediate
deep.
base
Through minor groov,1
Helix
Base inclination from 13.0
2.0
8.8
Helix
Occurrence
Occurs in dehydrated Occurs in hydrated Occurs in dehydral
conditions.
conditions.
high salt concentration
.
DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes
in prokaryotes. The set of chromosomes in a cell makes up its genome; According to the
report of human genome project, human genome has approximately 3 billion base pairs of
DNA arranged into 46 chromosomes. The information carried by DNA is held in the
sequence of pieces of DNA called genes. Transmission of genetic information in genes is
achieved via complementary base pairing. For example, in transcription, when a cell uses the
information in a gene, the DNA sequence is copied into a complementary RNA sequence
through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA
copy is then used to make a matching protein sequence in a process called translation which
depends on the same interaction between RNA nucleotides. Alternatively, a cell may simply
copy its genetic information in a process called DNA replication. Where using old DNA
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strand as a template the DNA is synthesised by using the semiconservative , conservative and
dispersive model of DNA replication.
Semi-Convservative
Replication
Stand - Sepration
Fig.2 REPLICATION PROCESS
1.3. LET US SUM UP
DNA is a polymer containing chains of nucleotide monomers. Each nucleotide
contains a sugar, a base and a phosphate group. Nucleotide triphosphates are joined to form
DNA polynucleotide chains. DNA molecules are composed of two polynucleotide strands
wrapped around each other to form a double helix. Hydrogen bonds between bases on the two
DNA strands stabilize the double helix. Variant DNA structures have been identified
including Z DNA which has a left handed helix.
1. Analyse why B and Z configuration occu as cellular DNA.
2. Critically analyse the correlationbetweent he x-ray diffraction pattern and the
double helical structure of DNA.
1. Write an essay on the structure and functions of DNA.
2. Why DNA needs to have a double helical structure to perform the functions attributed
to it?
3. explain the following :
(a) Complementary base pairing (b) polynucleotide chain has direction
(c) Left and right handed DNA (d) Double helical structure of DNA
(e) Phosphodiester bond
4. Write short notes on :
(a) Nucleotide; (b) Nucleoside
(c)Pyrimidines (d)Deoxyribizymes
4. Differentiate between the following :
(a) Nucleoside and nucleotide
(b) Polymerases and ligases
(c) Ribose and deoxyribose
5
(d) Major and minor grooves
1.6 REFERENCE
1. Genes VIII by Lewin.
6
LESSON 2: NUCLEIC ACIDS RNA
CONTENTS
2.1. INTRODUCTION
2.2. mRNA
2.3. tRNA
2.4. rRNA
2.5. LET US SUM UP
2.6. POINTS FOR DISCUSSION
2.8. REFERENCE
This chapter deals in detail with the structure of RNA and its component nucleic
acids.
2.1. INTRODUCTION
Ribonucleic acid or RNA is a nucleic acid polymer consisting of nucleotide
monomers that plays several important roles in the processes that translate genetic
information from deoxyribonucleic acid (DNA) into protein products; RNA acts as a
messenger between DNA and the protein synthesis complexes known as ribosomes, forms
vital portions of ribosomes, and acts as an essential carrier molecule for amino acids to be
used in protein synthesis.
RNA is very similar to DNA, but differs in a few important structural details: RNA
nucleotides contain ribose sugars while DNA contains deoxyribose and RNA uses
predominantly uracil instead of thymine present in DNA. RNA is transcribed (synthesized)
from DNA by enzymes called RNA polymerases and further processed by other enzymes.
RNA serves as the template for translation of genes into proteins, transferring amino acids to
the ribosome to form proteins, and also translating the transcript into proteins.
Nucleic acids were discovered in 1868 (some sources indicate 1869) by Johann
Friedrich Miescher (1844-1895), who called the material 'nuclein' since it was found in the
nucleus. It was later discovered that prokaryotic cells, which do not have a nucleus, also
contain nucleic acids. The role of RNA in protein synthesis had been suspected since 1939,
based on experiments carried out by Torbjrn Caspersson, Jean Brachet and Jack Schultz.
Hubert Chantrenne elucidated the messenger role played by RNA in the synthesis of proteins
in ribosome.Finally,Severo Ochoa discovered the RNA, winning Ochoa the 1959 Nobel Prize
for Medicine. The sequence of the 77 nucleotides of yeast RNA was found by Robert W.
Holley in 1964, winning Holley the 1968 Nobel Prize for Medicine. In 1976, Walter Fiers
and his team at the University of Ghent determined the complete nucleotide sequence of
bacteriophage MS2-RNA.
Fig.1 STRUCTURAL COMPARISON OF DNA AND RNA

2.2. mRNA
Messenger RNA is RNA that carries information from DNA to the ribosome sites of
protein synthesis in the cell. In eukaryotic cells, once mRNA has been transcribed from
DNA, it is "processed" before being exported from the nucleus into the cytoplasm, where it is
bound to ribosomes and translated into its corresponding protein form with the help of tRNA.
In prokaryotic cells, which have no partition into nucleus and cytoplasm compartments,
mRNA can bind to ribosomes while it is being transcribed from DNA. After certain amount
of time the mRNA degrades into its component nucleotides, usually with the assistance of
ribonucleases
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2.3. tRNA
Transfer RNA is a small RNA chain of about 74-95 nucleotides that transfers a specific
amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during
translation. It has sites for amino-acid attachment and an anticodon region for codon
recognition that binds to a specific sequence on the messenger RNA chain through hydrogen
bonding. It is a type of non-coding RNA.
Fig.2 CLOVER LEAF STRUCTURE OF tRNA
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2.4. rRNA
Ribosomal RNA is the catalytic component of the ribosomes, the protein synthetic
factories in the cell. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S,
28S, and 5S rRNA. Three of the rRNA molecules are synthesized in the nucleolus, and one is
synthesized elsewhere. rRNA molecules are extremely abundant and make up at least 80% of
the RNA molecules found in a typical eukaryotic cell.
In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a
ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes
may be attached to a single mRNA at any time.
Ribosome Structure
Fig.3 STRUCTURE OF RIBOSOME
Table of Standard Genetic Code
T
TTT Phe (F)
TTC "
T
TTA Leu (L)
TTG "
C
TCT Ser (S)
TCC "
TCA "
TCG "
A
TAT Tyr (Y)
TAC
TAA Ter
TAG Ter
G
TGT Cys (C)
TGC
TGA Ter
TGG Trp (W)
CTT Leu (L)

CTC "
C
CTA "
CTG "
CCT Pro (P)

CCC "
CCA "
CCG "
CAT His (H)

CAC "
CAA Gln (Q)
CAG "
CGT Arg (R)

CGC "
CGA "
CGG "
ATT Ile (I)

ATC "
A
ATA "
ATG Met (M)
ACT Thr (T)

ACC "
ACA "
ACG "
AAT Asn (N)

AAC "
AAA Lys (K)
AAG "
AGT Ser (S)

AGC "
AGA Arg (R)
AGG "
GTT Val (V)

GTC "
G
GTA "
GTG "
GCT Ala (A)

GCC "
GCA "
GCG "
GAT Asp (D)

GAC "
GAA Glu (E)
GAG "
GGT Gly (G)

GGC "
GGA "
GGG "
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2.5. LET US SUM UP
The structure of RNA is similar to that of DNA but a number of important differences
exist. In RNA ribose replaces 2- deoxyribose and the base thymine is replaced by another
base, uracil , which can also base pair with adenine In addition, RNA molecules normally
exist as a single polynucletide strand and do not form a double helix. However, it is possible
for base pairing to occur between complementary parts of the same RNA strand resulting in
short double stranded regions. tRNA s are small molecules that bring amino acids together
for protein synthesis in an order specified by a messenger RNA sequence. Cells contain a
number of different tRNAs each of which binds a specific amino acid. Each tRNA also binds
a specific codon in the mRNA allowing it to place its amino acid in the correct position.
Prokaryotic ribosomes are 70S with 50S and 30S sub units. They contain three rNRAs (
23s,16s and 5s). Eukaryotic ribosomes are 80S and have 60S and 40S subunits. They contain
four rRNAs ( 28S, 18S, 5.8S and 5S). The three rRNA s in E.coli are transcribed from a
single gene. Seven copies of the gene occur in the genome. Eukaryotic rRNA are transcribed
from a single gene present in multiple opies arranged as a series of clusters. The gene are
transcribed in the nucleolus by RNA polymerase I. A single pre-rRNA is synthesized which
is processed to give mature 28S, 18S and 5.8S rRNAs. The 5 S rRNA is transcribed
separately from unlinked genes by RNA polymerase III.
a) Compare and contrast the function of different RNAs.
b) Substantiate the structural component of ribosome with its function.
1.
2.
3.
4.
What is RNA? Discuss its structure. How does it differ from DNA?
Describe structure of mRNA. Write a note on its biogenesis.
Describe clover leaf model of tRNA.
Give differences between different types of RNAs found in the cell.
2.8 REFERENCE
1. Genes VII by Lewin
11
LESSON 3: PROTEIN SYNTHESIS AND REGULATION
CONTENTS
3.1. INTRODUCTION
3.2. TRANSCRIPTION
3.3. TRANSLATION
3.4. LET US SUM UP
3.6. REFERENE
The chapter deals in detail with transcription, translation (protein synthesis) and regulation of
protein.
3.1. INTRODUCTION
The genetic information coded in the DNA has to be converted in to proteins to carry
out all the functions of Nucleic acids, this has to be proceeded in a step wise conversion of
DNA in to mRNA by a Process called Transcription and subsequently the mRNA is
processed to form Protein by the process known as Translation. This concept generally
known as Central Dogma of living Organism.
DNA RNA Protein
(Central Dogma)
3.2. TRANSCRIPTION
A molecule which allows the genetic material to be realized as a protein was first
hypothesized by Jacob and Monad. RNA synthesis by RNA polymerase was established.
However, the RNA synthesized by these enzymes had properties that suggested the existence
of an additional factor needed to terminate transcription correctly.
Recently, Roger D. Kornberg won the 2006 Nobel Prize in Chemistry "for his studies
of the molecular basis of eukaryotic transcription".
Transcription is the process through which a DNA sequence is enzymatically copied
by an RNA polymerase to produce a complementary RNA. So to say, it is the transfer of
genetic information from DNA into RNA. In the case of protein-encoding DNA, transcription
is the beginning of the process that ultimately leads to the translation of the genetic code (via
the mRNA intermediate) into a functional peptide or protein. The stretch of DNA that is
transcribed into an RNA molecule is called a transcription unit.
Transcription has some proofreading mechanisms, but they are fewer and less
effective than the controls used for copying DNA; therefore, transcription has a lower
copying fidelity than DNA replication.
As in DNA replication, transcription proceeds in the 5' 3' direction (i.e. the old
polymer is read in the 3' 5' direction and the new, complementary fragments are generated
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in the 5' 3' direction). Transcription is divided into 3 stages: initiation, elongation and
termination.
3.2.1. INITIATION
In transcription, one strand of DNA, the coding strand, is used as a template for RNA
synthesis. As transcription proceeds in the 5' 3' direction, and uses base pairing
complimentarity with the DNA template to specify the correct copying, the DNA coding
strand is that oriented in the 3' 5' direction. The strand that is not used as the template is
called the non-coding strand, and has the DNA sequence that reflects that of the RNA
produced.
Transcription begins with the binding of RNA polymerase to the promoter in DNA. In
prokaryotes, the RNA polymerase is a core enzyme consisting of five subunits: 2 subunits,
1 subunit, 1 ' subunit, and 1 subunit. At the start of initiation, the core enzyme is
associated with a sigma factor (number 70) that aids in finding the appropriate -35 and -10
basepairs downstream of promoter sequences. Transcription initiation is far more complex in
eukaryotes, the main difference being that eukaryotic polymerases do not recognize directly
their core promoter sequences.
Unlike DNA replication, transcription does not need a primer to start. The DNA
unwinds and produces a small open complex and synthesis begins on only the template
strand.
3.2.2. ELONGATION
Unlike DNA replication, mRNA transcription can involve multiple RNA
polymerases, so many mRNA molecules can be produced from a single copy of the gene.
This step also involves a proofreading mechanism that can replace an incorrectly added RNA.
3.2.3. TERMINATION
Bacteria use two different strategies for transcription termination: in Rho-independent
transcription termination, RNA transcription stops when the newly synthesized RNA
molecule forms a hairpin loop, followed by a run of Us, which makes it detach from the DNA
template. In the "Rho-dependent" type of termination, a protein factor called "Rho"
destabilizes the interaction between the template and the mRNA, thus releasing the newly
synthesized mRNA from the elongation complex. Transcription termination in eukaryotes is
less well understood. It involves cleavage of the nascent transcript, followed by templateindependent addition of As at its new 3' end, in a process called polyadenylation.
13
Fig.1 PROTEIN SYNTHESIS

3.3. TRANSLATION
The mRNA carries genetic information encoded as a ribonucleotide sequence from
the chromosomes to the ribosomes. The ribonucleotides are "read" by translational machinery
in a sequence of nucleotide triplets called codons. Each of those triplets codes for a specific
amino acid.
The ribosome and tRNA molecules translate this code to produce proteins. The
ribosome is a multisubunit structure containing rRNA and proteins. It is the "factory" where
amino acids are assembled into proteins.
tRNAs are small noncoding RNA chains (74-93 nucleotides) that transport amino
acids to the ribosome. tRNAs have a site for amino acid attachment, and a site called an
anticodon. The anticodon is an RNA triplet complementary to the mRNA triplet that codes
for their cargo amino acid.
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Aminoacyl tRNA synthetase (an enzyme) catalyzes the bonding between specific
tRNAs and the amino acids that their anticodons sequences call for.
The product of this reaction is an aminoacyl-tRNA molecule. This aminoacyl-tRNA
travels inside the ribosome, where mRNA codons are matched through complementary base
pairing to specific tRNA anticodons. The amino acids that the tRNAs carry are then used to
assemble a protein.
The energy required for translation of proteins is significant. For a protein containing
n amino acids, the number of high-energy Phosphate bonds required to translate it is 4n-1.
It is also the process whereby ribosomes use the sequence of codons in mRNA to produce a
polypeptide with a particular sequence of amino acids.
3.4. LET US SUM UP
Genetic information is encoded in the base sequence of DNA molecules as a series of
genes. Gene expression is the term used to describe how cells decode the information to
synthesize proteins required for cellular function. The expression of a gene involves the
synthesis of a complementary RNA molecule whose sequence specifies the amino acid
sequence of a protein. The DNA sequence of the gene is collinear with the amino acid
sequence of the polypeptide. Amino acids are encoded by 64 base triplets called codons
which encode the 20 amino acids. Most amino acids have more than one codon. This is
known as the degeneracy of the genetic code and it helps to minimize the effect of mutations.
Codons that specify the same amino acid are known as synonyms and differ at their third
base, known as wobble position. AUG is the initiation codon and encodes methionine. There
are three stop codons: UAG, UGA and UAA. An open reading frame is a sequence of
codons, bounded by start codons and stop codons. The genetic code applies universally with
all organisms using the same codons for each amino acid. Translation is similar in
prokaryotes and eukaryotes and occurs in three stages (initiation, elongation and termination).
Each stage involves a set of accessory proteins. Energy is provided by hydrolysis of Adenine
triphosphate and Guanosine triphosphate.
Justify the different processes of transcription heeling DNA and translation healing
mRNA.
1.Explain the mechanism of protein synthesis.
2.Give the difference between transcription and translation.
3.Name the three codons on mRNA that are not recognized by tRNA. What are these called?
4.Name the type of bond that links the following molecule.
(a) Nucleotide to Nucleotide in Nucleic acids
(b) Amino acid to tRNA
(c) Codon in mRNA to anticodon in aminoacyl tRNA
(d) Amino acid to amino acid in polypeptide chain
3.6 REFERENCE
1. Genes VII by Lewin.
15
LESSON 4: REGULATION OF PROTEIN EXPRESSION IN PROKARYOTES
CONTENTS
4.1. INTRODUCTION
4.2. REGULATION OF GENE EXPRESSION
4.3. THE LAC OPERON
4.4. THE TRP OPERON
4.5. LET US SUM UP
4.8. REFERENCE
The chapter in detail deals with the protein expression and its regulation.
4.1. INTRODUCTION
After the synthesis of the protein, the modification and expression of the protein is
very important. The various non coding regions are spliced during the process. It is necessary
to study the expression and regulation of proteins for efficient manipulation or modification
of the protein.
4.2. REGULATION OF GENE EXPRESSION
For a Bacterium to function it is not necessary that all of its genes are transcribed at
all times. To conserve energy and resources bacteria regulate hi activity of their genes so that
only those gene products necessary for the cell functions are produced. For example, it would
be wasteful for a bacterium to produce enzymes required to synthesize an amino acid that
was already available to it from its environment. Regulation of gene expression allows
bacteria to respond to changes in their environment, typically to the presence or absence of
nutrients.
Bacteria regulate expression of their genes in order to control the amount of gene
product present. The steady state concentration of a gene product is determined by the
balance between the rate of synthesis and the rate of degradation of the expressed protein. In
practice, changes in the rate of synthesis are wlm alter the amount of gene product. The rate
of synthesis could potentially by altered by a number of factors:
changes in the rate of gene transcription;
changes in mRNA turnover time;
changes in the rate of translation.
4.3. THE lac OPERON
The lac operon is an operon required for the transport and metabolism of lactose in
Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes,
a promoter, a terminator, and an operator. The lac operon is regulated by several factors
including the availability of glucose and of lactose. Gene regulation of the lac operon was the
16
first genetic regulatory mechanism to be elucidated and is often used as the canonical
example of prokaryotic gene regulation.
Fig.1 LAC OPERON

The lac operon consists of three structural genes, a promoter, a terminator, and an
operator. The three structural genes are:: lacZ, lacY, and lacA.
lacZ encodes -galactosidase (LacZ), an intracellular enzyme that cleaves the

disaccharide lactose into glucose and galactose.
lacY encodes -galactoside permease (LacY), a membrane bound transport protein

that pumps lactose into the cell.
lacA encodes -galactoside transacetylase (LacA), an enzyme that transfers an acetyl

group from acetyl-CoA to -galactosides.
Only lacZ and lacY appear to be necessary for lactose catabolism.
Specific control of the lac genes depends on the availability of the substrate lactose to
the bacterium. The proteins are not produced by the bacterium when lactose is unavailable as
a carbon source. The lac genes are organized into an operon; that is, they are oriented in the
same direction immediately adjacent on the chromosome and are co-transcribed into a single
polycistronic mRNA molecule. Transcription of all genes starts with the binding of the
enzyme RNA polymerase (RNAP), a DNA-binding protein, to a specific DNA binding site
immediately upstream of the genes, the promoter. From this position RNAP proceeds to
transcribe all three genes (lac ZYA) into mRNA.
The regulatory response to lactose requires an intracellular regulatory protein called
the lactose repressor. The lacI gene encoding repressor lies nearby the lac operon and is
always expressed (constitutive). If lactose is missing from the growth medium, the repressor
binds very tightly to a short DNA sequence just downstream of the promoter near the
beginning of lacZ called the lac operator. Repressor bound to the operator interferes with
binding of RNAP to the promoter, and therefore mRNA encoding LacZ and LacY is only
made at very low levels. When cells are grown in the presence of lactose, a lactose metabolite
called allolactose binds to the repressor, causing a change in its shape. Thus altered, the
repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and
thereby leading to high levels of the encoded proteins.
The diagram below summarizes these statements.
17
Fig.2 lac OPERON IN DETAIL

4.3.1. USE IN MOLECULAR BIOLOGY
The lac gene and its derivatives are amenable to use as a reporter gene in a number of
bacterial-based selection techniques such as two hybrid analysis, in which the successful
binding of a transcriptional activator to a specific promoter sequence must be determined. In
LB plates containing X-gal, the colour change from white colonies to a shade of blue,
corresponds to about 20-100 -galactosidase units, while tetrazolium lactose and MacConkey
lactose media have a range of 100-1000 units, being most sensitive in the high and low parts
of this range respectively. Since MacConkey lactose and tetrazolium lactose media both rely
on the products of lactose breakdown, they require the presence of both lacZ and lacY genes.
The many lac fusion techniques which include only the lacZ gene are thus suited to the X-gal
plates or ONPG liquid broths.
Trp operon is an anabolic operon which promotes the production of tryptophan in the
absence of tryptophan in the environment. This is regulated both by repression and by
attenuation. Prokaryotes vs. eukaryotes
In prokaryotes regulation of transcription is needed for the cell to quickly adapt to the
ever changing outer environment. The presence of the quantity and type of nutrients
determines which genes are expressed; in order to do that, genes must be regulated in some
fashion. In prokaryotes, repressors bind to regions called operators that are generally located
downstream from and near the promoter (normally part of the transcript). Activators bind to
the upstream portion of the promoter, such as the CAP region (completely upstream from the
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transcript). A combination of activators, repressors and rarely enhancers (in prokaryotes)
determines whether a gene is transcribed.
In eukaryotes, transcriptional regulation tends to involve combinatorial interactions
between several transcription factors, which allow for a sophisticated response to multiple
conditions in the environment. This permits spatial and temporal differences in gene
expression. Eukaryotes also make use of enhancers, distant regions of DNA that can loop
back to the promoter. A major difference between eukaryotes and prokaryotes is the fact the
eukaryotes have a nuclear membrane, which prevents simultaneous transcription and
translation.
The Histidine operon leader is an RNA element found in the bacterial histidine
operon. At least 6 amino acid operons are known to be regulated by attenuation. In each a
leader sequence of 150-200 bp is found upstream of the first gene in the operon. This leader
sequence can assume two different secondary structures known as the terminator and the antiterminator structure. In each case the leader also codes for very short peptide sequence that is
rich in the end product amino acid of the operon. The terminator structure is recognised as a
termination signal for RNA polymerase and the operon is not transcribed. This structure
forms when the cell has an excess of the regulatory amino acid and ribosome movement over
the leader transcript is not impeded. When there is a deficiency of the charged tRNA of the
regulatory amino acid the ribosome translating the leader peptide stalls and the antiterminator
structure can form. This allows RNA polymerase to transcribe the operon.
When E. coli bacteria are subjected to heat stress, the 32 subunit of its RNA
polymerase changes such that the enzyme binds to a specialized set of promoters that precede
genes for heat-shock response proteins.
When a cell contains a surplus amount of the amino acid tryptophan, the acid binds to
a specialized repressor protein (tryptophan repressor). The binding changes the structural
conformity of the repressor such that it binds to the operator region for the operon that
synthesizes tryptophan, preventing their expression and thus suspending production. This is a
form of negative feedback.
In bacteria, the lac repressor protein blocks the synthesis of enzymes that digest
lactose when there is no lactose to feed on. When lactose is present, it binds to the repressor,
causing it to detach from the DNA strand.
4.4. THE TRP OPERON
This operon contains five genes encoding enzymes involved in biosynthesis of the
amino acid tryptophan. The genes are expressed as a single mRNA transcribed from an
upstream promoter. Expression of the operon is regulated by the level of tryptophan in the
cell. A regulatory gene upstream of the trp operon encodes a protein called the trp repressor.
This protein binds a DNA sequence called the trp operator which lies just downstream of the
trp promoter partly overlapping it. When tryptophan is present in the cell it binds to the trp
repressor protein enabling it to bind the trp operator sequence, obstructing binding of the
RNA polymerase to the trp promoter and preventing transcription of the operon. In the
absence of tryptophan the trp repressor is incapable of binding the trp operator and
transcription of the operon proceeds. Tryptophan, the end product of the enzymes encoded by
the trp operon, thus acts as a corepressor with the trp repressor protein and inhibits its own
synthesis by end product inhibition.
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Fig.3 TRYPTOPHAN OPERON

4.4.1. ATTENUATION
The trp operon makes use of an alternative strategy for controlling transcription called
attenuation which can finely tune expression levels (Fig. 4). The transcribed mRNA sequence
between. the trp promoter and the first trp gene is capable of forming two stem-loop
structures. The relative positions of the sequences mean that both stem-loops cannot form at
once just one or the other may be present at any time. The larger, more stable structure does
not influence transcription and occurs upstream of the smaller stem-loop which acts as a
transcription terminator, If this structure forms it will terminate transcription before the first
gene is reached eliminating gene expression, Attenuation depends on the fact that
transcription and translation are linked in bacterial ribosomes attach to mRNAs as they are
being synthesized and begin translating them into protein. An mRNA that is being transcribed
may already have one or more ribosomes attached to it. Binding of ribosomes to the trp
mRNA influences which of the two stem-loop structures can form and so determire whether
or not termination occurs, Immediately upstream of the stem-I region is a short open reading
frame containing 14 codons followed by a start codon which is translated before the structural
genes; two out of these 14 codons are for tryptophan, If levels of tryptophan are adequate the
ribosome will translate the coding region following closely behind the RNA polymerase, In
the circumstances the presence of the ribosome prevents formation of the large stem-loop
allowing the terminator loop to form and transcription ends, If tryptophan is lacking, the
ribosome will be stalled as it translates the coding region.
20
Fig.4 TRANSCRIPTIONAL ATTENUATION OF TRYPTOPHAN OPERON

4.4.2. TERMINATOR LOOP
The RNA polymerase will move ahead and the first stem-loop will be free to form.
Formation of the terminator loop is then blocked and transcription of the operon can proceed.
The speed at which the ribosome translates, the coding region will not be the same for each
transcript. When tryptophan is present in the medium at intermediate levels some transcripts
will terminate and others will not, thus allowing fine adjustments in the levels of transcription
of the operon. Overall, the trp repressor determines whether the operon is switched on or off
and attenuation determines how efficiently it is transcribed, both depend on the level of
tryptophan in the cell. Attenuation allows the cell to synthesize tryptophan according to its
exact requirements. Attenuation is not restricted to the trp operon and occurs in at least six
other operons that encode amino acid biosynthetic enzymes. Some operons such as the trp
and phe operons are regulated by repressors and attenuation and others such as the his, leu
and thr operons rely only on attenuation.
21
4.5. LET US SUM UP
Bacteria regulate the activity of their genes so that only gene products necessary for
the cells functions are produced. This allows bacteria to respond to environmental changes.
Alterations in the amount of gene product may potentially be achieved by varying rates of
transcription, mRNA turn over, mRNA processing and translation. Of these the mechanisms
that alter gene transcription are the best characterized. Many bacterial genes are arranged as
coordinately regulated operons that encode proteins with related functions. Inducible operon
such as the lac operon encodes enzymes involved in metabolic pathways and is induced by
the substrate for the pathway. Repressible operons such as the trp operon encode enzymes
involved in biosynthetic pathways. Gene expressions are regulated by the end product or by
attenuation.
Justify the relationship betweent he concentration of the tryptophan in the cell
movement of ribosomes and formation of stem loop structures in the mRNA.
1.
2.
3.
4.
Define an operon. Who gave this concept? Explain this concept with regard to lac operon.
Distinguish between induction and repression.
How does an excess of tryptophan cause switching off of the tryptophan operon?
What is the positive and negative control of gene activity? Discuss with example.
4.8. REFERENCE
Genes VII by Lewin.
22
UNIT II: GENE CLONING VECTORS
LESSON 5: BIOLOGY OF VECTORS
CONTENTS
5.1. INTRODUCTION
5.2. PLASMIDS
5.3. BACTERIOPHAGES
5.4. COSMIDS
5.5. PHASMIDS
5.6. LET US SUM UP
5.9. REFERENCES
The chapter is focused on the vector biology and its uses.
5.1. INTRODUCTION
A cloning vector is a small DNA vehicle that carries a foreign DNA fragment. The
insertion of the fragment into the cloning vector is carried out by treating the vehicle and the
foreign DNA with the same restriction enzyme, then ligating the fragments together. There
are many types of cloning vectors. Plasmids and bacteriophages (such as phage ) are perhaps
most commonly used for this purpose. Other types of cloning vectors include bacterial
artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).
5.2. PLASMIDS
Plasmids are double stranded closed circular DNA molecule, which exist in cell as
extra chromosomal units. They are self replicated, and found in variety of bacterial species.
They replicate and are inherited independently.
There are 3 general types of plasmids:1. Virulence plasmid - codes for toxin genes
2. Drug resistance plasmid - confers resistance to antibiotic
3. Conjugation plasmid codes for bacterial conjugation
Plasmids range in size from 1 to 200 Kb and depends on host for certain proteins for
maintenance and replication Copy number: the amount of plasmid found to be present in the
bacterial cell. Single copy plasmid is found only one plasmid in the cell. Multiple copy
plasmids are found many in one cell. There are also plasmids under relaxed replication
control permitting accumulation of very large number of plasmids - up to 1000 copies per
cell. Most plasmids used in cloning have a relaxed mode of replication as that of E.coli
Plasmid colE1, whose antibacterial proteins called colicins. These plasmids are used as
cloning vectors due to their increased potential. Most plasmid vectors in current use carry a
replicon derived from a plasmid pMBI, which is originally isolated from a clinical specimen.
23
Under normal condition of growth, 15-20 copies of plasmid carrying this replicon are
maintained in each bacterial cell. Replication of plasmid is carried out by a subset of enzyme
which is used to replicate the bacterial plasmid. The F plasmid encodes the gene required for
bacterial conjugation. This extra chromosomal genetic element is more or less 95 Kb, and is
named so as it involves in bacterial fertility. It is present as 1 copy per cell and codes for
nearly 100 genes some of which are required during F-plasmid transfers during conjugation
(tra gene) As a result of integration into E.coli chromosome and then an imprecise excision
resulting in the reformation of the circular plasmid, the F plasmid is able to transfer a portion
of the E.coli into another bacterium via conjugation F plasmid containing E.coli strains are
required in molecular cloning involving M13 filamentous bacteriophage. The F plasmid
carries the sex pili gene which encodes the cell surface binding site for M13 phages.
A plasmid vector used for cloning is specifically developed by adding certain features
1. Reduction in size of vector in order to increase its capacity to clone large fragments
preferably 3-4 Kb. In this way a insert of 10-12 Kb can be accommodated. Since the
efficiency of transformation bacterial cells drops drastically when plasmids larger than 15 Kb
are used,
2. It should have a origin of replication that operates in the organism into which the clone is
to be introduced
3. They must posses at least 1, but preferably several markers such as resistance to
antibiotics.
5.2.1. PLASMID CLASSIFICATION
Fertility or F-plasmid carry only tra-gene and have no conjugate character of plasmid
character eg; F plasmid of E.coli Resistance or R plasmids carry genes conferring on the host
bacterium resistance to 1 or more antibacterial agents, such as chloramphenicol. Plasmids are
very important in clinical microbiology as their spread through natural populations can have
profound consequences in the treatment of bacterial infections Ex.. RP 4, commonly found in
pseudomonas but also occurring in many other bacteria. Col plasmid code for colicin proteins
that kill other bacteria Eg. Col E1 of E.coli. Degraditive plasmid : allow the host bacterium to
metabolize unusual molecules such as toluene and salicylic acid Eg: Tol plasmid of
pseudomonas putida. Virulence plasmids confer pathogenicity on the host bacterium Eg. Ti
plasmids of Agrobacterium tumefaciens, which induce Crown Gall disease on dicotyledonous
plants.
5.2.2. PLASMIDS IN ORGANISMS OTHER THAN BACTERIA

Although it is common in bacteria, they also found in yeast (Saccharomyces
cerevisiae). The eukaryotic 2 m circle. Search of plasmids in other eukaryotes (Fungi,plant
& animal) have disappointed.
The constructed vectors:
5.2.2.1. pBR 322
This was one of the first artificial cloning vectors to be constructed and the most
widely using vector upto now.
24
It is a 4.3 Kb double stranded cloning vector. This plasmid vector has been put
together from fragments originating from three different naturally occurring plasmids.
The first useful feature of pBR322 is its size, it was stated that cloning vector ought to
be less than 10 Kb, to avoid problems such as DNA break down during purification.
pBR322 contains two antibiotic resistance genes , one is the Ampicillin resistance
gene coding for -lactamase (which modifies ampicillin into a form that is non toxic to
E.coli) and other is the Tetracycline resistance gene. (a set of genes coding for enzymes that
detoxify tetracycline). The gene determining tetracycline resistance was derived from the
naturally occurring plasmid pSC101.
The complete nucleotide sequence of pBR322 has been determined. The plasmids
contains 20 unique recognition sites for restriction enzymes. Six of these sites for restriction
enzymes i.e., EcoR1, BamH1, Sph1, Sal1,XmaIII and NruI, are located with in the gene
coding for tetracycline.
pBR327- a higher copy number plasmid-it was constructed by removing a 1089 bp
from pBR322.
5.2.2.2. pUC 8
The name pUC is derived because it was developed in the university of California by
Messings and his colleagues (Norrander etal 1983).
These plasmids are of 2.7 K bp and possess the Col E1 ori of replication. These
vectors contain amp R gene and a new gene called lac Z, which was derived from the lac
operon of E.coli that encodes for B- galactosidase enzyme .
This enzyme splits lactose into glucose and galactose. A recombinant pUC molecule
is constructed by inserting new DNA in to one of the restriction site that are clustered near
the the start of the lac Z.
By using X-gal assay the blue colonies are developed in recombinant. Recombinants
can therefore be selected by the colour of colonies and there will be no need of replica plating
as done in the case of pBR 322.
5.2.2.3. pUN 121
This is a 4.4 Kbp vector derived from pBR 322. This vector has been developed for
rapid selection of bacteria containing a recombinant plasmid. This is required when genomic
libraries are constructed, which involves the construction of several thousands of clones and
handpicking of individual clones with inserted DNA would be extremely tedious. In these
vectors, the original promoter region of the tetracycline resistance gene is exchanged for the
promoter.
Plasmids serve as important tools in genetic engineering, it is a circular extra
chromosomal structure found in bacteria and in some yeast. Where they are commonly used
to multiply (make many copies of) or express particular genes and used to transform the
desired genes from one organism to another organism. Many plasmids are commercially
available for such uses. The gene to be replicated is inserted into copies of a plasmid which
25
contains genes that make cells resistant to particular antibiotics. Next, the plasmids are
inserted into bacteria by a process called transformation. Then, the bacteria are exposed to
the particular antibiotics. Only bacteria which take up copies of the plasmid survive the
antibiotic, since the plasmid makes them resistant. In particular, the protecting genes are
expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In
this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria
can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to
isolate the plasmid of interest.
Another major use of plasmids is to make large amounts of proteins. In this case,
researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the
bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce
large amounts of proteins from the inserted gene. This is a cheap and easy way of massproducing a gene or the protein it then codes for, for example, insulin or even antibiotics.
5.3. BACTERIOPHAGES
A bacteriophage (from 'bacteria' and Greek phagein, 'to eat') is any one of a number of
virus-like agents that infect bacteria. Phages are ubiquitous and can be found in all reservoirs
populated by bacterial hosts, such as soil or the intestine of animals. One of the densest
natural sources for phages and other viruses is sea water, where up to 109 virions per milliliter
have been found at the surface, and up to 70% of marine bacteria may be infected by phages.
They are also found in drinking water and in some foods, including fermented vegetables and
meats e.g. pickles, where they serve the function of controlling any growth of bacteria.
Phages are very specific to bacteria, and thus are more accurate and potent than antibiotics
5.4. COSMIDS
Plasmids with cos site are known as cos site. They have been developed in the late
1970s and have been improved significantly since.They are predominantly plasmids with a
bacterial oriV, an antibiotic selection marker and a cloning site, but they carry one, or more
recently two cos sites derived from bacteriophage lambda. Depending on the particular aim of
the experiment broad host range cosmids, shuttle cosmids or 'mammalian' cosmids (linked to
SV40 oriV and mammalian selection markers) are available. The loading capacity of cosmids
varies depending on the size of the vector itself but usually lies around 40-45 kb. The cloning
procedure involves the generation of two vector arms which are then joined to the foreign
DNA. cosmids always form colonies and not plaques! Also clone density is much lower with
around 105 - 106 cfu per g of ligated DNA.
After the construction of recombinant lambda or cosmid libraries the total DNA is
transfered into an appropriate E.coli host via a technique called in vitro packaging. The
necessary packaging extracts are derived from E.coli cI857 lysogens (red- gam- Sam and
Dam (head assembly) and Eam (tail assembly) respectively). These extracts will recognize
and package the recombinant molecules in vitro, generating either mature phage particles
(lambda-based vectors) or recombinant plasmids contained in phage shells (cosmids). These
differences are reflected in the different infection frequencies seen in favour of lambdareplacement vectors. This compensates for their slightly lower loading capacity. Phage
library are also stored and can be screened easier than cosmid libraries.
26
Target DNA: the genomic DNA to be cloned has to be cut into the appropriate size
range of restriction fragments. This is usually done by partial restriction followed by either
size fractionation or dephosphorylation (using calf-intestine phosphatase ) in order to avoid
chromosome scrambling, ie the ligation of physically unlinked fragments.
5.5. PHASMIDS
A phagemid or phasmid is a type of cloning vector developed as a co-infection of the
M13 helper phage and plasmids (phage + plasmid) to produce a smaller version of the virus.
An example is PRS314 which is of 4.78 kb size. Phagemids contain an ori for double
stranded replication as well as an ori for single stranded replication, mostly not comprising
the entire phagemid (i.e., only a small part of the phagemid is copied as a single strand.
Fig.1 STRUCTURE OF pBLUESCRIPT (PHAGEMID)

A phagemid is different from a closely related cloning vector called a cosmid. While a
cosmid can enter a host cell very effectively, almost all of the viral genes have been removed
to make room for exogenous DNA; therefore the virus cannot replicate itself. It is also having
an origin of replication. This means that in the presence of a 'helper' virus such as f1, the rest
of the genes to replicate viral proteins are present, therefore more virus particles can be
created using the host cell's resources.
In genetics, pBluescript (pBS) or pBluescript II is a commercially available phagemid
containing several useful sequences for use in cloning with bacteriophage. The sequences
27
include a polylinker sequence, antibiotic resistance sequence and an E. coli and f1 helper
phage origin of replication. The polylinker sequence is located within a LacZ controlled gene
designed to provide a blue coloration when expressed in bacteria. If the gene is disrupted by
successful insertion of a DNA sequence into to gene, the bacteria exhibit a white coloration in
Blue white screening, distinguishing successful recombination from those phagemids which
were not altered.
Fig.2 STRUCTURE OF pBLUESCRIPT

5.6. LET US SUM UP
DNA cloning technique allows individual DNA sequences in complex mixtures to be
isolated and copied permitting detailed analysis and manipulation. The DNA to be cloned is
recombined with vector DNA and introduced in to host cells where it is copied. Recombinant
vector purified from cultures of host cells and the cloned DNA can be recovered for analysis.
Plasmids are circular DNA molecules found in bacteria that are frequently used as cloning
vectors. Plasmids are easily purified and confer antibiotic resistance to host bacteria allowing
easy identification of recombinants. Later plasmids such as pUC identify recombinants by
blue/white selection based on disruption of the lac Z gene by insertion of the foreign DNA.
Additional plasmid modifications include multiple cloning sites, phage promoter sequences
for in vitro transcription and the ability to express cloned sequences as protein. Cosmids
resemble plasmids but contain lambda phage cos sequences which allow them to be packed
in to capsids. Packaged cosmids insert E.coli and are replicated. Cosmids do not contain
genes and so produce bacterial colonies instead of plaques. Cosmids can accommodate large
inserts upto about 44 kbp.
28
5.7. POINTS FOR DISUCSSION
1. Differentiate between cosmids and phasmids.
2. Discuss the advantages and disadvantages of cosmids.
1. What are plasmids and how do you classify them?
2. Describe different types of plasmids in detail.
3. Write about cosmids and phasmids.
4. Describe briefly on bacteriophages.
5.9. REFERENCE
1. Genes to Clones by L. Winnecker.
29
LESSON 6: SPECIALIZED VECTORS
CONTENTS
6.1. INTRODUCTION
6.2. SHUTTLE VECTORS
6.3. PLANT VIRAL VECTORS
6.4. ANIMAL VIRAL VECTORS
6.5. LET US SUM UP
6.8. REFERENCE
The chapter deals about the specialized vectors.
6.1. INTRODUCTION
An expression vector is generally a plasmid that is used to introduce and express a
specific gene into a target cell. Expression vector allows production of large amounts of
stable mRNA. Once the expression vector is inside the cell, the protein that is encoded by the
gene is produced by the cellular transcription and translation machinery. The plasmid is
engineered such that it contains a highly active promoter which causes the production of large
amounts of mRNA.
After expression of the gene product, the purification of the protein is required; but
since the vector is introduced to a host cell, the protein of interest should be purified from the
proteins of the host cell. Therefore, to make purification process easy, the cloned gene should
have a tag. This tag could be histidine (His) tag or any other marker protein.
Expression vectors are used for molecular biology techniques such as site-directed
mutagenesis. In general, DNA vectors that are used in many molecular biology gene cloning
experiments need not result in the expression of a protein. Expression vectors are often
specifically designed to contain regulatory sequences that act as enhancer and promoter
regions, and lead to efficient transcription of the gene that is carried on the expression vector.
Expression vectors are basic tools for biotechnology and used for the production of proteins
such as insulin that are important for medical treatments of specific diseases like diabetes
6.2. SHUTTLE VECTORS
A shuttle vector is a vector (eg. plasmid) constructed so that it can propagate in two
different hosts species (Lodish et al. 2007). DNA recombined into a shuttle vector can be
tested or manipulated in several cell types.
Shuttle vectors include plasmids that can propagate in eukaryotes and prokaryotes (eg.
both Escherichia coli and Saccharomyces cerevisiae) and in different species of bacteria (eg.
both E. coli and Rhodococcus erythropolis). There are also adenovirus shuttle vectors, which
can propagate in mammals.
Shuttle vectors are frequently used for efficient amplification. They can also used for
in vitro experiments.
30
One of the most common types of shuttle vectors is the yeast shuttle vector. Yeast
shuttle vectors have components that allow for replication and selection in both E. coli cells
and yeast cells. The E. coli component of a yeast shuttle vector includes an origin of transfer
and a selectable marker (eg. antibiotic resistance). The yeast component of a yeast shuttle
vector includes an autonomously replicating sequence (ARS), a yeast centromere (CEN), and
a yeast selectable marker (eg. URA3, a gene that encodes an enzyme for uracil synthesis)
(Lodish et al. 2007).
Fig.1 STRATEGY FOR CONSTRUCTION OF CLONINNG VECTOR
31
Viral vectors are a tool commonly used by molecular biologists to deliver genetic
thzmaterial into cells. This process can be performed inside a living organism (in vivo) or in
cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently
transport their genomes inside the cells they infect. Delivery of genes by a virus is termed
transduction and the infected cells are described as transduced. Molecular biologists first
harnessed this machinery in the 1970s. As an alternative to stable transformation using
Agrobacterium or direct DNA transfer, plant viruses can be employed as gene transfer and
expression vectors. There are several advantages to the use of viruses. First, viruses are able
to adsorb to and introduce their nucleic acid into intact plant cells. However, for many
viruses, naked DNA or RNA is also infectious, allowing recombinant vectors to be
introduced directly into plants by methods such as leaf rubbing. Second, infected cells yield
large amounts of virus, so recombinant viral vectors have the potential for high-level
transgene expression. Third, viral infections are often systemic. The virus spreads throughout
the plant allowing transgene expression in all cells. Fourth, viral infections are rapid, so large
amounts of recombinant protein can be produced in a few weeks. Finally, all known plant
viruses replicate episomally, therefore the transgenes they carry are not subject to the position
effects that often influence the expression of integrated trans genes. Since plant viruses
neither integrate nor pass through the germline, plants cannot be stably transformed by viral
infection and transgenic lines cannot be generated. However, this limitation can also be
advantageous in terms of containment.
A complete copy of a viral genome can also be introduced into isolated plant cells or
whole plants by Agrobacterium or direct DNA transfer. In this manner, it is possible to
generate transiently transformed cell lines or transgenic plants carrying an integrated
recombinant viral genome. In the case of RNA viruses, transcription of an integrated cDNA
copy of the genome yields replication-competent viral RNA, which is amplified episomally,
facilitating high-level transgene expression. Transgenic plants are persistently infected by the
virus DNA can produce large amounts of recombinant protein. In the case of DNA viruses,
Agrobacterium-mediated transient or stable transformation with T-DKA containing a
partially duplicated viral genome can lead to the "escape" of intact genomes, which then
replicate episomally. The latter process, known as "agroinfection" or "agroinoculation"
provides a very sensitive assay for gene transfer. More recently, the Agrobacterium-mediated
delivery of viral genomes has been enhanced through a process called magnifection, in which
amplification of the vector occurs in all infected leaves (Marrilonnet et al. 2005), As well as
their use for the expression of whole foreign proteins, certain plant viruses have recently been
developed to present short peptides on their surfaces. Epitope-display systems based on
cowpea mosaic virus, alfalfa mosaic virus, potato virus X, and tomato bushy stunt virus have
been developed as a potential source of vaccines, particularly against animal viruses
(reviewed by Lomonossoff & Hamilton 1999, Pogue et al. 2002, Yusibov& Rabindran 2004,
Khalsa et al. 2004, Twyman et al. 2005).
32
The genomic map of CaMV

Fig.2 GENOMIC MAP OF CaMV
The first plant viral vectors were based on DNA viruses because of their small and
simple genomes. The vast majority of plant viruses have RNA genomes. However, the two
groups of DNA viruses that are known to infect plants - the caulimoviruses and the
geminiviruses - were the first to be developed as vectors because of the ease with which their
small, DNA genomes could be manipulated in plasmid vectors. The type member of the
caulimoviruses is cauliflower mosaic virus (CaMV). The 8-kb dsDNA genome of several
isolates has been completely sequenced, revealing an unusual structure characterized by the
presence of three discontinuities in the duplex, There are eight tightly packed genes,
expressed as two major transcripts: the 35S RNA (which essen. tially represents the entire
genome) and the 19S RNA (which contains the coding region for gene VI), As discussed
earlier in the chapter, the promoter terminator sequences for both transcripts have lutilized in
plant expression vectors. And the 3SS water is particularly widely used only two of the genes
in the CaMV genome are essential for replication (gene II and gene VII). Since CaMV has an
icosahedral capsid, the size of be genome cannot be increased greatly without tiding the
efficiency of packaging. The maximum capacity of the CaMV capsid has been defined as
t3kb, and with the removal of all non-essential FOes. this represents a maximum insert size of
less !ban I kb (Daubert et al. 1983). This restriction in die capacity for foreign DNA
represents a major Imitation of CaMV vectors. Thus far it has been possible 10 express a
number of very small transgenes, ch as the 240-bp bacterial dhfr gene (Brisson et aI. 19841,
the 200-bp murine metallothionein cDNA /Ilfebm et al. 1987) and a SOO-bp human interkron
cD!\A (de Zoeten et al. 1989). SV40 a virus that infects primate cells through the
33
development of replicon vectors and helper viruses or complementary cell lines supplying
essential funclionin trans. Unfortunately. such an approach is not possible with CaMV due to
the high level of recombination that occurs, leading to rapid excision of the foreign DNA
(Gronenborn & Matzeit 1989). Geminiviruses are characterized by their twin (geminate)
virions comprising two partially fused icosahedral capsids. The small single-stranded DNA
genome is circular and in some species is divided into two segments called DNA A and DNA
B. Interest in geminivirus vector development was stimulated by the discovery that such
viruses use a DNA replicative intermediate suggesting they could be more stable than CaMV,
whose RNA-dependent replication cycle is rather error-prone (Stenger et al. 1991). of the
three genera of geminiviruses. two have been developed as vectors. The begomoviruses have
predominantly bipartite genomes; they are transmitted by the whitefly Bemisia tabaci and
infect dicots. Species that have been developed as vectors include African cassava mosaic
virus (ACMV) and tomato golden mosaic virus (TGMV). The mastreviruses have
monopartite genomes; they are transmitted by leafhoppers and predominantly infect
monocots. Species that have been developed as vectors include maize streak virus (MSV) and
wheat dwarf virus (WDV).
An important additional distinction between these genera is that mastreviruses are not
mechanically transmissible. MSV for example has never been introduced successfully into
plants as native or cloned DNA. Grimsley et aI. (1987) were able to overcome this problem
using Agrobacterium. and were the first to demonstrate the principle of agroinfection. They
constructed a plasmid containing a tandem dimer of the MSV genome. This dimer was
inserted into binary vector. and maize plants were infected with A. tumefaciens containing
the recombinant T-DNA. Viral symptoms appeared within two weeks of inoculation.
Agroinfection has been used to introduce the genomes of a number of different viruses into
plants. It can be demonstrated that if the T -DNA contains partially or completely duplicated
genomes single copies of the genome can escape and initiate infections. This may be
mediated by homologous recombination or a replicative mechanism (Stenger et aI.1991). The
study of Grimsley et aI. (1987) incidentally provided the first evidence that Agrobacterium
could transfer T -DNA to maize. Agroinfection is a very sensitive assay for transfer to the
plant cell because of the amplification inherent in the virus infection and the resulting visible
symptoms.
A number of geminiviruses have been developed as expression vectors because of the
possibility of achieving high-level recombinant protein expression as a function of viral
replication (reviewed by Stanley 1993. Timmermans et aI. 1994. Palmer & Rybicki 1997). A
generally useful strategy is the replacement of the coat protein gene, since this is not required
for replication and the strong promoter can be used to drive transgene expression. In the case
of begomoviruses, which have bipartite genomes, the coat protein gene is located on DNA A
along with all the functions required for DNA replication. Replicons based on DNA are
therefore capable of autonomous replication in protoplasts (e.g. Townsend et al. 1986).
Geminivirus replicon vectors can facilitate the high-level transient expression of foreign
genes in protoplasts. There appears to be no intrinsic limitation to the size of the insert,
although larger transgenes tend to reduce the replicon copy number (e.g. Laufs et al. 1990,
Matzeit et al. 1991). Generally, it appears that mastrevirus replicons can achieve a much
higher copy number in protoplasts than replicons based on begomoviruses. A WDV shuttle
vector capable of replicating in both E. coli and plants was shown to achieve a copy number
of greater than 3 x 104 in protoplasts derived from cultured maize endosperm cells
(Timmermans et al. 1992), whereas the typical copy number achieved by TGMV replicons in
tobacco protoplasts is less than 1000 (Kanevski et al. 1992). This may, however reflect
34
differences in the respective host cells rather than the intrinsic efficiencies of the vectors
themselves. Geminiviruses are also valuable as expression vectors in whole plants. In the
case of the mastreviruses, all viral genes appear to be essential for systemic infection, so coat
protein replacement vectors cannot be used in this manner. In contrast. the coat protein genes
of ACMV and TGMV are nonessential for systemic infection. but they are required for insect
transmission (Briddon et al. 1990) Therefore replicon vectors based on these viruses provide
an in planta contained transient expression system. Note that viral movement functions are
supplied by DNA B, so systemic infections occur only if DNA B is also present in the plant.
In an early study, Ward et al. (1988) replaced most of the ACMV A VI gene with the cat
reporter gene. In infected tobacco plants, high-level CAT activity was detected for up to four
weeks. Interestingly, they found that deletion of the coat protein gene caused a loss of
infectivity in plants, but this was restored upon replacement with cat, which is approximately
the same size as the deleted gene. This and many subsequent reports indicated that. while
there may be no intrinsic limit to the size of replicon vectors in protoplasts. systemic infection
is dependent on preserving the size of the wild-type DNA A component. A further limitation
to this system is that the transmissibility of the recombinant genomes is poor, probably
because they are not packaged in which this can be addressed is to generate transgenic plants
in which recombinant viral gene are produced in every cell. This is achieved by forming
plants with DNA constructs contain partially duplicated viral genome (Meyer elill.) Intact
replicons can excise from the delivered gene in the same way as the MSV genome during
agroinfection, and autonomously re ing episomal copies can be detected. Tran tobacco plants
have also been produced car integrated copy ofDNA B (Hayes et al. 1988,1'1 In the presence
of replication functions s by DNA A, the DNA B sequence is rescued the trans gene and can
replicate episomally, D can then provide movement functions to Dr facilitating the systemic
spread of the vector. Most plant virus expression vectors are b on RNA viruses because they
can accept larger transgenes than DNA viruses Most plant RNA viruses have a filamentous
morphology, so the packaging constraints affect use of DNA viruses such CaMV.
A breakthrough was made in 1984, when full-length clone corresponding to the genome
brome mosaic virus (BMV) was obtained. Infecting RNA could be produced from this cDNA
by transcription (Ahlquist & Janda 1984, Ahlquist 1984). The BMV genome comprises three
segments RNAl, RNA2, and RNA3. Only RNA land RNA necessary for replication. RNA3,
which encodes the viral coat protein and movement protein During BMV infection, a
subgenomic RNA tnagment is synthesized from RNA3, containing the coati tein gene alone.
It is therefore possible to raise the coat protein gene with foreign DNA and generate a
productive infection (Ahlquist et al.). This was demonstrated by French et al. in an
experiment where the coat protein genewa stituted with the cat reporter gene. Followi
introduction of recombinant RNA3 into barl toplasts along with the essential RNAI and
segments, high-level CAT activity was achiev This experiment showed that brame mosal was
a potentially useful vector for foreign gene expression. However, to date, BMV has bel
Franconi et al. 1999, Ziegler et al. 2000) and for the expression of genes that affect plant
physiology (e.g. the fungal avirulence gene avr9, Hammondbosack et al. 1995). The stable
transformation of plants with cDNA copies of the PVX genome potentially provides a
strategy for extremely high-level transgene expression, because transcripts should be
amplified to a high copy number during the viral replication cycle. However, instead of highlevel expression, this strategy leads to potent and consistent transgene silencing, as well as
resistance to viral infection (English et al. 1996. English & Baulcombe 1997). In terms of
vector development. however. it is notable that PVX-based vectors are probably most widely
used to study virus-induced gene silencing and related phenomena (Dalmay et al. 2000) and
to deliberately induce silencing of homologous plant genes (reviewed by Baulcombe 1999).
35
Retroviruses are the one of mainstays of current gene therapy approaches. The
recombinant retroviruses such as the Moloney murine leukemia virus have the ability to
integrate into the host genome in a stable fashion. They contain a reverse transcriptase which
allows integration into the host genome. They have been used in a number of FDA-approved
clinical trials such as the SCID-X1 trial.The primary drawback to use of retroviruses such as
the Moloney retrovirus involves the requirement for cells to be actively dividing for
transduction. As a result, cells such as neurons are very resistant to infection and transduction
by retroviruses. There is a concern for insertional mutagensis due to the integration into the
host genome which can lead to cancer or leukemia.
Lentiviruses are a subclass of retroviruses. They are widely adapted as vectors and
has the ability to integrate into the genome of non-dividing as well as dividing cells. The viral
genome in the form of RNA is reverse-transcribed when the virus enters the cell to produce
DNA, which is then inserted into the genome at a random position by the viral integrase
enzyme. The vector, now called a provirus, remains in the genome and is passed on to the
progeny of the cell when it divides. The site of integration is unpredictable, which can pose a
problem. The provirus can disturb the function of cellular genes and lead to activation of
oncogenes promoting the development of cancer, which raises concerns for possible
applications of lentiviruses in gene therapy.
Lentiviral vectors lacks the genes for their replication. To produce a lentivirus, several
plasmids are transfected into a so-called packaging cell line, commonly HEK 293. One or
more plasmids, generally referred to as packaging plasmids, encode the virion proteins, such
as the capsid and the reverse transcriptase. Another plasmid contains the genetic material to
be delivered by the vector. It is transcribed to produce the single-stranded RNA viral genome
and is marked by the presence of the (psi) sequence. This sequence is used to package the
genome into the virion.
As opposed to lentiviruses, adenoviral DNA does not integrate into the genome and is
not replicated during cell division. This limits their use in basic research, although adenoviral
vectors are occasionally used in in vitro experiments. Their primary applications are in gene
therapy and vaccination. Since humans commonly come in contact with adenoviruses, which
cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response
with potentially dangerous consequences. To overcome this problem scientists are currently
investigating adenoviruses to which humans do not have immunity.
6.5. LET US SUM UP
A number of plant and animal viruses have also been used as vectors both for
introducing foreign genes into cells and for gene amplification and expression in host cells.
Following are some of the viruses that are commonly used as vectors: (i) plant viruses
cauliflower mosaic virus and gemini viruses (ii) Animal viruses baculo virus, adeno virus
papova viruses, herpes virus, retro virus.
36
1. Analyse the need for shuttle vector.
2. Describe the genome map of caMv.
1. How shuttle vectors differ from plasmids?
2. Describe about plant vectors?
3. Give details about animal cloning vectors.
4. How lenti viruses can be used as vectors?
6.8. REFERENCE
Genes to Clones by L. Winnecker.
37
LESSON 7: CLONING STRATEGIES
CONTENTS
7.1. INTRODUCTION
7.2. cDNA CLONING
7.3. LET US SUM UP
7.6. REFERENCE
The chapter discuss about the cloning strategies and cDNA.
7.1. INTRODUCTION
Cloning of any DNA fragment essentially involves four steps: fragmentation, ligation,
transfection, and screening/selection. Although these steps are invariable among cloning
procedures a number of alternative routes can be selected, these are summarized as a cloning
strategy.
Initially, the DNA of interest needs to be isolated to provide a relevant DNA segment
of suitable size. Subsequently, a ligation procedure is employed whereby the amplified
fragment is inserted into a vector. The vector (which is frequently circular) is linearised by
means of restriction enzymes, and incubated with the fragment of interest under appropriate
conditions with an enzyme called DNA ligase. Following ligation the vector with the insert of
interest is transfected into cells. A number of alternative techniques are available, such as
chemical sensitivation of cells, electroporation and biolistics. Finally, the transfected cells are
cultured. As the aforementioned procedures are of particularly low efficiency, there is a need
to identify the cells that have been successfully transfected with the vector construct
containing the desired insertion sequence in the required orientation. Modern cloning vectors
include selectable antibiotic resistance markers, which allow only cells in which the vector
has been transfected, to grow. Additionally, the cloning vectors may contain colour selection
markers which provide blue/white screening (-factor complementation) on X-gal medium.
Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present
in the cells obtained. Further investigation of the resulting colonies is required to confirm that
cloning was successful. This may be accomplished by means of PCR, restriction fragment
analysis and/or DNA sequencing.
7.2. cDNA CLONING
One of the more powerful approaches which has been developed over the last twenty
years is the ability to make, isolate and use DNA that are complementary (cDNA) to
messenger RNAs (mRNAs) that encode proteins of interest.
Cloning refers to the isolation of a genetically homogeneous strain of any organism.
Within a clone, all organisms are identical to all other organisms at a genetic level. It
is possible to clone bacteria or phage or even higher plants by isolating a single cell
and allowing that single cell to produce a colony, or a plaque, or an entire plant. Since
38
most plants are derived from a single cell with a unique genotype, the act of rooting
leaves to produce a collection of identical African violets is cloning.
o cDNA cloning is isolating and amplifying a single, self-replicating organism
that includes within its DNA, a cDNA that is of interest.
o In some cases cDNA cloning may simply refer to the isolation of any single
cDNA, since, in some circumstances, an experimentalist may be interested in
any cDNA produced by a particular tissue. More frequently, the challenge of
cDNA cloning is not the isolation of any cDNA but the selection of a single
cDNA that is of interest to the experimentalist for a particular reason. In the
same way it is possible to isolate clones that are not cDNA clones but rather
are genomic clones.
o Genomic clones are simply DNA derived directly from a genome. Genomic
DNA would incorporate some sequences such as introns or regulatory
sequences that would not be found in cDNAs.
o Likewise, the isolation of a monoclonal antibody refers to the isolation of a
single cell that expresses a mRNA for a unique antibody. Thus, making
monoclonal antibodies an exercise in cloning.
Library. The second concept that is important in understanding the strategy needed to
isolate a cDNA clone, a genomic clone, or even a monoclonal antibody is the idea of a
library.* A library is defined simply as a collection of different DNA sequences that
have been incorporated into a vector.
Vector. A vector is simply a self-replicating organism which is usually designed for
the convenience of its experimental purpose. For experimental convenience, vectors
are usually derivatives of viruses (plasmids, bacteriophages, animal viruses,
retroviruses). Since the essence of being able to isolate clones is the ability to
replicate to make large amounts of biological material, the essence of a vector is that
it must incorporate some mechanism of reproduction. (i.e., one must expand the clone
to make many copies of the same organism). Thus, one would expect that vectors
would incorporate an origin of DNA replication. Since vectors are an important
experimental approach, a considerable amount of effort has gone into designing
vectors that are particularly easy to use in an experimental sense. It would be
impossible to provide even a brief description of the tricks that have been
incorporated into various classes of vectors. The incorporation of selectable markers
is certainly a significant experimental advantage in many cases.
7.2.1. CLONING STRATEGY

The underlying experimental approach to cloning can be divided into four parts.
First, it is necessary to produce or obtain a library including the sequence of interest.

Second, it is necessary to isolate clones that may be of interest.
Third, it is essential to develop a formal test to ensure that the clones that have been
isolated are indeed the correct clones.
Fourth, it is essential to put the cDNA that has been isolated to some interesting
biological use.
7.2.2. cDNA LIBRARIES

Let's consider the important aspects of constructing a cDNA library. A cDNA library
simply contains sequences that are complementary to mRNAs. There are a number of
39
different criteria that might be used to judge the quality of a cDNA library. A cDNA library is
generally better if the size of the inserts (that is the amount of continuous cDNA in each
clone) is large, ideally full-length. Ideally, no member of the library should include cDNAs
derived from different mRNAs (this could be confusing). The library should be sufficiently
large that it contains the cDNA of interest (or,more precisely, it should have enough
independently derived clones that it contains the cDNA of interest). In general this means that
it should be representative of all the mRNAs present in a particular tissue. Of course,
choosing a tissue that has a relatively large amount of the mRNA of interest is an important
experimental choice. In general it is easier to isolate a cDNA from a library where it is
represented many times than from a library where it is present rarely. Some characteristics of
a library depend on the vector chosen. Some vectors are designed to express only the cDNAs,
while others have been modified to express not only the cDNA but also to express it in a
context so the cDNA is made into a protein or a fusion protein. Before using a cDNA library
it is wise to determine if it is a good quality library.
7.2.3. MAKING cDNA
Generation of cDNAs can also be done by a wide variety of processes, but, in
virtually all cases, cDNA is generated by the enzyme reverse transcriptase* (RT) which has
the ability to use the information in an RNA to generate a complementary DNA. Thus,
reverse transcriptase is a RNA-dependent DNA polymerase. Like all DNA polymerases it
cannot initiate synthesis de novo but depends on the presence of a primer. Since many
mRNAs have a poly-A tail at the 3' end, oligo-dT is frequently used to prime DNA synthesis.
Once the initial cDNA has been generated it is generally necessary to produce a second
strand of DNA. Again, there are many strategies for doing this, but a convenient mechanism
involves exposure of the DNA/RNA hybrid to a combination of RNAase-H and DNA
polymerase. RNAase-H has the ability to cause single-stranded nicks in the RNA, and DNA
polymerase can then use these single-stranded nicks to initiate " second strand" DNA
synthesis. This two-step procedure has been optimized to maximize fidelity and length of
cDNAs.
7.2.4. INCORPORATING cDNA INTO THE VECTOR
The next challenge is to incorporate this collection of cDNA s into a vector so that it
can be manipulated. One of the most convenient ways of doing this is to attempt to
manipulate the cDNAs so that each one has a unique restriction site at those ends. To do this,
the cDNAs are frequently methylated with a specific methyl transferase that incorporates a
methyl group into particular restriction site to protect them from the restriction enzyme that
will be used later. Any 3' or 5' extensions must be then either eliminated by nuclease
treatment or filled in with polymerase. This produces a "blunt ended" molecule in which the
3' and 5' bases are in "register". It is then possible to ligate a synthetic oligonucleotide to the
ends of this cDNA . Blunt end ligation is generally a low efficiency process; but, by using a
high concentration of these synthetic oligonucleotides, it is possible to drive the reaction to
near completion. These synthetic oligonucleotides can either be 'linkers' (which are
synthesized to have one blunt end and one end that have an 'overhang' (i.e., region of single
stranded DNA) that is complementary to that produced by restriction enzymes or they can be
'adapters' (which are a double-stranded DNA molecule that can be treated with a nuclease to
produce the appropriate overhang).
40
The value of producing an overhang is that it will facilitate the introduction of the
cDNA into a vector. The vector can also be prepared by treating it with the same nuclease, or
a nuclease that produces the same restriction site, to produce a single-stranded region that is
complementary to the single-stranded region in the cDNA. Mixing the cDNA of interest with
the vector in the presence of ligase allows incorporation of the cDNA into the vector. One of
the experimental difficulties in doing this is that the vector itself will have a high tendency to
re-ligate to form a vector without any cDNA insert. This is frequently minimized by treating
the vector with the phosphatase to remove the terminal phosphates. These phosphates are
required for ligase to act, so this strategy prevents this unwanted side reaction.
The choice of the vector used also has an important impact on experimental outcome.
Initially, plasmids were chosen as vectors and were modified to include markers that could be
used to determine whether a plasmid had been introduced into a bacterial cell or whether
there was a cDNA insert in the cloning site. More recently, derivatives of bacteria phage
lambda has been made that can be effective vectors for cDNA cloning. The advantage of
bacteria phage lambda is that it is possible to isolate more independent clones from a given
amount of mRNA/cDNA and to screen a higher number of clones using hybridization
techniques. The extent of understanding of lambda and lambda genetics has made it possible
to isolate lambda derivatives where some non-essential genes have been removed making it
possible to carry inserts of up to 11 kb of cDNA, which is a convenient size and sufficient for
the isolation of most cDNAs. The lambda genome is a linear molecule when it is packaged
into the bacteriophage and the cDNA can be incorporated into the central region of the DNA.
The lambda "arms" (the more distal parts of the DNA) encode all the essential information
for replication of lambda in an infectious cycle. The cloning site in lambda -gt10 was chosen
to interrupt genes that are essential for lambda to undergo lysogeny. If the lambda arms religate in the absence of an insert, and an appropriate host is chosen (hfl, for high frequency of
lysogeny), then these particles will not form plaques. Thus only particles carrying an insert
will form plaques. The remarkable power of bacteriophage lambda as a vector is that once the
cDNA has been ligated into the lambda arms, the DNA can then be incorporated into a phage
particle in vitro. Extracts prepared from cells that have all the necessary proteins for the
assembly of lambda can then be mixed with the library DNA and ATP and particles will be
assembled. These particles can then be used to infect E coli and each individual plaque is an
independent clonal population which represents a single cDNA species. This ability can be
used both to amplify the cDNA library (which is somewhat dangerous because repeated
amplification can lead to a loss of some cDNA sequences) and for the screening of the cDNA
library to isolate the cDNA of interest.
7.3. LET US SUM UP
Gene cloning has made important contributions to many areas of research in biology.
These include: identification of genes involved in disease process; construction of genome
maps; production of recombinant proteins; and the creation of genetically modified
organisms. All DNA cloning experiments are based on the construction of rDNA molecules.
This involves joining different DNA molecules together. The DNA molecule to be cloned is
inserted in to another, usually circular DNA molecule called a Vector. The recombinant
vector is introduced in to a host cell, usually the bacterium E.coli, where it produces multiple
copies of itself. When the host cell divides copies of the recombinant are passed on to the
daughter cells. Large amounts of the vector are produced which can be purified from cultures
of the host cells and used for analysis of the foreign DNA insert.
41
1. Sustify the need for cDNA.
2. Critically analyse role of cDNA in cloning.
7.4. LESSON END ACTIVITIES
1.
2.
3.
What are the usual cloning strategies?

How will you make a cDNA library?
What is the method for incorporating cDNA in to a vector?
7.5. REFERENCES
1. Gene to Clones by L. Winnecker.
2. Reference papers cited within the lesson.
42
LESSON 8: SCREENING STRATEGIES
CONTENTS
8.1. INTRODUCTION
8.2. DESIGNING A PROBE
8.3. GETTING FULL LENGTH cDNAs
8.4. CLONING FROM EXPRESSION LIBRARIES
8.5. COMPLEMENTATION
8.6. EXPRESSION ON THE CELL SURFACE WITH ANTIBODY SCREENING
8.7. FUNCTIONAL CLONING OF RECEPTORS
8.8. HOMOLOGY SCREENING
8.9. PCR-BASED SCREENS
8.10. PLUS/MINUS SCREENING AND DIFFERENTIAL DISPLAY
8.11. TWO HYBRID SCREENING
8.12. SCREENING BY DATABASES
8.13. cDNAs AND EXPERIMENTAL DESIGN
8.14. LET US SUM UP
8.17. REFERENCES
The chapter deals with the screening strategies of the cloned DNA.
8.1. INTRODUCTION
A lambda gt10 library can be conveniently screened by plating it at relatively high
concentrations on a bacterial lawn of E coli. High density screening allows the
experimentalist to screen between 100,000 and 1,000,000 independent plaques on a single
plate and makes it theoretically possible to screen for a cDNA that is present only at one copy
per cell in a particular tissue. Screening is done by a "replica plating" procedure. After the
phage infect E coli and form individual plaques, a perfect spatial representation of the
infected plaque can be produced by placing a piece of nitrocellulose on top of the lawn of Ecoli. Nitrocellulose binds DNA with great avidity and so some of the DNA of each plaque
can be transferred to nitrocellulose paper or even several different nitrocellulose papers. each
nitrocellulose sheet should have a representation of the original pattern of infected cells on a
petri dish. The DNA from the library can then be cross-linked to the filter and extraneous
protein can be washed off. The plaques of interest can then be screened using a hybridization
assay.
A probe is an oligonucleotide that is designed to be complementary to the mRNA of
interest so that it can be used to screen a library. Of course, any mRNA produces a unique
polypeptide when it is translated; but the reverse is not true. Because the triplet code is
degenerate, there are many mRNA sequences that might produce the same amino acid
sequences. Because of this the design of an oligonucleotide probe is not straight forward, but
a clever experimentalist can make good choices in designing a probe. There are basically two
43
strategies that can be used. Either the experimentalist can choose to design a relatively short
oligonucleotide that hopefully will have a high degree of homology to the mRNA of interest
or the experimentalist can choose to design a longer probe that is more likely to have some
regions that are not complementary to the mRNA of interest but hopefully will have at least
some sequences that can form a stable duplex. In many cases it makes sense to make a
mixture of different probes, which are homologous, but have different bases in positions
where it is not possible to make a good prediction of which one should be present. This is
called degeneracy. A probe can frequently be 64 or 128 fold degenerate; but too much
degeneracy reduces the specific activity of a probe and increases the chance of hybridization
with the 'wrong' cDNA. The choice of which strategy depends on the amino acid sequences
that are available.
Testing of any probe for its correspondence to known sequences in the data base is,
thus, essential. Using amino acids or amino acid combinations that have fewer potential
triplet coding sequences or lower degree of degeneration (i.e., potential sequences) is of great
importance. If multiple related probes are possible, it is often sensible to screen with a
degenerate oligonucleotide. Once a probe or a series of probes are designed they can be
synthesized chemically and labeled to high specific activity with 32 P. The oligonucleotide
probes can then be incubated with the nitrocellulose filters to allow hybridization. Then the
filters are washed to remove unlabeled or non-specifically bound oligonucleotides. The filters
can then be autoradiographed to identify the regions of the filter corresponding to a,
hopefully, specific signal. Since the filter is a replicate of the original plate, the
experimentalist can then return to this plate and isolate the original plaque or group of
plaques responsible for the signal. The plaques can then be re-plated on fresh E coli
(remember each plaque contains phage that can infect and replicate in E. coli), and the
process is repeated to eventually isolate a single plaque that is responsible for the signal.
8.2.1. TEST FOR SPECIFICITY
While the isolation of a plaque that gives a strong signal is clearly an exciting step, it
is only the first step. The next question must be asked: is the isolated cDNA really the one of
interest? It could certainly be a cDNA for a related protein or a completely unrelated protein
that just happened to have a sequence that would hybridize to the probe that was chosen.
There are a number of criteria that will fulfill this need. The simplest takes advantage of
sequence information that can be obtained from the isolated cDNA. In contrast to proteins
where getting sequence information is experimentally difficult, it is relatively straight
forward to get sequence information from DNA. The cDNA can be subcloned into a
convenient vector and sequence information can be obtained. If the sequence of the cDNA
that has been isolated also encodes the sequence of some of the peptides that had been
sequenced but not used to design a probe, this is certainly persuasive evidence that the correct
cDNA has been isolated.
Frequently, there are also elements of the structure of the predicted protein that can be
used to help confirm the correctness of the cloning procedure. During the characterization of
the protein, it is frequently known that the protein for example may be a membrane protein in
which case one might predict the existence of transmembrane sequences. Some proteins are
known to be phosphoproteins which suggest the presence of either serines or threonines in
particular context that will allow kinase to phosphorylate them. Likewise, some proteins are
glycosylated and the presence of amino acid sequences that are associated with glycosylation
will also support the correctness of the cloning approach. Again, it must be emphasized that
44
all of these are simply criteria that the correct cDNA has been isolated. These must be used
by the experimentalist to develop a convincing case, but none are absolutely fool-proof. In
some cases, the pattern of expression of a protein (a tissue-specific manner) or a change in
mRNA in organisms that carry a particular mutation that is known to influence the activity of
the protein of interest can be a powerful criterion that will allow the experimentalist to make
a persuasive case that the correct cDNA has been isolated.
8.3. GETTING FULL LENGTH cDNAs
In some cases, indeed in most cases, the cDNA that is isolated will not be full-length,
i.e. it will correspond only to parts of mRNA but not the entire sequence. In this case it is
necessary to re-screen the library, generally using the cDNA that already has been isolated to
identify either a full-length cDNA or a series of partial cDNAs that would encompass the
entire cDNA of interest. This brings up the interesting question of how an experimentalist
knows whether a full-length cDNA has indeed been isolated. Consideration of basic
molecular biology can provide a number of clues in this question. The molecular weight of a
mRNA can be estimated by northern analysis and this can be compared to the size of the
cDNA that has been isolated. Of it is possible that several mRNAs may be generated from a
single gene by alternative splicing and this should be remembered. A mRNA should include
both a coding region which has a long open-reading frame as well as non-coding sequences
(frequently called UTRs, for untranslated regions) at both the 3' and 5' ends. An open reading
frame is simply an un-interrupted series of triplets that does not contain stop codons. Such a
coding sequence should predict a protein of an appropriate molecular weight which can often
be compared to the molecular weight of the known protein. Upstream of the translation start
site are frequently, but not always, found stop codons. The 3' end of the message frequently
has a poly-A tail. There is almost always special interest in clearly identifying the 5' end of
the mRNA. This sequence is often most difficult to obtain from a cDNA library since it
requires effective reverse transcriptase to the extreme end of the mRNA. Often it is necessary
to return to a cDNA library repeatedly or use specialized approaches to isolate an authentic 5'
end. Often, the best way to identify sequences at the 5' end of a cDNA is to use RACE, which
is a PCR based technique to amplify DNA sequences near either the 5' or the 3' end of a
DNA. The authenticity of a particular 5' end can be confirmed by doing 'primer extension'
experiments. In this technique, reverse transcriptase is used to extend an oligonucleotide
primer which has been designed to hybridize near the predicted 5' end of a mRNA. The
extension of such a primer should produce a polymer of a specific and predicted size.
8.3.1. OTHER METHODS OF ISOLATING cDNAs
The choice of how to isolate cDNAs depends on the interest of the investigator and
the tools that are available. Design of an oligonucleotide probe has been used effectively in
many cases but there are many other additional approaches that can be used. A few of them
will be listed and described in this section.
8.4. CLONING FROM EXPRESSION LIBRARIES
In many cases a vector can be designed so that the cDNA will be expressed,
frequently as a fusion protein. In this case the cDNA has been incorporated into a vector in a
position where it is within a coding sequence of another protein. The vector also incorporates
promoter sequences that allows the protein to be expressed (both transcribed and translated).
When such a vector is used to make a library it is called an expression library. Expression
45
libraries have the advantage and disadvantage that the protein is present. In some cases this
may mean that there may be selective pressure against the expression a cDNA of interest, but
in many cases this expression allows for a novel screening approaches. The most straight
forward of these is the use of antibodies to screen a library.
Screening with an antibody is quite similar to screening with an oligonucleotide
probe, but in this case an antibody to the protein is the reagent that is available. This antibody
can be generated experimentally, but it can also be available because of interesting
autoimmune response in an animal model or in a human population. For example, some
cancer patients develop an autoimmune disease that leads to neuronal degeneration. The
antisera from these patients can then be used to isolate a gene which produces a protein that is
recognized by this antibody. The antibody can be added to nitrocellulose filters under
conditions where it binds specifically and the antibody can be then detected by a secondary
antibody that is either labeled with an isotope or covalently attached to an enzyme like horse
radish peroxidase that can be detected using standard enzymatic reactions.
If a cDNA is thought to encode a soluble factor that has a known biological effect,
and if that effect can be easily assayed, then the assay could be a way to screen the library,
although it may be difficult to screen a large number of independent isolates.
8.5. COMPLEMENTATION
Some genes can be isolated by a classic genetic complementation approach. If there is
a method to select for the expression of a particular gene then this selection can be used to
isolate a cDNA that encodes for that gene. For example it is relatively straight forward to
select either for or against the presence of the enzyme HGPRTase (hypoxanthine guanine
phospho ribosol transferase) in E. coli or in eukaryotic cells. If HGPRTase-deficient E. coli
can be isolated and then transformed with an expression vector, those cells expressing the
appropriate activity would become HGPRTase+. Since it is possible to select for such
colonies, this would be an easy way to isolate a cDNA for HGPRTase from any organism.
Complementation has been used to isolate many types of cDNAs including some that
regulate complex phenomenon like the cell cycle or membrane trafficking. The power of this
approach is that it provides such strong evidence for specific in vivo function. Of course, it is
essential to independently establish that the correct clone has been isolated.
8.6. EXPRESSION ON THE CELL SURFACE WITH ANTIBODY SCREENING
Cell surface receptors are special interest in biology and they can sometimes be
isolated using an expression strategy. Cell surface molecules on lymphocytes for example
have been identified by the isolation of specific monoclonal antibodies. Likewise, the ligand
for many receptors has been isolated before the nature of the receptor is established. In both
cases a cDNA for the cell surface molecule when expressed, will lead to the presence of a
binding site on the cell surface. This binding site can be used to screen a library either by a
method analogous to the antibody screening mentioned above or by using the ligand or
antibody as an affinity reagent to "pan" for cells that express the binding site.
8.7. FUNCTIONAL CLONING OF RECEPTORS
One of the more interesting classes of cell surface molecules are molecules that
encode ionic channels. Because of the tremendous power and sensitivity of the
46
electrophysiology (electrophysiologists can even measure the function of a single molecule),
the presence of one or a few mRNA molecules in a single cell can produce enough ion
channels to be detected relatively easily using an electrophysiological approach. Injection of
mRNAs in frog oocytes can lead to the appearance of particular ion channels that can be
detected either because of their responsiveness to electrical signals or the presence of
extracellular ligands. This approach provided a straight forward assay for the cell-surface
receptor for glutamic acid (glutamate), which is the most common neurotransmitter receptor
in the central nervous system. This type of approach can either rely on expression vectors that
can produce the mRNA of interest or it can rely on a negative criterion. Co-injection of
cDNAs can quench a signal by hybridizing specifically with the mRNA. Of course the
difficulty with any of these approaches is that it becomes more difficult to screen a large
number of mRNAs. This problem has been successfully conquered by using strategies
involving 'sib (for sibling) selection'. In this strategy, thousands of independent clones are
screened at once, and, once a signal is identified in any one pool, the pool itself can then be
subdivided until an individual clone can be isolated.
8.8. HOMOLOGY SCREENING
One of the most productive, although perhaps less creative approaches to isolating
cDNAs is homology screening. Once an interesting gene has been isolated from one species,
it is relatively straight forward to use a low stringency hybridization strategy to isolate
cDNAs from another species. Likewise, additional family members from the same species
can frequently be identified. The power of this approach should not be underestimated.
Interesting mutants are frequently obtained in Drosophila by using genetic screens and
identifying the existence of corresponding genes in humans can be tremendously important.
Likewise, because of the large population of humans in the careful monitoring of their
medical care, human genetic diseases are proving an abundant source of interesting genes and
eventually interesting cDNAs. Determining the existence of such cDNAs in model systems
can then be extremely valuable. Good examples of this come from the field of apoptosis.
Some of the original genes like the ICE protease were originally identified in studies of C.
elegans and subsequently human homologs of this genes were isolated. Likewise, the human
oncogene, bcl 2, was initially isolated by genetic studies which led to the isolation of the
cDNA and subsequently homologs were identified in model systems.
8.9. PCR-BASED SCREENS
PCR-based screening is also a method to isolate novel cDNAs. After two or more
members of a family have been isolated, regions of homology can be identified. These
regions of homology are conserved within the family; PCR primers can be designed and used
to amplify reverse transcriptase products of mRNAs in an appropriate tissue. The molecular
weight of known members of the family can be predicted and novel mRNAs may give rise to
novel amplification products. These amplification products in turn can be used to screen
cDNA libraries. In some cases even a single region of conserved structure may be sufficient
to isolate novel genes using the following strategy. Reverse transcriptase can be used to
extend a primer which has been made to a conserved sequence. Such products of course
could be heterogeneous because different reverse transcriptase molecules would extend to
different degrees. However, some restriction enzymes are capable of cleaving single stranded
DNA and treatment of such a product with an enzyme of this type would produce a fragment
of a unique size. Such a fragment can then be homo-polymer tailed (i.e. a sequence of Cs can
be added to the end of the molecule) using terminal transferase. This sequence of Cs can then
47
be used as a site to anchor an oligonucleotide primer containing a stretch of Gs. If this primer
is extended the resulting product will be suitable for PCR amplification between the two
primers that were used in its creation.
8.10. PLUS/MINUS SCREENING AND DIFFERENTIAL DISPLAY

Another useful approach to isolating cDNAs of interest relies not on knowledge of their
primary structure, but rather on assumptions about their expression. Both plus/minus
screening and differential display rely on strategies that seek to isolate cDNAs that are
expressed in one situation but not another. For example, growth factors like NGF or PDGF
and hormones like estrogen are known to induce the expression of novel genes. Thus, a
population of cells that are cultured or grown in the presence and absence of such an
experimental manipulation (e.g., +/-NGF, +/-estrogen, + /- retinoic acid) should express some
genes in common, but have some distinct mRNAs. Likewise, tissues at different
developmental stages may have expression patterns that are of special interest. Tissues that
are related but distinct may also express interesting subset of genes. There are presumably
interesting genes that are expressed in cerebellum, but not basal ganglia; or in T cells, but not
B cells. An isolation of those genes may give a clue to the function of those tissues or the way
gene expression is regulated.
In plus/minus screening, mRNA is isolated from two populations of cells and reverse
transcribed to produce a population of cDNAs. Aliquots of these cDNAs can then be
converted to probe by random hexamer priming and used to screen duplicate lifts
from a library (i.e., two nitrocellulose filters produced from the same plate of plaques
or cells. Any plaque or colony that hybridizes duplicate lifts from a library to one
probe but not the other is a potential candidate for interest, and differential expression
can be tested by northern analysis or a related approach.
Differential display is a simple modification of PCR amplification. In this approach,
mRNA is reverse transcribed using a series of primers. Frequently primers are chosen
to have a random set of oligonucleotide and an oligo-dT section that would hybridize
to a poly-A tail. mRNAs that are homologous to the randomly chosen sequence
should be reverse transcribed, producing a single-stranded cDNA. The addition of
another primer, again, randomly chosen will allow amplification of a subset of the
reverse transcribed cDNAs. Depending on the distance between the two primers,
fragments of varying molecular weights will be obtained. By doing this procedure
with mRNA that has been isolated from two different cell populations, the pattern of
expression between the two cell types or cell states can be determined. Again, an
amplified product that is thought to be unique to a particular cell type, can then be
used a probe to screen a library or test expression by northern analysis. Both of these
methods have been used to isolate large number of interesting genes using only their
expression pattern.
8.11. TWO HYBRID SCREENING

One of the more active approaches to isolating cDNA are the two-hybrid screens. These
screens are named because they take advantage of a specific protein -protein interaction that
occurs between two proteins each of which is itself a hybrid protein. The entire assay relies
on the ability of one part of each of the hybrid protein to form a specific interaction that is
48
reasonably stable under physiological conditions with the other. A number of variations of
this approach have been developed, but they all rely on the same feature.
In the most straight forward version a test cell which expresses an easily assayed
gene, like beta-galactosidase, under the control of a well characterized promoter is
produced. The promoter is chosen so that it has a low basal activity in the absence of
stimulation from a specific regulatory element.
The same cell is then transfected with an expression vector for a hybrid protein. One
part of the hybrid protein is derived from a transcription factor which is designed to
recognize the DNA regulatory element. Binding to the site, however, is not sufficient
to induce gene expression; rather, a specific mechanism to activate transcription is
required. A second part of this hybrid protein includes a sequence isolated from a
particular gene of interest. This protein can be derived from another transcription
factor, from a structural protein, or from an intracellular signaling protein. The only
requirement is that the hybrid itself is not sufficient to activate expression of the
reporter gene.
This cell system is then transfected with an expression library that also expresses a
collection of fused protein. In this case the fusion protein consists of two part. One
part of the fusion protein is coded by the collection of the cDNA libraries that the
experimentalist hopes may encode a protein which will interact with target in the
hybrid protein already expressed in the cell. The second part of this fusion protein is
an activator of transcription, frequently the activating region of VP16, a potent
transcription factor. If a cell is transfected with a hybrid protein that does not
recognize the hybrid protein already present in the cell nothing should happen.
In the rare case where VP16 is expressed as a hybrid with the protein that interacts
with the hybrid already present in the cell, this should result in activation of betagalactosidase. Thus, the screen serves as an initial assay for protein-protein interaction
and is structured in such a way that a large number of members of a cDNA library can
be quickly screened and selected for testing for specific interaction. Of course, there
are always the possibility that activation can occur by a non-specific mechanism, but
this possibility can be tested for without too much difficulty.
8.12. SCREENING BY DATABASES

The rapid accumulation of sequence information and genetic data often allows
scientists to bypass the steps required to isolate cDNAs. For example, if partial protein
sequence or partial cDNA sequence is available, searching data bases may result in
identifying candidate clones that can be ordered and tested to determine if they are the 'right'
clone. Databases include the sequence of entire genomes as well as short sequences from
cDNAs that serve to tag individual clones (ESTs, or expressed sequence tags).
The importance of isolating the cDNAs :1. Isolating cDNAs allows the experimentalist to use the cDNA to develop expression
vectors so proteins of interest can be produced in high quantities, greatly simplifying the task
of protein purification (e.g. see baculovirus expression.)
2. Knowing the sequence of an amino acid immediately gives access to the sequence
of the protein. By appreciating protein structure and studying the common motifs present in
known proteins, a great deal of information can be deduced about the possible structure
49
and/or function of the protein encoded by a known cDNA. Presence of sequences can easily
suggest the protein product may be phosphorylated or may bind a particular small
biochemical molecule, like GTP.
3. The availability of a mRNA allows one to quickly design assays for studying the
expression of the mRNA; labeling cDNA can be used to determine the expression of mRNA
using both northern analysis and RNase protection and the subcellular distribution of RNase
can be determined by in situ hybridization. Each of these approaches provides a specific
value.
4. Availability of a cDNA makes production of both polyclonal and monoclonal
antibodies much easier. Knowing the sequence of a protein allows one to design and
synthesize a peptide that can be used as an antigen (anti-peptide antigen). Thus, in some
cases, an antibody that recognizes a specific protein can be produced without ever purifying
that protein. It also allows the expression and purification of a protein to be used as an
antigen.
8.13. cDNAs AND EXPERIMENTAL DESIGN
The effort invested in isolating and characterizing a cDNA is well rewarded by the
large number of uses that can be made of such a reagent.
1. The most obvious use of a cDNA is to study expression of mRNA. This can be
done by northern analysis, by RNase protection assay, by PCR-based detection, or by in situ
hybridization. To detect mRNAs by northern analysis*, mRNA must be prepared and
fractionated by gel electrophoresis to separate mRNAs of different molecular weight. The
RNA on the gel can then be transferred to nitrocellulose and detected by hybridization with a
labeled cDNA. Label is generally incorporated into cDNAs by primer extension using a
random selection of oligonucleotide hexemers. This technique has the ability to distinguish
mRNAs of different molecular weights and so may reveal alternatively spliced products. RTPCR*is frequently chosen because it is a more sensitive method of identifying mRNAs. In
this technique, a probe is generated by using a vector that incorporates a promoter for an
RNA polymerase. This promoter can then transcribe in vitro a high specific activity RNA,
part of which can be designed to be homologous to any cDNA. This synthesized RNA is of
course extremely sensitive to ribonuclease treatment; however, if it is hybridized to a
preparation of mRNA that includes a complementary sequence, a hybrid will be formed and
this will render the RNA resistant to RNase digestion. In some cases the amount of protected
RNA can be measured directly but it can also be fractionated on gels to determine the
molecular weight of the protected species. Another useful approach is to take advantage of
sequence information and use RT-PCR (reverse transcription-polymerase chain reaction). In
this approach, mRNA is isolated, reverse transcribed to generate a complementary DNA, and
this complementary DNA is then amplified using PCR primers. Again this is a sensitive
method of detecting mRNA, but care is required to make quantitative claims about the
amount of mRNA present in various samples. Finally, hybridization can be carried out in
fixed tissues to determine what cell types express mRNA. Again, mRNA can be detected by
virtue of its hybridization with a labeled probe. Alternatively, a modified RT-PCR protocol
can also be done in situ. Thus all of these methods have the ability to detect mRNA
abundance and changes in mRNA among various cell types in response to development, and
in response to hormones or other signaling molecules.
50
2. The sequence of a mRNA is the quickest and most reliable way to identify the
sequence of the encoded protein. The sequence of an cloned DNA can be determined
relatively quickly by either Maxam-Gilbert Sequencing or Sanger Sequencing. With the
rapidly expanding DNA database and the appreciation of how specific amino acid sequences
can be used to define particular domains in proteins, the sequence information can be used
extremely profitably. For example, the sequence of a protein can be used to determine the
likelihood that particular regions of a protein will adopt an alpha-helix configuration.
Likewise particular sequences are associated with particular functions or particular structures.
The zinc finger motif is a particular protein structure that can bind zinc atoms with high
affinity and this structure is frequently found in DNA-binding proteins. Likewise, the helixloop-helix structure which includes two alpha-helices connected by a loop, is frequently
found in transcription factors. The catalytic triad is a sequence of 3 amino acids that is found
in many proteases. Protein sequence will also reveal the presence of particular sites for posttranslational modification. The sequences for addition of carbohydrates, fatty acids, or
phosphate groups are reasonably well conserved and the presence of these sequences is
strong indicator about the post-translational modification of a protein. If alpha-helices are
predicted and show a high concentration of hydrophobic groups on their surface, this is a
strong indication that protein may have a transmembrane segment. A repeated pattern of such
a motif is found in many signaling molecules. For example, the classic seven transmembrane
pattern that was originally found in bacterial rhodopsin is also present in many cell-surface
receptors. Of course, any prediction made on the basis of amino acid sequence must be
confirmed, but primary sequence is often a powerful indication of what experiments should
be done.
3. The availability of a cDNA clone allows the protein to be expressed in a variety of
contexts. A cDNA can be inserted into a variety of expression vectors for different purposes.
Perhaps the most obvious use of such an approach is to drive expressions to extremely high
levels. This produces a rich source of protein that considerably eases the difficulty of protein
purification. This can make available abundant supplies of protein for physiological testing or
use as a reagent. A more striking use of expression system was in the ability to express
mutant proteins. Since it is possible to mutate DNA sequences essentially at will, it is
possible to express not only the wild type proteins but also related proteins that have
particular mutations. These mutations, if well designed, can be used to test particular
structure-function relationships within a protein. They can determine whether a particular
residue is important for catalytic activity or for association with another protein. In a related
and more practical way, proteins can be modified for specific uses. One can incorporate
disulfide bonds to increase the thermal stability of proteins that have industrial and
commercial applications. Reagents that are used in molecular applications can be modified so
unwanted activities are suppressed. For example, nuclease activities can be dissociated from
polymerase activities in DNA plolymerases. One of the most interesting examples of
expressing mutant proteins can be found in the design of dominant negative mutant of a
protein that can interfere with the activity of an endogenous protein. For example, if it is
possible to separate the DNA-binding domain and the RNA polymerase activating domain
from a transcription factor, expression of the DNA-binding domain in the absence of the
activating domain might be expected to interfere with the activity of the endogenous domain.
Many proteins function a multimers, so expression of a mutant protein can frequently
interfere with the activity of an endogenous protein by interfering with protein-protein
dimerization. This strategy has been extremely useful in study of specific transcription
factors. Likewise, intracellular signaling requires sequential interactions of a series of
proteins. Expression of a mutant protein that can interact with one member of the cascade but
51
not the subsequent downstream members can interfere with the function of endogenous
protein. This strategy has been used very profitably by making truncated mutants of receptors
that express only the extracellular but not the intracellular domain of a protein or by
expressing mutant version of ras or other GTP-binding proteins that transduce the signal
within the cell.
4. The availability of mRNA sequence also opens the possibility of taking a genetic
approach to understanding protein function. In many cases, expression of an antisense
oligonucleotide or the presence of a high concentration of synthetic anti-sense
oligonucleotides can suppress the translation of an endogenous mRNA, leading to a cell that
is depleted of a protein of interest. Analysis of such a cell or tissue can help establish the
function of a protein in vivo. Likewise, information about the sequence of a cDNA or the
gene encoding it can be used to develop a strategy to disrupt or modify the gene encoding the
c-DNA. Using homologous recombination it is possible to either disrupt and eliminate
expression of a gene or to force the expression of an altered gene product.
5. Knowing the cDNA sequence of a protein will frequently facilitate the
development of antibodies and monoclonal antibodies. Most simply, an over expressed
protein can be purified and used as an antigen. Alternatively, careful consideration of a
cDNA sequence and the likely structure of the encoded protein can be used to design peptides
that can be used as antigens for the production of either polyclonal or monoclonal antibodies,
and this will be discussed in the page devoted to antibodies.
6. Lastly, a cDNA sequence can be used as a probe to screen genomic libraries and
isolate the gene encoding a particular cDNA. This is an extremely valuable approach because
it provides a bridge between cDNA cloning and classic genetic analysis. Once the gene for a
cDNA has been mapped, it can be tested for its association with a particular developmental or
disease phenotypes. It is possible to use classic genetic approaches to determine whether
mutations in a particular gene co-segregate with alterations in the gene or its cDNA.
In conclusion, the availability of a cDNA opens such a wide variety of experimental
approaches and cDNA cloning is such a powerful technology that isolating a cDNA should
be considered, regardless of the ultimate experimental goal.
8.14. LET US SUM UP
A lambda gt10 library can be conveniently screened by plating it at relatively high
concentrations on a bacterial lawn of E coli. A probe is an oligonucleotide that is designed to
be complementary to the mRNA of interest so that it can be used to screen a library. The
molecular weight of a mRNA can be estimated by northern analysis and this can be compared
to the size of the cDNA that has been isolated. Screening with an antibody is quite similar to
screening with an oligonucleotide probe. One of the most productive, although perhaps less
creative approaches to isolating cDNAs is homology screening. PCR-based screening is also
a method to isolate novel cDNAs. Another useful approach to isolating cDNAs of interest
relies not on knowledge of their primary structure, but rather on assumptions about their
expression. Both plus/minus screening and differential display rely on strategies that seek to
isolate cDNAs that are expressed in one situation but not another. Each of these methods of
screening a cDNA library provides a specific screen or assay for cDNAs that may be of
interest. Just as it is true that when purifying a protein, one is likely to get what one assays
for, in screening a cDNA library one is likely to get what one screens for. Determining
52
whether the cDNA that has been isolated is indeed the one that is of most interest to the
experimentalist requires additional tests. In the absence of understanding of what those tests
should be, it will makes little sense to do initial screenings. Likewise, careful consideration of
what are the best screens for a specific purpose is likely to result in a more fruitful search
with a higher percentage of successes.
1. Substantiate the need for the probe.
2. Justify the use of complementation as a tool.
1. What are the common screening strategies? Describe details of two?
2. What are the significances of cDNA?
3. Write a short note on differential display.
4. Write about genomic DNA library and cDNA library.
8.17. REFERENCES
1. Principles of Gene Manipulation by R.W. Old and S.B. Primrose, Third Edition,
Blackwell Scientific Publication, 1985.
53
UNIT III: RECOMBINANT TECHNIQUES

LESSON 9: BLOTTING TECHNIQUES
CONTENTS
9.1. INTRODUCTION
9.2. BLOTTING TECHNIQUES
9.3. LET US SUM UP
9.6. REFERENCES
In this lesson, we will be discussion on blotting techniques, employed in Molecular
biology. Basically, the lesson will be focused on the following aspects viz.
Why Blotting?
How to perform Blotting.
Probe preparations
Blotting methods employed for RNA, DNA and Proteins
9.1. INTRODUCTION
In considering the potential utility of blotting techniques it is important to understand
some of the fundamental attributes of nucleic acids and proteins. It will add up ones
knowledge on functions that are happening in vivo and in vitro. Several such techniques often
mimic natural functions of nucleic acids and proteins such as hybridization, protein-protein
interaction etc. This chapter is intended to provide an overview of some common blotting
techniques that are involved in various facets of molecular biology.
9.2. BLOTTING TECHNIQUES
Blotting describes the immobilization of sample nucleic acids/proteins on to the solid
support generally nylon or nitrocellulose membranes. The blotted samples are then used as
targets in subsequent hybridization experiments.
54
Fig. BLOTTING TECHNIQUE

Electrophoresis of digested/denatured Nucleic acids/Protein allows separation based
on size to be carried out. However, it provides no indication of the presence of a specific
desired fragment among the complex sample. This can be achieved by transferring the DNA
from the intact gel onto a piece of nitrocellulose or nylon membrane placed in contact with it.
This provides a more permanent record of the sample, since the DNA/Protein begins to
diffuse out of a gel that is left for a few hours. The bound DNA/protein to membrane will be
exactly the same pattern as that of originally on the gel. Thus upon hybridization/coupling of
appropriate molecules which is used as a probe, one can detect the desired target from the
battery of bands obtained on the gel.
Essentially blotting procedure is adopted in several ways viz.
Blotting of from gels (nucleic acid/protein)

Dot and slot blotting (nucleic acids)
Colony and plaque blotting (whole cell/DNA)
55
The original method of blotting was developed by D.M.Southern (1975) for detecting
fragments in an agarose gel that are complementary to a given RNA or DNA sequence. Thus
the procedure for transfer of DNA from gel to the nitrocellulose filter resembling blotting was
named as Southern blotting. This technique has since been extended to the analysis of RMA
(Northern blotting) and proteins (Western blotting. These names are only jargon terms i.e.
Reverse of Southern being northern and so and do not reflect any functional or historical
significance.
BLOTTING METHODS
Blotting consists of the transfer of macromolecules like proteins and nucleic acids
from the gel onto the surface of an immobilizing membrane in the same relative positions as
they occur in the gel. The chief objective of blotting is to make the macromolecules separated
in the gel freely available for and amenable to detection and analysis by their interactions
with specific ligands, e.g. Antigens, antibodies, lectins and DNA/RNA probes. It is important
that the free sites present on the membrane for binding of the ligands are blocked by suitable
treatments before the ligands are used for the detection. Some of the transfer methods are
discussed below:
i) Diffusion blotting
The blotting membrane is placed on the top of the gel surface and the molecules
diffuse from the gel on to the surface of the membrane. Diffusion can also be accelerated by
increasing temperature (thermoblotting). This method is mostly used with gels having large
pores
ii) Capillary Blotting.
Here the gel is place do the top of a buffer-saturated filter paper. Then the blotting
membrane is placed on top of the gel, and finally dry filer papers are placed on the top of this
membrane. The buffer moves from the bottom filter paper through the gel carrying the
macromolecules along with it. T he latter get trapped on to the blotting membrane while
buffer is absorbed by the dry filet papers placed on the top of the blotting membranes. This
buffer movement is basically by capillary action where it will be slow which will require
overnight transfer. This can be used for DNA, RNA and large proteins.
iii) Vacuum Blotting
A controlled method where low vacuum is used to faster the movement of solution
through the gel and blotting membrane. Vacuum blotter which is employed here consists of a
vacuum chamber and a buffer reservoir. Both are separated by a porous vacuum plate. This
is relatively faster (30-90 min), gives high resolution, reduces mechanical stress on gel and
ultimately economic.
iv) Electrophoretic blotting
The technique of electrophoretic blotting was mainly developed for protein transfers
following SDS-PAGE and rarely used for nucleic acids also. There are two main variations
viz. Tank/ wet and semi-dry blotting.
56
Wet blotting is performed in a cassette where gel and membrane are clamped together
between filter papers and sponge pads. This is immersed in a vertical tank filled with
appropriate buffers. Electrodes fixed on either sides of the tank creates electrical field
enhancing transfer. This will occur overnight and requires cooled condition.
Semi-dry blotting apparatus consists of two horizontal plates one for anode and another for
cathode. A couple of filter papers soaked in buffer is kept on anode Blotting membrane is
placed onto this followed by gel. This is again covered with few filter papers to touch the
cathode plate. There should not be air bubbles in between these set up. Usually this is
performed with a constant current of 0.8 to 1 mA per cm2 for 1 hour.
Blotting membranes
There are a range of blotting membranes available for performing blotting and few to
mention are nitrocellulose membrane, Nylon membrane, and Polyvinylidenedifluoride
(PVDF) membranes.
Detection methods:
There are several detection methods employed after blotting and some of the predominant
ones are discussed hereunder:
Hybridization: Useful in DNA/RNA deterciont where probes labeled with radioactivity or
chemiluminiscence are used for detection of the complementary sequences
Enzyme blotting: By employing enzyme-substrate interactions which are coupled with
staining reactions, k detection is made easier.
Immunoblotting: By employing specific antibodies detections is facilitated with enzyme
coupling which develops colour once the substrate is added.
Lectin blotting: Those glycoproteins and specific carbohydrate moieties can be detected by
lections were development is facilitated by aldehyde detection or avidin-biotinperoexidase/alkaline phosphatase complex.
9.2.1. PROBE PREPARATION
To perform hybridization, one has to prepare probes that are specific for detection.
There are radioactive probes or non radioactive (chemiluminoscent) probes employed for this
purpose
Probes are short piece of DNA of known sequence (black bases) which has a
reporter substance attached to it. Generally probes span 10-30 bases long which is used to
detect the presence of complementary sequence in nucleic acid samples. This is achieved by
permitting the probes to base pair with the sample nucleic acids and then identifying the
samples that show base pairing with the probes. i.e. hybridization. The detection of
hybridization is highly precise and extremely sensitive provided the probes are suitably
labeled for an easy detection. Clearly, hybridization can occur only when the base sequence
of a probe is present with in the gene or DNA segment, which it is aimed to detect. Both
DNA and RNA are used as probes. Single stranded DNA probes are more convenient and
preferable, but denatured double stranded DNA molecules can also be used. RNA probes are
ordinarily single stranded.
Preparation of Probes:
Some of the sources to prepare probes for hybridization are:
57
Highly purified mRNA (naturally single stranded) can be used as probe;

Single stranded RNA probes can be prepared by cloning the corresponding DNA
sequence into vectors like pGEM, pBluescript upon transcription in suitable host.
DNA segments from genome/cDNA molecules can be cloned in E.coli and used as
probes (essentially double stranded!).
Cloning of DNA in phage M13 vectors of E.coli.
ss cDNA probes can be prepared by limiting copying of mRNA by Reverse
transcriptase to only one strand.
PCR generated single stranded copies of DNA sequence (Asymmetric PCR)
Synthetic oligonucleotides can be prepared for use as probes
Labeling of probes: Preparation of the probes essentially needs a label to be incorporated

into it.
The probes can be labeled with (1) Radioactivity e.g. 32P or (2) non radio active labels
e.g. Biotin, digoxigenin, fluroscent labels.
Radioactive labeling: The various techniques for labeling of nucleic acids are nick
translation, primer extension; RNA polymerase based and end labelling.
Nick translation:
It is a traditional method of labelling DNA. Low concentrations of DNase I are used
to make occasional single-stranded nicks in the double stranded DNA that is to be used as the
gene probe. DNE polymerase then fills in the nicks, using an appropriate
deoxyribonucleosided triphosphate (dNTPs), at the same time making a new nick to the 3
end o the precious once. In this way the nick is translated along ether DNA. If labeled dNTPs
are added to the reaction mixture, they will be used to fill the nicks, and also the DNA can be
labeled to a very high specific activity.
Nick translation. Pancreatic DNase I introduces single-stranded nicks by cleaving internal

phosphodiester bonds (p), generating a 5 phosphate group and a 3 hydroxyl terminus.
Addition of the multisubunit enzyme E. coli DNA polymerase I contributes two enzyme
58
activities: (i) a 5
3 exonuclease attacks the exposed 5 termini of a nick and sequentially
removes nucleotides in the 5
3 direction; (ii) a DNA polymerase adds new nucleotides to
the exposed 3 hydroxyl group, continuing in the 5
3 direction, thereby replacing
nucleotides removed by the exonuclease and causing lateral displacement (translation) of the
nick.
ii) Random Priming:
This method consists of hybridizing small random polymers to a template for the
probe and then allow DNA synthesis using radio active nucleotides as substrates. Typically,
DNA is denatured by heating and mixed with random sequence hexamer primers. Since a six
nucleotide sequences such a short sequences it normally finds a region of complementarity
somewhere in the probe template. If we now add the Klenow fragment (DNA polymerase I
without the 3 5 exonuclease activity) and all dNTPs, one of which caries a 32P label, the
enzyme will synthesise the probe and the probe will be radio active. The probe DNA is
boiede immediately before use.
Random primed labeling. The Klenow subunit of E. coli DNA polymerase I can synthesize
new radio labeled DNA strands using as a template separated strands of DNA, and random
hexanucleotide primers.
iii) End labeling.
The simplest form of labeling DNA is by 5 or 3 end labeling. 5 end labeling
involves a phosphate transfer or exchange reaction where the 5 phosphate of the DNA is to
be used as the probe is removed and in its place a labeled phosphate 32P is added. This is
carried our by alkaline phosphatase (removes existing PO4 group from DNA) and
polynucleotide kinase (catalyses transfer of a PO4 group with 32P to 5 end). The newly
labeled probe can be column purified to remove the unincorporated radiolabel. Alternatively
3 end labeling can also be done which is slightly complex which involves terminal
transferase. The addition of only one label is a potential disadvantage of this method.
59
(A) Kinase end-labeling of oligonucleotides. The 5 -terminal phosphate of the oligonucleotide
is replaced in an exchange reaction by the 32P-labeled g-phosphate of [g-32P] ATP. The same
procedure can be used to label the two 5 termini of double-stranded DNA. (B) Fill-in endlabeling by Klenow. The DNA of interest is cleaved with a suitable restriction nuclease to
generate 5 overhangs. The overhangs act as a primer for Klenow DNA polymerase to
incorporate labeled nucleotides complementary to the overhang. Fragments labeled at one
end only can be generated by internal cleavage with a suitable restriction site to generate two
differently sized fragments which can easily be size-fractionated.
iv) Non radioactive labeling:
There are several strategies for non radioactive labeling of nucleic acids, e.g. Labeling
with biotin, digoxigenin, fluorescent molecules etc. The relative shorter half life, risk,
disposal problems of radio active labels can over come by non-radio active labels which is
usually stored for long periods of time.
60
insitu HYBRIDIZATION
Cell or tissue specific gene expression can be determined by insitu hybridization. Tissues
are fixed on a microscopic slide and hybridized with cDNA probes. The cells expressing
the gene of interest are detected by Fluorescence or autoradiography. This is widely used
in developmental biology for determining developmentally regulated gene expression.
Uses of Probes:
Some of the applications of probes include,
Identification of the recombinant clone carrying the desired DNA insert

Confirmation of integration of DNA insert to host genome (by southern)
Development of RFLP maps
DNA fingerprinting for the unequivocal identification of plant varieties, criminals,
parental relationships, etc
In situ hybridization for determining the locations of specific sequences in specific
chromosomes
Accurate diagnosis of diseases caused by parasites, pathogens or defective viruses.
Preparation of genome maps of eukaryotes, including man.
9.2.2. SOUTHERN BLOTTING

Southern blotting is designed to locate a particular sequence of DNA within a
complex mixture. It can be used to locate a particular gene within an entire genome.
The transfer can be performed electorphoretically or by drawing large volumes of
buffer through both gel and membranes thus transferring DNA from one to the other by
capillary action. The membrane can be treated with a labeled DNA molecule. Thus single
stranded DNA probe will hybridize under the right conditions to complementary fragments
immobilized onto the membrane. The temperature and salt concentrations are critical for
hybridization.
The amount of DNA needed for this technique is dependent on the size and specific
activity of the probe. Short probes tend to be more specific. Under optimal conditions, one
can expect to detect 0.1 pg of the DNA for which you are probing.
Basic steps involved in a Southern blot included
1. Digestion of the DNA: This is to be done with an appropriate restriction enzyme.
2. Agarose gel electrophoresis: Running the digest on an agarose gel.
3. Denaturation of the DNA (usually while it is still on the gel):
For example, by soaking it in about 0.5M NaOH, which would separate doublestranded DNA into single-stranded DNA. Only ssDNA can transfer.
4. Depurination (optional): Fragments greater than 15 kb are hard to transfer to the
blotting membrane. Depurination with HCl (about 0.2M HCl for 15 minutes) takes the
purines out, cutting the DNA into smaller fragments. If it is not depurinated,
neutralization is usually required.
61
5. Transfer of the denatured DNA to the membrane. Nitrocellulose membrane is
usually used, although nylon or a positively charged nylon membrane is in practice.
Nitrocellulose typically has a binding capacity of about 100g/cm, while nylon has a
binding capacity of about 500 g/cm. Nylon is better since it binds more and is less
fragile. Transfer is usually done by capillary action, which takes several hours.
Capillary action transfer draws the buffer up by capillary action through the gel into
the membrane, which will bind ssDNA.
Fig. SOUTHERN BLOTTING
6. Cross linking: After the transfer of DNA to the membrane cross linking by UV
exposure is done to (via covalent bonds) the DNA to the membrane. Alternatively one
can also bake nitrocellulose at about 80C for a couple of hours.
7. Hybridization: it is nothing but probing the membrane with labeled ssDNA. This
process relies on the ssDNA hybridizing (annealing) to the DNA on the membrane
due
to
the
binding
of
complementary
strands.
Probing is often done with radioactive or non-radioactive probe.
8.Prehybridization: Generally a prehybridization step is required before

hybridization to block non-specific sites, since you don't want your single-stranded
probe binding just anywhere on the membrane.
9. Development: The radioactive labeled target sequence can be visualized after

exposure. If radio labeled 32P probe was used, then one can visualize the results by
62
autoradiograph. Biotin/streptavidin detection is done by colorimetric methods, and
bioluminescent visualization uses luminescence.
Non specific DNA: You can literally milk the salmon for sperm. If you squeeze a
salmon, they give up their sperm, which is DNA rich. Since most people aren't
probing for salmon sperm sequences, it's a good source for nonspecific DNA
3.1.2.3. NORTHERN BLOTTING
Northern analysis remains a standard method for detection and quantitation of mRNA
levels despite the advent of powerful techniques, such as RT-PCR, gene array analysis and
nuclease protection assays. Northern analysis provides a direct relative comparison of
message abundance between samples on a single membrane. It is the preferred method for
determining transcript size and for detecting alternatively spliced transcripts.
The Northern blotting procedure is straightforward and provides opportunities to
evaluate progress at various points (e.g., integrity of the RNA sample and how efficiently it
has transferred to the membrane). RNA samples are first separated by size via electrophoresis
in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization is exceptionally
versatile in that radio labeled or nonisotopically labeled DNA, in vitro transcribed RNA and
oligonucleotides can all be used as hybridization probes. Additionally, sequences with only
partial homology (e.g., cDNA from a different species or genomic DNA fragments that might
contain an intron) may be used as probes.
Fig. NORTHERN BLOTTING
63
LIMITATIONS
If RNA samples are even slightly degraded, the quality of the data and the ability to
quantitate expression are severely compromised. For example, even a single cleavage in 20%
of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents
and techniques are essential.
A standard Northern procedure is, in general, less sensitive than nuclease protection
assays and RT-PCR, although improvements in sensitivity can be achieved by using high
specific activity antisense RNA probes, optimized hybridization buffers and positively
charged nylon membranes. Sensitivity can be further improved with oligo dT selection for
enrichment of mRNA, since physical constraints of gel electrophoresis and membrane
transfer limit the amount of RNA that can be analyzed without loss of resolution and
saturation of the transfer membrane.
9.2.4. WESTERN BLOTTING
Western blot analysis can detect one protein in a mixture of any number of proteins
while giving you information about the size of the protein. It does not matter whether the
protein has been synthesized in vivo or in vitro.
METHODOLOGY
1. Separate the proteins using SDS-polyacrylamide gel electrophoresis.
2. Blot to a nitrocellulose membrane through wet/semi-dry method to drive the protein bands
onto the nitrocellulose membrane.
64
3. Transfer of proteins can be analyzed for their uniformity and overall effectiveness of
transfer by various protein staining methods. For example, membranes may be stained with
Coomassie or Ponceau S dyes.
4. Incubate the nitrocellulose membrane with a primary antibody.
5. Block the remaining area with Nonfat Dry Milk
6. Incubate the nitrocellulose membrane with a secondary antibody. This antibody should be
an antibody-enzyme conjugate. The secondary antibody should be an antibody against the
primary antibody. This means the secondary antibody will "stick" to the primary antibody,
just like the primary antibody "stuck" to the protein. The conjugated enzyme is there to allow
you to visualize all of this.
5. Add substrate to see bands wherever there is a protein-primary antibody-secondary
antibody-enzyme complex, or, in other words, wherever your protein is.
MAKING A PRIMARY ANTIBODY
This description assumes you have available purified protein. There are several
methods to prepare the antigen. However here a relatively common and easier method is
discussed.
Run the protein on an SDS-PAGE gel. Stain the gel with KCl. The KCl forms a
precipitate with the SDS. Since the area with the protein has a low concentration of SDS, the
area with the protein will not show a precipitate. This will allow you to see the protein band
as a clear band against a milky white precipitate on the rest of the gel.
Carefully cut out the band and soak it in 1 mL PBS buffer. Crush it and make an
emulsion with 1 mL Freund's Complete Adjuvant (which is an oily substance). The complete
adjuvant contains mycobacterium (an immune stimulant) to increase the immune response.
Immunise by injecting this subscapularly into a rabbit (or mice or rat or any animal system).
Only use the complete adjuvant for the first inoculation. NEVER inject a rabbit with
complete adjuvant more than one time.
After one month, repeat immunization (booster) the process using an incomplete
adjuvant. Usually good antibody titers will be developed in about 10 days after the second
booster.
Bleed the rabbit to obtain about 30 to 40 mL per bleeding, and about 50 percent of the
volume is serum which is the rabbit antisera.
To get your primary antibody, dilute the rabbit antisera in blotto (aka Carnation
Nonfat Dry Instant Milk) and apply it to your nitrocellulose blot. Make sure you dilute 1:500
to 1:100 in blotto; less dilution will give you background binding and really muddy up your
results.
65
SECONDARY ANTIBODY
The secondary antibody essentially need to be raised with the serum of the test animal
species (rabbit) in another animal system. Generally the goat-anti-rabbit is the preferred
secondary antibody and the HRP is the conjugated enzyme that will allow one to visualize the
protein of interest.
Traditionally western blot detection takes place with a two-step process; however
there are now some one-step detection methods available.
ALTERNATE DEVELOPING STRATEGY
Chemiluminescence: By far the most common method, but which may be replace
chemiluminescent detection for western blots require incubation of the membrane a substrate
that will luminescence when exposed to the enzyme present on the (modified) secondary
antibody. The localized light which is emitted from the bands is detected by photosensitive
photographic film.
Developments recently have allowed CCD camera capturing of the light emission
from the membrane and a more quantitative analysis of the membrane as a digital image of
the exposed western blot membrane.
Colorimetric detection protocols for western blot membranes depend on the incubation of the
membrane with a substrate that reacts with the reporter enzyme present on the secondary
antibody. The enzyme then catalyzes the conversion of the substrate into an insoluble
coloured dye that precipitates in a localized area in the vicinity of the bound antibody. The
reaction is stopped by washing or removing the substrate from the enzyme.
The colour stains the nitrocellulose membrane and can its intensity can be detected by
densitometry methods similar to ECL or spectrophotometry.
Instead of enzyme-linked antibodies, the secondary or even primary antibodies
contain a radioactive label. This radioactive label can be 32P, 125 I or similar radioactivity
emitting element. The radioactive emissions are then detected by medical or X-ray sensitive
film which is placed against the western blot membrane.
The film is developed in the dark, similarly to chemiluminescent western blot
detection. The film is exposed to the membrane, and the localized radioactivity emitted from
the antibodies bound to the proteins exposed the film in the region to create a dark band.
9.3. LET US SUM UP
Blotting can be performed in several ways viz, capillary, electroblotting, wet and semidry
basis.
Generally for Nucleic acid studies probe preparation is the key experiment before blotting.
DNA can be easily blotted through capillary methods employing protocol suggested by
Southern.
Northern employing RNA is relatively risky but possible and used extensively be researchers
66
Western blotting for Proteins generally uses Immunodetection which is widely performed by
molecular biologists.
Establish the principles behind the binding and interaction of probes in southern,
northern and western blot methods.
1. How will your prepare a probe to detect a 16S rRNA gene of Bacillus subtilis from a soil
DNA sample?
2. Why Northern is not widely performed when compared to Southern?
3. What are the blotting techniques followed for biomolecules?
4. How will you perform western blotting with a radio active label?
9.6. REFERENCES
67
LESSON 10: TRANSFORMATION IN E.coli
CONTENTS
10.1. INTRODUCTION
10.2. PREPARATION OF COMPETANT CELL
10.3. TRANSFORMATION OF E. COLI BY CACL2 METHOD:
10.4. ELECTORPHORATION
10.5. TRANSFECTION
10.6. LET US SUM UP
10.9. REFERENCES
In this unit discussions will be on
Why transformation in E.coli?
Different methods of transformation.
Screening techniques
Alternate strategies.
10.1. INTRODUCTION
This is a very basic technique that is used on a daily basis in a molecular biological
laboratory. The purpose of this technique is to introduce a foreign plasmid into a bacterium
and to use those bacteria to amplify the plasmid in order to make large quantities of it.
Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial
cell's membrane. In order to make bacteria take in the plasmid, they must first be made
"competent" to take up DNA. This is done by creating small holes in the bacterial cells which
is done chemically with calcium chloride or by employing alternate strategies.
To amplify/express large quantities of a DNA of our interest, it tis to be ligated to a
vector and has to be transformed into a bacterial host. Most methods for bacterial
transformation are based the uptake of plasmid /bacteriophage enhanced by a chemical or
electro completant cells. Many variations in this basic technique have been described, all
directed towards optimizing the efficiency of transformation for different bacterial strains.
Various steps involved in this techniques are as follows:
The first step in transformation is to select a piece of DNA to be inserted

into a vector.
The second step is to cut that piece of DNA with a restriction
enzyme and then ligate the DNA insert into the vector with DNA Ligase.
The third step is insertion of vector into a host cell, in a process called transformation.
Selection by markers which can be for antibiotic resistance, color changes, or any
other characteristic which can distinguish transformed hosts from untransformed
hosts.
68
10.2. PREPARATION OF COMPETANT CELLS
Generally most of the bacterial cells are competant to receive the forgien DNA.
Especially some of the Bacillus species are naturally competant. However to increase the
efficiencey of those bacterial like E.coli, there are many approaches of which chemcial and
elecrical method is promising and widely used.
For most cloning applications, we use DH5 host cells. These cells are compatible
with lacZ blue/white selection procedures, are easily transformed, and good quality plasmid
DNA can be recovered from transformants. However several other E.coli strains also are in
use such as JM 109, JM 101, and BL21 etc.
For maximum transformation efficiency, it is very important that the bacterial culture
is in the logarithmic phase of growth and that cell density is low at the preparation time.
During this period, the efficiency of transformation increases.
The ability of bacteria to take up DNA is generally short-lived. Hence after exposure
to agents that enhance uptake, most strains of bacteria remain in a competent state for only 12 days. However storage of competant cells in deep freeze (-70C) condition is common in
molecular biology laboratories.
There are two major methods to transform E. coli cells with plasmid DNA - chemical
transformation and electroporation. For chemical transformation, cells are grown to mid-log
phase, harvested and treated with divalent cations such as CaCl2. Cells treated in such a way
are said to be competent.. Usually, glycerol is added to the water to a final concentration of
10% so that the cells can be stored frozen and saved for future experiments.
For electroporation, cells are also grown to mid-log phase but are then washed extensively
with water to eliminate all salts To electroporate DNA into cells, washed E. coli are mixed
with the DNA to be transformed and then pipetted into a plastic cuvette containing
electrodes. A short electric pulse, about 2400 volts/cm, is applied to the cells causing smalls
holes in the membrane through which the DNA enters. The cells are then incubated with
broth as above before plating.
Chemically competent E.coli cells methodology
1. The pre inoculum need to be prepared by using a single colony of (DH5 / JM109)
which is to be inoculated to 2 ml of LB broth and left overnight at 37C on shaker.
2. From the overnight inoculum, about 1% culture need to be inoculated to 25 ml of LB
broth.
3. The media is to be left on shaker at 37C for 2-3 hours until the O.D reaches 0.5-0.6
at 600nm. OD is to be checked at regular intervals from 2 hours.
4. After reaching the required growth, the culture need to be placed at ice/cold room for
30 min.
5. Cells need to be recovered by centrifugation at 4000rpm for 10 min at 4C.
6. The pellet is to be resuspended in 4ml of 0.1 M CaCl2 by aspirating gently with a
micropipette with wide mouthed tip.
7. The mixture is to be again centrifuged at 4000rpm for 10 min at 4C.
8. Finally the pellet need to be resuspended in 1 M CaCl2 and alliquoted into 200l each
in microfuge tubes.
9. The contents should be frozen immediately in liquid nitrogen.
69
10. Further storage is to be done in -70C freezer where it can be stored for indefinite
period.
Chemical competence is conferred to E.coli by re-suspension in CaCl2 solution at 0C.
Under these conditions, the Ca2+ ion is thought to create pores in the membrane, assist
binding of the DNA to the cell membrane and mask the negative charge on the DNA, easing
its passage through the hydrophobic cell membrane. The DNA is forced into the cells by
applying a short 42C heat shock, which results in a thermal current that sweeps the DNA
into the cells.
ELECTROCOMPETANT CELLS METHODOLOGY
A widely used methodology for making electrocompetant cells is given hereunder:
1. Inoculate a colony of DH5 (or other appropriate host strain) to inoculate 5 ml of SOB/
medium and leave in shaker with vigorous shaking for aeration (300rpm) overnight at 37C.
2. Add 1% of the preinoculum into 250 ml of SOB /LB in a 1 liter flask. Grow for 2 to 3
hours with vigorous shaking as above at 37C until the cells reach an OD of 0.8 is reached at
600nm.
3. Harvest cells by centrifugation at 5000 RPM for 10 min in sterile centrifuge bottles. At
4C.
4. Wash the cell pellet in 250 ml of ice-cold 10% glycerol in sterile water.
5. Centrifuge the cell suspension at 5,000 RPM for 15 min and retain the pellet.
6. Wash the cell pellet a second time by resuspending in 250 ml of sterile ice-cold Glycerolwater buffer.
7. Resuspend the cell pellet in the WB and the final volume should be about 1 ml. Cells can
be used immediately or can be frozen in 0.2 ml aliquots in freezer vials using a dry iceethanol bath.
8. Store frozen cells at -70C.
Electroporation is less cumbersome than chemical transformation and generally gives
higher transformation efficiencies (measured in colonies formed per microgram of DNA).
However, it is more expensive, requiring specialized apparatus to deliver the charge and
cuvettes to transfer the charge to the cell suspension. Electroporation is sensitive to salt, so
precious samples can be lost if excess salt is carried over into the cuvette.
10.3. TRANSFORMATION OF E. coli BY CACL2 METHOD
1. Gently mix the DNA with the bacteria (after thawing) and incubate the mixture on ice
for 20 minutes.
2. Heat shock the mixture for 45 to 90 seconds at 42C.
3. Transfer the bacterial suspension to 10 ml of LB Broth without any antibiotic.
4. Leave on shaker for 45 minutes in a 37C incubator.
5. Gently spin the contents and discard the supernatant by retaining cell pellet.
6. Resuspend the bacteria in 100 l of LB broth and plate on LB agar plate with
antibiotic (for selection).
7. Determine the transformation efficiency the next day by counting the number of
colonies and scaling up this number per microgram of vector.
70
Good transformation efficiencies are > 105 transformants/g of vector DNA for this
protocol. Higher transformation efficiencies may be obtained with RbCl2-treated cells, and
with commercially available cells.
10.4. ELECTORPHORATION
For chemical transformation, there is no need to pre-treat the DNA. For
electroporation, the DNA must be free of all salts so the ligations are first precipitated with
alcohol before they are used.
TRANSFORMATION EFFICIENCY
To calculate the competency of your cells, divide the number of colonies on your
plate by the amount of DNA (in g) you added to the transformation.
To determine the efficiency of transformation, a positive control transformation
should be done using 1 ng of uncut plasmid DNA, e.g. pUC19. The efficiency of
transformation is calculated as the number of transformants/g of input DNA. A negative
control should also be included that contains cells with no added DNA.
A negative control with cells only (no added DNA) should also be included.
10.5. TRANSFECTION
Phage introduction is the process of transfection, which is equivalent to
transformation,except a phage is used instead of bacteria. In vitro packaging of a vector is
used. This uses lambda or MI3 phages to produce phage plaques which contain recombinants.
The recombinants that are created can be identified by differences in the
recombinants and non-recombinants using various selection methods.
10.6. LET US SUM UP
Thus, Foreign DNA can be introduced into competent E.coli using chemical
transformation or electroporation.
Electroporation involves the application of an electric current across the cell, which is
thought to create momentary pores in the cell membranes and force the negatively charged
DNA into the cells by an electrophoresis-type effect.
Evaluate the advantages and disadvantages of calcium chloride and electroporation methods.
71
1. Find out what are the difference between a Blue and white colony in a Blue-White
selection.
2. Understand what is meant by ultracompetant cells
3. Know the etiquettes in preparing competent cells.
4. In what way electrocompetant method is risky and discuss on its advantages over the
chemical method.
5. How can you make an E.coli cell chemically competent to perform transformation
experiment?
6. What are all the ways with which you can transform DNA into a host cell?
7. What is transformation efficiency? How will you calculate?
8. What is transfection? How often it is followed in microbial transformations?
10.9. REFERENCES
72
LESSON 11: POLYMERASE CHAIN REACTION (PCR)
CONTENTS
11.1. INTRODUCTION
11.2. PCR METHODOLOGY
11.4. PCR OPTIMIZATION
11.5. PCR AUTOMATION
11.6. VARIATIONS IN PCR
11.7. LET US SUM UP
11.10. REFERENCES
11.0 AIMS AND OBJECTIVES
In this lesson we will be discussing the polymerase chain reaction in detail.
How the technique PCR works
What are the critical elements involved in PCR
Variations in PCR
11.1. INTRODUCTION
The polymerase chain reaction (PCR) is the cardinal laboratory technology of
molecular biology. Arguably one of the most powerful laboratory techniques ever discovered,
PCR combines the unique attributes of being very sensitive and specific with a great degree
of flexibility. With the PCR it is possible to specifically address a particular DNA sequence
and to amplify this sequence to extremely high copy numbers.
The polymerase chain reaction won its discoverer Kary Mullis a Nobel Prize in 1993.
He has recorded the thought process he went through as he conceived of the idea and he
states that he knew immediately that he was going to become very famous because of this
discovery.
Since its initial development in the early 1980s, dozens of variations in the basic
theme of PCR have successfully been carried out. In fact, the very flexibility and applicationspecific variation of PCR make it seem like there are as many ways to do a PCR reaction as
there are researchers doing them.
The success of the technology is mainly due to the variety of thermo stable
polymerases and automation which are now available for the molecular biologists. Though
several enzymes are in use the DNA polymerase, known as 'Taq polymerase' is common
which is named after the hot-spring bacterium Thermus aquaticus from which it was
originally isolated. The enzyme can withstand the high temperatures needed for DNA-strand
separation, and can be left in the reaction tube.
In a PCR, the cycle of heating and cooling is repeated over and over, stimulating the
primers to bind to the original sequences and to newly synthesised sequences. The enzyme
will again extend primer sequences. This cycling of temperatures results in copying and then
copying of copies, and so on, leading to an exponential increase in the number of copies of
73
specific sequences. Because the amount of DNA placed in the tube at the beginning is very
small, almost all the DNA at the end of the reaction cycles is copied sequences.
The reaction products are separated by gel electrophoresis. Depending on the quantity
produced and the size of the amplified fragment, the reaction products can be visualized
directly by staining with ethidium bromide or rarely by means of silver-staining or
autoradiography.
11.2 PCR METHODOLOGY
The polymerase chain reaction (PCR) is a technique for copying a piece of DNA a
billion-fold. As the name suggests, the process creates a chain of many pieces, in this case the
pieces are nucleotides, and the chain is a strand of DNA.
PCR is an enzyme-mediated reaction, and as with any enzyme, the reaction must
occur at the enzyme's ideal operating temperature. The enzymes that are used for the PCR are
DNA-dependent DNA polymerases (DDDP) derived from thermophilic (heat-loving)
bacteria.
There are several critical parameters to be noted for a PCR reaction.
1. Choosing Target Substrates and PCR Primers
The choice of the target DNA is, of course, dictated by the specific experiment.
However, one thing is common to all substrate DNAs and that is they must be as clean as
Possible and uncontaminated with other DNAs.
2. Setting Up the Reaction
Once you have chosen the appropriate substrate and your PCR primer sequences and
You have them on hand; the basic reaction components are as follows:
Water
10x Reaction Buffer
MgCl2
dNTPs
Forward Primer
Reverse Primer
Target DNA
Polymerase enzyme
3. Choosing the Reaction Conditions
The reaction conditions of PCR amplification are composed of the total number of
Cycles to be run and the temperature and duration of each step in those cycles. In
General, 25 to 35 cycles is the standard for a PCR reaction. This results in from
Approximately 34 million to 34 billion copies of the desired sequence. However, reactions in
excess of 45 cycles are quite rare. Also, increasing the number of cycles for larger amounts of
starting material is counter productive because the presence of very high concentrations of
the PCR product is itself inhibitory. Once the number of cycles is selected, it is necessary to
choose the temperature and duration of each step in the cycles.
74
The first step is the DNA denaturation step that renders the entire DNA in the reaction
single stranded. This is routinely accomplished at 94C or 95C for 30 seconds. The second
step is the primer annealing step during which the PCR primers find their complementary
targets and attach themselves to those sequences. Here the choice of temperature is largely
determined by the melting temperature (Tm) of the two PCR primers).Again, the usual
duration is 30 seconds. Finally, the last step in a PCR cycle is the polymerase extension step
during which the DNA polymerase is producing a
complementary copy of the target DNA strand starting from the PCR primer sequence
(Thus the term primer). The usual temperature of this step is 72 C, considered to be a
Good optimum temperature for thermal-stable polymerases. A common rule of thumb for
The duration of this step has been 30 seconds for every 500 bases in the PCR product.
In addition to these cycling conditions, it is often desirable to place a single initial
denaturation step of three to five minutes at 94C or 95C at the beginning of the
Reaction and a final extension step of a few minutes at 72C.
4. Validating the Reaction
Once your PCR reaction has run, there are two ways of determining success or failure.
The first is to simply take some of the final reaction and run it out on an agarose gel with an
appropriate molecular weight marker to make sure that the reaction was successful and
If the amplified product is the expected size relative to the maker
The ultimate validation of a PCR reaction is to directly sequence the amplicon. This is
often a choice that is not readily available since not everyone has access to a DNA sequencer
nor will they have either the time or the funds to carry out such an analysis.
CRITICAL STEPS IN A PCR (PHYSICAL CONDITIONS)
Broadly speaking, there are three steps identified by incubating at different temperatures.
The three steps make up a PCR "cycle".
1. Double-stranded DNA separation or denaturation (D in Figure 1)
2. Primer annealing to template DNA (A in Figure 1)
3. Primer extension (E in Figure 1)
75
Figure. A PCR cycle.

The three temperatures which make up a single cycle. The DNA denaturation section
(D), oligonucleotide annealing section (A) and the primer extension (E) section are marked.
The temperature range over which dsDNA duplexes can denature (TD) or 'melt', and the range
over which the oligonucleotide primer can hybridize (TM) are also marked.
DENATURATION
At temperatures above 90C, double-stranded DNA denatures or "melts". That means
the weak hydrogen bonds that usually hold the two complementary strands together at normal
temperatures are disrupted resulting in two single stranded DNA strands (shown below in an
idealized form).
PRIMER ANNEALING
At the annealing temperature (TA), primers that collide with their complementary
sequence can hybridize or "bind" to it. The chance of such an encounter happening is
increased because we use a vast excess of each primer in the reaction mixture compared to
the number of template molecules present.
76
The assay in the example below has been designed to amplify a region of the template
spanned by and including, the primer sequences.
PRIMER EXTENSION
At the extension temperature (TE), the DNA polymerase binds to the hybridized
primer and begins to add complementary nucleotides (i.e. every time the polymerase reads a
"G" on the template strand, its adds a "C"; an "A" for a "T"; a "G" for a "C" and a "T" for
an"A"), chemically binding each new addition to the last to form a growing chain. The
process only occurs in one direction. In our example, the green primer is binding to its
complementary template sequence and is facing toward the right (this is called the 5' (fiveprime) to 3' (three prime) direction. Extension occurs in the direction that the primer faces.
The result is a new double-stranded PCR product we usually call an "amplicon". An amplicon
can be defined as an amplified molecule of a single type, in this case, an exact replicate of the
original template.
EXPONENTIAL TEMPLATE DUPLICATION

The process is then repeated by cycling through the temperatures over and over again
(35 times). Each cycle results in a new DNA duplex, each strand acting as a potential
template for one or other primer.
77
Considerations:
CRITICAL ELEMENTS IN PCR (CHEMICAL CONDITIONS)
Template DNA. Nearly any standard method is suitable for template DNA purification. An
adequate amount of template DNA is between 0.1 and 1 g for genomic DNA for a total
reaction mixture of 100 l. Larger template DNA amounts usually increase the yield of nonspecific PCR products.
78
Primers.
(1) PCR primers should be 10-24 nucleotides in length.
(2) The GC content should be 40%-60%.
(3) The primer should not be self-complementary or complementary to any other primer in
the reaction mixture, to prevent primer-dimer and hairpin formation.
(4) Melting temperatures of primer pairs should not differ by more than 5C, so that the GC
content and length must be chosen accordingly.
(5) The melting and annealing temperatures of a primer are estimated as follows: if the
primer is shorter than 25 nucleotides, the approximate melting temperature is calculated with
the formula: Tm = 4 (G + C) + 2 (A + T).
(6) The annealing temperature should be about 5C lower than the melting temperature.
MgCl2 concentration. Because Mg2 + ions form complexes with dNTPs, primers and DNA
templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too
few Mg2 + ions result in a low yield of PCR product, and too many will increase the yield of
non-specific products. The recommended range of MgCl2 concentration is 1 to 3 mM, under
the standard reaction conditions specified.
Taq DNA polymerase. Higher Taq DNA polymerase concentrations than needed may cause
synthesis of non-specific products.
dNTPs. The concentration of each dNTP (dATP, dCTP, dGTP, dTTP) in the reaction mixture
is usually 200 M. These concentrations must be checked as being equal, because
inaccuracies will increase the degree of misincorporation.
11.4. PCR OPTIMIZATION
There are several PCR reaction conditions that must be optimized. Some of them are:
Primer annealing temperature:
Increases specificity of primer annealing;
Decrease Increases the sensitivity of the reaction
DNA polymerase Enzyme
Low enzyme concentration produces insufficient;
Product and high concentration decreases the specificity;
Taq - most efficient but has highest error rate;
pfu - has a low error rate but least amount of product
Magnesium Concentration
Low concentration increases specificity;
High concentration increases sensitivity, but decrease primer specificity
79
Number of Cycle
Elevated denaturation temperature can increase sensitivity;
Taq polymerase activity decreases above 93C;
Longer primer extension times increase sensitivity in long distance PCR;
Assay sensitivity is depended on the enzyme concentration and the number of cycles;
Low concentration of templates requires higher number of cycles.
Some technical tips:
DNA extraction and PCR reaction mixing and processing should be performed in separate
areas.
Use of sole-purpose vessels and positive displacement pipettes or tips for DNA sample and
reaction mixture preparation is strongly recommended.
All solutions, except dNTPs, primers and Taq DNA polymerase, should be autoclaved.
Where possible, solutions should be aliquoted in small quantities and stored in designated
PCR areas.
A good practice, to confirm absence of contamination, is to add a control reaction without
template DNA.
11.5. PCR AUTOMATION
The very success of this technique is the automation which eventually facilitated
scientist to explore it to a maximum extent. Earlier liquid cooled/heated modules were
introduced. However, soon those were replaced by Heating and cooling blocks using
refrigerants. Due to the heavy nature and space occupying units, an alternative using peltier
block came into market. Such peltier blocks facilitated gradient feature of temperature which
enabled scientist to reduce the standardization protocols.
Nowadays even simpler air cooled systems are available which is relatively economic
and maintained free. Some of the firms who are widely marketing PCR machines include,
Perkin Elmer, Eppendorf, Corbett and Syngene.
There are three basic designs for PCR dedicated thermocycler, which differ in low
temperature ramping, are achieved.
1. Mechanized Robot with programmable arm that move the set of PCR tubes between three
separate heating blocks
2. Conventional thermocycler that uses refrigeration and heating elements to change the
temperature
3. Thermocycler that uses heating cooling pump principles of the Peltier effect.
Yet another revolution in PCR automation is the Real time PCR (RT-PCR) which has
facility to monitor the PCR products formed for every cycle. Such machines widely find
applications in expression studies and disease diagnostics at an advanced level.
80
11.6. VARIATIONS IN PCR
Hot-start PCR - to reduce non-specific amplification.
Touch-down PCR - start at high annealing temperature, and then decrease annealing
temperature in steps to reduce non-specific PCR product.
Nested PCR - PCR using an outer set of primers and the product of this PCR is used for
further PCR reaction using an inner set of primers to synthesize more reliable product.
Inverse PCR - for amplification of regions flanking a known sequence. DNA is digested, the
desired fragment is circularised by ligation, then PCR using primer complementary to the
known sequence extending outwards.
AP-PCR (arbitrary primed) / RAPD (random amplified polymorphic DNA) - methods for
creating genomic fingerprints from species with little-known target sequences by amplifying
using arbitrary oligonucleotides.
RT-PCR - using RNA-directed DNA polymerase to synthesize cDNAs, which is then used
for PCR and is extremely sensitive for detecting the expression of a specific sequence. It may
also be used to quantify mRNA transcripts.
RACE - (rapid amplification of cDNA ends) - used where information about DNA/protein
sequence is limited.
Amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer
each (+ one adaptor primer). Overlapping RACE products can then be combined to produce
full cDNA.
DD-PCR (differential display) - used to identify differentially expressed genes in different
tissues. First step involves RT-PCR, then amplification using short, intentionally nonspecific
primers. Get series of band in a high-resolution gel and compare to that from other tissues,
any bands unique to single samples are considered to be differentially expressed.
Multiplex-PCR - 2 or more unique targets of DNA sequences in the same specimen are
amplified simultaneously. One can be use as control to verify the integrity of PCR. Can be
used for mutational analysis and identification of pathogens.
Q/C-PCR (Quantitative comparative) - uses an internal control DNA sequence (but of
different size) that competes with the target DNA (competitive PCR) for the same set of
primers. Used to determine the amount of target template in the reaction.
11.7. LET US SUM UP
PCR technique amplifies specific DNA fragments from minute quantities of source
DNA material, even when that source DNA is of relatively poor quality.
The PCR steps are all carried out, one after the other, in bouts of cycling. Each cycle
essentially consist of Denaturation, annealing and extension.
81
During denaturation (about 1 min at 95C), the DNA strands separate to form single
strands.
During annealing (about 1 min at temperatures ranging between 45C and 60C), one
primer binds to one DNA strand and another binds to the complementary strand. The
annealing sites of the primers are chosen so that they will prime DNA synthesis in the region
of interest during extension.
During extension (about 1 min at 72C), the DNA synthesis proceeds through the
target region and for variable distances into the flanking region, giving rise to 'long
fragments' of variable lengths
Critical components of PCR include MgCl2, dNTPs, Primers, Template DNA and the
Thermostable Polymerase enzyme.
1. Differentiate between a manual PCR and automated PCR. Justify hich one is the best.
2. Justify: optimization in required for each PCR amplification.
1. What are the critical steps in a PCR reaction?
2. How do calculate the number of products formed after nth cycle of a PCR relation started
with 100 molecules of DNA template?
3. Brief about PCR automation.
4. Which of the following is critical in PCR: Denaturation/annealing/extension;
time/temperature?
5. What are the variations in PCR?
11.10. REFERENCES
82
LESSON 12: DNA MARKERS
CONTENTS
12.1. INTRODUCTION
12.1. RAPD
12.2. RFLP
12.3. AFLP
12.5. SSCP
12.6. SNP
12.7. LET US SUM UP
12.10. REFERENCES
In this lesson some of the important tools in Marker analysis are discussed,which
include
How RAPD works
Principle and methodology involved in RFLP,AFLP,SSCP,SNP
What are all the limitations of molecular markers
12.1. INTRODUCTION
A genetic marker can be defined as an allelic difference/variation at a given locus in
the genome that can be observed at the level of Morphology, Isozyme or DNA/RNA.
A good useful genetic marker must be discrete, easily distinguishable between parents and
individuals. It also should accurately reproduce the difference in the progeny.
Though classical markers like morpho-physiological characteristics or isoenzymes or
protein variants are easy to study, they have some problems. These are very limited in
number (low genetic resolution), influenced by environment, analysis limited to specific
development stages and difficult to score/interpret.
Alternatively DNA based markers are readily detectable variations in the DNA. These
have all the characteristics that are desired in a high resolution approach for genetic
discrimination.
12.2. RAPD
Random Amplified Polymorphic DNA (RAPD) markers are DNA fragments from
PCR amplification of random segments of genomic DNA with single primer of arbitrary
nucleotide sequence. The primer may be designed specifically, but could be chosen randomly
and is used to amplify a series of samples which will include both the material of interest as
well as other control samples with which the experimental material needs to be compared.
Choice of primer length will be critical to the determination of band complexity in the
resulting amplification pattern. Eventually a particular probe will be found that is able to
distinguish between the sample of interest and those that are different.
83
How It Works
Unlike traditional PCR analysis, RAPD (pronounced "rapid") does not require any
specific knowledge of the DNA sequence of the target organism: the identical 10-mer primers
will or will not amplify a segment of DNA, depending on positions that are complementary to
the primers' sequence. For example, no fragment is produced if primers annealed too far apart
or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in
the template DNA at the site that was previously complementary to the primer, a PCR
product will not be produced, resulting in a different pattern of amplified DNA segments on
the gel. Selecting the right sequence for the primer is very important because different
sequences will produce different band patterns and possibly allow for a more specific
recognition of individual strains.
Thus in RAPD,
The primers must anneal in a particular orientation (such that they point towards each
other).
The primers must anneal within a reasonable distance of one another.
In this figure which depicts a RAPD reaction, a large fragment of DNA is used the
template in a PCR reaction containing many copies of a single arbitrary primer.
RAPD
Reaction
#1:
The arrows represent multiple copies of a primer (all primers (arrows) have the same
sequence). The direction of the arrow also indicates the direction in which DNA
synthesis will occur.
The numbers represent locations on the DNA template to which the primers anneal.
Primers anneal to sites 1, 2, and 3 on the bottom strand of the DNA template and
primers anneal to sites 4, 5, and 6 on the top strand of the DNA template.
In this example, only 2 RAPD PCR products are formed:

1) Product A is produced by PCR amplification of the DNA sequence which lies in between
the primers bound at positions 2 and 5.
2) Product B is the produced by PCR amplification of the DNA sequence which lies in
between the primers bound at positions 3 and 6.
84
Note that no PCR product is produced by the primers bound at positions 1 and 4
because these primers are too far apart to allow completion of the PCR reaction.
Note that no PCR products are produced by the primers bound at positions 4 and 2 or
positions 5 and 3 because these primer pairs are not oriented towards each other
Suppose there was a change in sequence at primer annealing site #2:
RAPD
Reaction
#2:
Molecular polymorphisms can be identified at random and used for genetic mapping.
LIMITATIONS OF RAPD
Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a
DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2
copies). Co-dominant RAPD markers, observed as different-sized DNA segments amplified
from the same locus, are detected only rarely.
PCR is an enzymatic reaction, therefore the quality and concentration of template
DNA, concentrations of PCR components, and the PCR cycling conditions may greatly
influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and
needs carefully developed laboratory protocols to be reproducible.
Mismatches between the primer and the template may result in the total absence of
PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results
can be difficult to interpret.
FACTS
The complexity of eukaryotic nuclear DNA is sufficiently high that by chance pairs of
sites complementary to single octa- or decanucleotides may exist in the correct orientation
and close enough to one another for PCR amplification.
With some randomly chosen decanucleotides no sequences are amplified. With others, the
same length products are generated from DNAs of different individuals. With still others,
patterns of bands (such as those illustrated) are not the same for every individual in a
85
population. The variable bands are commonly called random amplified polymorphic DNA
(RAPD) bands.
12.3. RFLP
Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous
DNA sequences that can be detected by the presence of fragments of different lengths after
digestion of the DNA samples in question with specific restriction endonucleases. RFLP, as a
molecular marker, is specific to a single clone/restriction enzyme combination.
Most RFLP markers are co-dominant (both alleles in heterozygous sample will be
detected) and highly locus-specific.
An RFLP probe is a labeled DNA sequence that hybridizes with one or more
fragments of the digested DNA sample after they were separated by gel electrophoresis, thus
revealing a unique blotting pattern characteristic to a specific genotype at a specific locus.
Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes.
The RFLP probes are frequently used in genome mapping and in variation analysis
(genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.).
PRINCIPLE
For example, let's follow a particular RFLP that is defined by the restriction enzyme
EcoRI and the target sequence of 20 bases GCATGCATGCATGCATGCAT. EcoRI binds to
its recognition sequence GAATTC and cuts the double-stranded DNA as shown:
In the segment of DNA shown below, you can see the elements of an RFLP; a target
sequence flanked by a pair of restriction sites. When this segment of DNA is cut by EcoRI,
three restriction fragments are produced, but only one contains the target sequence which can
be bound by the complementary probe sequence (dull).
Let's look at two people and the segments of DNA they carry that contain this RFLP
(for clarity, we will only see one of the two stands of DNA). Since Jack and Jill are both
diploid organisms, they have two copies of this RFLP. When we examine one copy from Jack
and one copy from Jill, we see that they are identical:
86
Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTCJill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-
When we examine their second copies of this RFLP, we see that they are not
identical. Jack 2 lacks an EcoR I restriction site that Jill has 1.2 kb upstream of the target
sequence (difference in italics).
Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)GAATTCJill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)GAATTC-
Therefore, when Jack and Jill have their DNA subject to RFLP analysis, they will
have one band in common and one band that does not match the other's in molecular weight:
METHODOLOGY
o Total DNA is digested with a methylation-sensitive enzyme (for example, PstI),
thereby enriching the library for single- or low-copy expressed sequences (PstI clones
are based on the suggestion that expressed genes are not methylated).
o The digested DNA is size-fractionated on a preparative agarose gel, and fragments
ranging from 500 to 2000 bp are excised, eluted and cloned into a plasmid vector (for
example, pUC18).
o Digests of the plasmids are screened to check for inserts.
o Southern blots of the inserts can be probed with total sheared DNA to select clones
that hybridize to single- and low-copy sequences.
o The probes are screened for RFLPs using genomic DNA of different genotypes
digested with restriction endonucleases. Typically, in species with moderate to high
polymorphism rates, two to four restriction endonucleases are used such as EcoRI,
EcoRV, and HindIII. In species with low polymorphism rates, additional restriction
endonucleases can be tested to increase the chance of finding polymorphism.
87
PCR-RFLP
Isolation of sufficient DNA for RFLP analysis is time consuming and labor intensive.
However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to
the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter
time. An alternative name for the technique is Cleaved Amplified Polymorphic Sequence
(CAPS) assay.
12.4. AFLP
Amplified Fragment Length Polymorphisms (AFLPs) are differences in restriction
fragment lengths caused by SNPs that create or abolish restriction endonuclease recognition
sites.
The AFLP technique is based on the selective PCR amplification of restriction
fragments from a total digest of genomic DNA. AFLP-PCR was originally described by
Zabeau & Vos in 1993.
PRINCIPLE
The procedure of this technique is divided into three steps:
1) Digestion of total cellular DNA with one or more restriction enzymes and ligation of
restriction half-site specific adaptors to all restriction fragments.
2) Selective amplification of some of these fragments with two PCR primers that have
corresponding adaptor and restriction site specific sequences.
3) Electrophoretic separation and amplicons on a gel matrix, followed by visualisation of the
band pattern.
88
METHODOLOGY
In the first step of AFLP analysis, genomic DNA is digested with both a restriction
enzyme that cuts frequently (MseI, 4 bp recognition sequence) and one that cuts less
frequently (EcoRI, 6 bp recognition sequence).
The resulting fragments are ligated to end-specific adaptor molecules.
A preselective PCR amplification is done using primers complementary to each of the
two adaptor sequences, except for the presence of one additional base at the 3' end.
Which base is chosen by the user. Amplification of only 1/16th of EcoRI-MseI
fragments occurs.
In a second, "selective", PCR, using the products of the first as template, primers
containing two further additional bases, chosen by the user, are used. The EcoRIadaptor specific primer used bears a label (fluorescent or radioactive).
Gel electrophoretic analysis reveals a pattern (fingerprint) of fragments representing
about 1/4000 th of the EcoRI-MseI fragments
89
LIMITATIONS
Proprietary technology is needed to score heterozygotes and ++ homozygotes.
Otherwise, AFLP must be dominantly scored.
Developing locus-specific markers from individual fragments can be difficult.
Need to use different kits adapted to the size of the genome being analyzed.
12.5. SSCP
It is a technique for detecting point mutations in genes by amplifying a region of
genomic DNA (using asymmetric PCR) and running the resulting product on a high quality
gel. Single base substitutions can alter the secondary structure of the fragment in the gel,
producing a visible shift in its mobility.
SSCP is the electrophoretic separation of single-stranded nucleic acids based on
subtle differences in sequence (often a single base pair) which results in a different secondary
structure and a measurable difference in mobility through a gel.
METHODOLOGY
The procedure used during the development of SSCP was as follows:

digestion of genomic DNA with restriction endonucleases
denaturation in an alkaline (basic) solution
electrophoresis on a neutral polyacrylamide gel
transfer to a nylon membrane
hybridization with either DNA fragments or more clearly with RNA
copies synthesized on each strand as probes
Since then, more convenient procedures have been developed, taking into account
other molecular techniques, although sometimes it is simpler to amplify the double strand and
then denature it into single strands instead of trying to find suitable primers for the below
PCR method if the targeted sequence is unknown.
Most experiments involving SSCP are designed to evaluate polymorphisms at single
loci and compare the results from different individuals.
90
Figure. SSCP Procedure. The three equal-length double-stranded DNA fragments are
shown with the corresponding single-stranded structures, the red fragment folding into the
smallest molecule and the green the largest (Panel A). The desired polymorphism is selected
with PCR primers; primer A is in excess to amplify only a single strand (Panel B). Both the
double-stranded and single-stranded fragments are run through gel electrophoresis (Panel C).
If not for the color labels, there would be no distinction between the double-stranded
fragments. The single-stranded fragments, however, show considerable variation in mobility;
the small red molecule migrates more quickly through the gel than either the blue or the large
green molecule. Using this SSCP result, it becomes clear that the different lanes (red, blue,
or green) contain strands with different sequences; the more far apart the bands, the less
similar the nucleotide sequence.
Procedure as illustrated in Figure 1:
1. A specific pair of PCR primers (forward and reverse) is used to amplify the desired
DNA fragments from individuals.
2. Single-stranded DNA is produced by asymmetric PCR: the primer on one side of
the fragment is greatly in excess over the other primer. After the low- concentration primer
supply is exhausted, continued PCR produces only the target single strand.
3. The mobilities of the single stranded fragments are compared by electrophoresis
on a neutral polyacrylamide gel.
4. Bands are detected by radioactive labeling or (more often) silver staining, and the
pattern is interpreted.
Figure. Sample SSCP Gel Result and Interpretation. DNA was isolated and amplified from
sand flies (Lutzomyia longipalpis). SCCP analysis of the DNA shows multiple haplotypes or
sets of alleles usually inherited as a unit. Lanes 3 and 4 were identical haplotypes from two
individuals. The difference in band migration in adjacent lanes is associated with the number
of nucleotide differences (in parentheses): lanes 2-3 (2), lanes 3-4 (0), lanes 4-5 (3), lanes
5-6 (1), lanes 6-7 (3), lanes 7-8 (1), lanes 8-9 (1), and lanes 9-10 (4).
91
LIMITATIONS
Single-stranded DNA mobilities are dependent on temperature. For best results,

gel electrophoresis must be run in a constant temperature.
Sensitivity of SSCP is affected by pH. Double-stranded DNA fragments are

usually denatured by exposure to basic conditions: a high pH. Kukita et al. found that adding
glycerol to the polyacrylamide gel lowers the pH of the electrophoresis buffer--more
specifically, the Tris-borate buffer--and the result is increased SSCP sensitivity and clearer
data (1997).
Fragment length also affects SSCP analysis. For optimal results, DNA fragment
size should fall within the range of 150 to 300 bp, although SSCP analysis of RNA allows for
a larger fragment size (Wagner, 2002). The presence of glycerol in the gel may also allow a
larger DNA fragment size at acceptable sensitivity (Kukita et al., 1997).
Under optimal conditions, approximately 80 to 90% of the potential base exchanges

are detectable by SSCP (Wagner, 2002).
If the specific nucleotide responsible for the mobility difference is known, a similar
technique called Single Nucleotide Polymorphism (SNP) may be applied.
12.6. SNP
A Single Nucleotide Polymorphism (SNP) is the variation of a single base pair in the
DNA sequence between either the members of a species or between the paired chromosomes
of an individual. These polymorphisms may affect how organisms develop diseases and
respond to chemicals and drugs. SNPs are, therefore, of great value in biomedical research
and drug development. SNP detection and genotyping can be used to explain and diagnose
many diseases, to study the variation in drug responses, to establish the origin of biological
material and to study the relatedness between individuals.
Sequencing is a popular method for SNP genotyping but this method is more
expensive and time consuming as compared to the microarray based methods. DNA
microarrays represent a technological platform that enables SNP genotyping. Microarrays
allow simultaneous testing of up to hundreds of SNPs.
An example of a SNP is the alteration of the DNA segment AAGGTTA to
ATGGTTA, where the second "A" in the first snippet is replaced with a "T". On average,
SNPs occur in the human population more than 1 percent of the time. Because only about 3 to
5 percent of a person's DNA sequence codes for the production of proteins, most SNPs are
found outside of "coding sequences". SNPs found within a coding sequence are of particular
interest to researchers because they are more likely to alter the biological function of a
protein. Because of the recent advances in technology, coupled with the unique ability of
these genetic variations to facilitate gene identification, there has been a recent flurry of SNP
discovery and detection.
BACKGROUND
Single nucleotide polymorphisms or SNPs (pronounced "snips") are DNA sequence
variations that occur when a single nucleotide (A,T,C,or G) in the genome sequence is
altered. For example a SNP might change the DNA sequence AAGGCTAA to ATGGCTAA.
For a variation to be considered a SNP, it must occur in at least 1% of the population. SNPs,
which make up about 90% of all human genetic variation, occur every 100 to 300 bases along
92
the 3-billion-base human genome. Two of every three SNPs involve the replacement of
cytosine (C) with thymine (T). SNPs can occur in both coding (gene) and noncoding regions
of the genome. Many SNPs have no effect on cell function, but scientists believe others could
predispose people to disease or influence their response to a drug.
Although more than 99% of human DNA sequences are the same across the
population, variations in DNA sequence can have a major impact on how humans respond to
disease; environmental insults such as bacteria, viruses, toxins, and chemicals; and drugs and
other therapies. This makes SNPs of great value for biomedical research and for developing
pharmaceutical products or medical diagnostics. SNPs are also evolutionarily stable --not
changing much from generation to generation --making them easier to follow in population
studies.
Scientists believe SNP maps will help them identify the multiple genes associated
with such complex diseases as cancer, diabetes, vascular disease, and some forms of mental
illness. These associations are difficult to establish with conventional gene-hunting methods
because a single altered gene may make only a small contribution to the disease.
Degeneracy codes employed in SNP studies
IUPAC code
A
C
G
T
M
R
W
S
Y
K
V
H
D
B
N
Meaning
A
C
G
T
A or C
A or G
A or T
C or G
C or T
G or T
A or C or G
A or C or T
A or G or T
C or G or T
G or A or T or C
SNP CLASS:
Het variation has unknown sequence composition, but is observed to be heterozygous
In Del insertion deletion polymorphism, deletions represented by '-' in allele string
microsatellite / simple sequence repeat mixed map multiple nucleotide polymorphism (all
alleles same length where length>1) named allele sequences defined by name tag instead of
raw sequence, e.g. (Alu)/- no variation submission reports invariant region in surveyed
sequence snp true single nucleotide polymorphism
93
METHOD CLASS
Computed variation was mined from sequence alignment with software dhplc
Denaturing High Pressure Liquid Chromatography used to detect SNP hybridize
hybridization method (e.g. chip) was used to assay for variation rflp variation in enzyme
restriction site used to detect variation sequence samples were sequenced and resulting
alignment used to define variation sscp single stranded conformational polymorphism used to
detect variation
12.7. LET US SUM UP
RAPD
RAPDs exhibit polymorphism and thus can be used as genetic markers.
RAPDs are dominant in the sense that the presence of a RAPD band does not allow
distinction between hetero- and homozygous states.
Breeders should be able to identify RAPD bands closely linked to the marker they
wish to transfer. Scoring individuals (or groups of individuals) for the linked RAPD
marker should speed the breeding process.
RAPD markers linked to genes of interest can serve as starting points for chromosome
walks to isolate those genes.
RFLP
Isolation of sufficient DNA for RFLP analysis is time-consuming and labor intensive.
However, PCR can be used to amplify very small amounts of DNA, usually in 2-3
hours, to the levels required for RFLP analysis. Therefore, more samples can be
analyzed in a shorter time.
AFLP
AFLP's, can be codominant markers, like RFLP's. Codominance results when the
polymorphism is due to sequences within the amplified region. Yet, because of the
number of bands seen at one time, additional evidence is needed to establish that a set
of bands result from different alleles at the same locus.
If, however, the polymorphism is due to presence/absence of a priming site, the
relationship is dominance. The non-priming allele will not be detected as a band.
SSCP
The SSCP technique makes possible the detection of both known and unknown single
point mutations and polymorphisms in products of the PCR.
Optimization of SSCP analysis to detect the maximum number of mutations requires

electrophoresis under carefully controlled conditions at different temperatures and
using different gels.
94
SNP
A single nucleotide polymorphism, or SNP is a DNA sequence variation occurring

when a single nucleotide - A, T, C, or G - in the genome differs between members of
a species.
Single nucleotide polymorphisms may fall within coding sequences of genes, noncoding regions of genes, or in the intergenic regions between genes.
SNPs within a coding sequence will not necessarily change the amino acid sequence
of the protein that is produced, due to degeneracy of the genetic code.

1. Evaluate the differences between RELP and AFLP.
1.
2.
3.
4.
5.
Give an account of Genetic markers.

How SSCP differ from SNP?
How AFLP is better than RFLP?
Why RAPD is not accepted by scientific community in spite of its easiness?
Suggest an ideal marker for paternal disputes.
12.10. REFERENCES
95
LESSON 13: CONSTRUCTION OF CDNA LIBRARY
CONTENTS
13.1. INTRODUCTION
13.2. CDNA LIBRARY TERMS AND KEY WORDS
13.3. CLONING STRATEGY
13.4. SCREENING
13.6. GETTING FULL LENGTH CDNAS
13.7. OTHER METHODS OF ISOLATING CDNAS
13.8. APPLICATIONS
13.9. LET US SUM UP
13.12. REFERENCES
In this lesson we will be discussing construction of cDNA library in detail.
Key words in cDNA cloning

Cloning strategy involved
Probe designing
13.1. INTRODUCTION
Suppose you have known the partial sequence of a gene (e.g., from the sequence of a
homologous gene) and want to determine its entire sequence, then you may use the technique
described in this section.
DNA library is a collection of cloned DNA fragments. There are two types of DNA
library: The genomic library contains DNA fragments representing the entire genome of an
organism. The cDNA library contains only complementary DNA molecules synthesized
from mRNA molecules in a cell.
Genomic library is the total representative of total genes present in an organism,
whereas a cDNA library is representative of genes, which are expressed during a particular
stage of the cell. cDNA library is a collection of clones containing an insert obtained from
cDNA cloning.
The advantage of cDNA library is that it contains only the coding region of a
genome. To prepare a cDNA library, the first step is to isolate the total mRNA from the cell
type of interest. Because eukaryotic mRNAs consist of a poly-A tail, they can easily be
separated. Then the enzyme reverse transcriptase is used to synthesize a DNA strand
complementary to each mRNA molecule. After the single-stranded DNA molecules are
converted into double-stranded DNA molecules by DNA polymerase, they are inserted into
vectors and cloned.
96
Thus an insert in a cDNA library can express a protein under the presence of a
promoter. Genomic library contains introns which are not represented in the mRNA. It also
contains inserts, which do not code for any product or protein. Such inserts are obtained from
the extragenic DNA.
Hence, when the aim of the work is to obtain a gene with protein-making ability or to
analyse what genes are expressed to make one cell to convert into another cell type, it is best
to go for cDNA library construction than genomic library construction as cDNA library
generates a lesser number of recombinant clones, which can be screened very easily.
97
13.2. cDNA LIBRARY TERMS AND KEY WORDS
One of the more powerful approaches which has been developed over the last twenty years is
the ability to make, isolate and use DNA that are complementary (cDNA) to messenger
RNAs (mRNAs) that encode proteins of interest. At the end of the page we will briefly
summarize the reason that cDNAs can be extremely valuable in experimental design,
although many of these should already be obvious to most readers. It is also true that there are
dozens of different approaches to isolating cDNAs of interest and these will be briefly
described in the second part of the section. We will begin by describing how a cDNA for a
known protein can be isolated using amino acid sequence information, which, historically,
was the first way that a cDNA encoding for a known protein was isolated. In this first section
we will also consider how cDNAs are made and how cDNA libraries are constructed.
KEY WORDS IN CDNA CLONING
Clone. First, what does it mean to clone? Cloning refers to the isolation of a genetically
homogeneous strain of any organism. Within a clone, all organisms are identical to all other
organisms at a genetic level. It is possible to clone bacteria or phage or even higher plants by
isolating a single cell and allowing that single cell to produce a colony, or a plaque, or an
entire plant. Since most plants are derived from a single cell with a unique genotype, the act
of rooting leaves to produce a collection of identical African violets is cloning.
cDNA cloning is isolating and amplifying a single, self-replicating organism that
includes within its DNA, a cDNA that is of interest to the experimenter.
In some cases cDNA cloning may simply refer to the isolation of any single cDNA,
since, in some circumstances, an experimentalist may be interested in any cDNA produced by
a particular tissue. More frequently, the challenge of cDNA cloning is not the isolation of any
cDNA but the selection of a single cDNA that is of interest to the experimentalist for a
particular reason. In the same way it is possible to isolate clones that are not cDNA clones but
rather are genomic clones.
Genomic clones are simply DNA derived directly from a genome. Genomic DNA
would incorporate some sequences such as introns or regulatory sequences that would not be
found in cDNAs.
Likewise, the isolation of a monoclonal antibody refers to the isolation of a single cell
that expresses a mRNA for a unique antibody. Thus, making monoclonal antibodies an
exercise in cloning.
Library. The second concept that is important in understanding the strategy needed to isolate
a cDNA clone, a genomic clone, or even a monoclonal antibody is the idea of a library.* A
library is defined simply as a collection of different DNA sequences that have been
incorporated into a vector.
Vector. A vector* is simply a self-replicating organism which is usually designed for the
convenience of its experimental purpose. For experimental convenience, vectors are usually
derivatives of viruses (plasmids, bacteriophages, animal viruses, retroviruses). Since the
essence of being able to isolate clones is the ability to replicate to make large amounts of
biological material, the essence of a vector is that it must incorporate some mechanism of
reproduction. (i.e., one must expand the clone to make many copies of the same organism).
98
Thus, one would expect that vectors would incorporate an origin of DNA replication. Since
vectors are an important experimental approach, a considerable amount of effort has gone
into designing vectors that are particularly easy to use in an experimental sense. It would be
impossible to provide even a brief description of the tricks that have been incorporated into
various classes of vectors. The incorporation of selectable markers is certainly a significant
experimental advantage in many cases.
13.3. CLONING STRATEGY
The underlying experimental approach to cloning can be divided into four parts.
First, it is necessary to produce or obtain a library including the sequence of

interest.
Second, it is necessary to isolate clones that may be of interest.
Third, it is essential to develop a formal test to ensure that the clones that have been
isolated are indeed the correct clones.
Fourth, it is essential to put the cDNA that has been isolated to some interesting
biological use.
cDNA libraries. A cDNA library simply contains sequences that are complementary to
mRNAs. There are a number of different criteria that might be used to judge the quality of a
cDNA library. A cDNA library is generally better if the size of the inserts (that is the amount
of continuous cDNA in each clone) is large, ideally full-length. Ideally, no member of the
library should include cDNAs derived from different mRNAs (this could be confusing). The
library should be sufficiently large that it contains the cDNA of interest (or,more precisely, it
should have enough independently derived clones that it contains the cDNA of interest). In
general this means that it should be representative of all the mRNAs present in a particular
tissue. Of course, choosing a tissue that has a relatively large amount of the mRNA of interest
is an important experimental choice. In general it is easier to isolate a cDNA from a library
where it is represented many times than from a library where it is present rarely. Some
characteristics of a library depend on the vector chosen. Vectors are frequently chosen
because they allow the screening of a large number of independent members of the library
with experimental ease. Some vectors are designed to express only the cDNAs, while others
have been modified to express not only the cDNA but also to express it in a context so the
cDNA is made into a protein or a fusion protein. (Fusion proteins will be discussed below.)
Before using a cDNA library it is wise to determine if it is a good quality library. More than
one student has wasted months of time screening a library that had no inserts or inserts so
short that they were of little value.
Making cDNA. Generation of cDNAs can also be done by a wide variety of processes, but, in
virtually all cases, cDNA is generated by the enzyme reverse transcriptase (RT) which has
the ability to use the information in an RNA to generate a complementary DNA. Thus,
reverse transcriptase is a RNA-dependent DNA polymerase. Like all DNA polymerases it
cannot initiate synthesis de novo but depends on the presence of a primer. Since many
mRNAs have a poly-A tail at the 3' end, oligo-dT is frequently used to prime DNA synthesis
(it is also possible, and frequently essential, to generate cDNAs by using either random
primers or primers designed to amplify a specific mRNA). Once the initial cDNA has been
generated it is generally necessary to produce a second strand of DNA. Again, there are many
strategies for doing this, but a convenient mechanism involves exposure of the DNA/RNA
hybrid to a combination of RNAase-H and DNA polymerase. RNAase-H has the ability to
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cause single-stranded nicks in the RNA, and DNA polymerase can then use these singlestranded nicks to initiate " second strand" DNA synthesis. This two-step procedure has been
optimized to maximize fidelity and length of cDNAs.
Incorporating cDNA into the vector. The next challenge is to incorporate this collection of
cDNA s into a vector so that it can be manipulated. One of the most convenient ways of
doing this is to attempt to manipulate the cDNAs so that each one has a unique restriction site
at those ends. To do this, the cDNAs are frequently methylated with a specific methyl
transferase that incorporates a methyl group into particular restriction site to protect them
from the restriction enzyme that will be used later. Any 3' or 5' extensions must be then either
eliminated by nuclease treatment or filled in with polymerase. This produces a "blunt ended"
molecule in which the 3' and 5' bases are in "register". It is then possible to ligate a synthetic
oligonucleotide to the ends of this cDNA . Blunt end ligation is generally a low efficiency
process; but, by using a high concentration of these synthetic oligonucleotides, it is possible
to drive the reaction to near completion. These synthetic oligonucleotides can either be
'linkers' (which are synthesized to have one blunt end and one end that have an 'overhang'
(i.e., region of single stranded DNA) that is complementary to that produced by restriction
enzymes or they can be 'adapters' (which are a double-stranded DNA molecule that can be
treated with a nuclease to produce the appropriate overhang).
The value of producing an overhang is that it will facilitate the introduction of the
cDNA into a vector. The vector can also be prepared by treating it with the same nuclease, or
a nuclease that produces the same restriction site, to produce a single-stranded region that is
complementary to the single-stranded region in the cDNA. Mixing the cDNA of interest with
the vector in the presence of ligase allows incorporation of the cDNA into the vector. One of
the experimental difficulties in doing this is that the vector itself will have a high tendency to
re-ligate to form a vector without any cDNA insert. This is frequently minimized by treating
the vector with the phosphatase to remove the terminal phosphates. These phosphates are
required for ligase to act, so this strategy prevents this unwanted side reaction.
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The choice of the vector used also has an important impact on experimental outcome.
Initially, plasmids were chosen as vectors and were modified to include markers that could be
used to determine whether a plasmid had been introduced into a bacterial cell or whether
there was a cDNA insert in the cloning site. More recently, derivatives of bacteria phage
lambda has been made that can be effective vectors for cDNA cloning. The advantage of
bacteria phage lambda is that it is possible to isolate more independent clones from a given
amount of mRNA/cDNA and to screen a higher number of clones using hybridization
techniques. The extent of understanding of lambda and lambda genetics has made it possible
to isolate lambda derivatives where some non-essential genes have been removed making it
possible to carry inserts of up to 11 kb of cDNA, which is a convenient size and sufficient for
the isolation of most cDNAs. The lambda genome is a linear molecule when it is packaged
into the bacteriophage and the cDNA can be incorporated into the central region of the DNA.
The lambda "arms" (the more distal parts of the DNA) encode all the essential information
for replication of lambda in an infectious cycle. The cloning site in lambda -gt10 was chosen
to interrupt genes that are essential for lambda to undergo lysogeny*. If the lambda arms religate in the absence of an insert, and an appropriate host is chosen (hfl, for high frequency of
lysogeny), then these particles will not form plaques. Thus only particles carrying an insert
will form plaques. The remarkable power of bacteriophage lambda as a vector is that once the
cDNA has been ligated into the lambda arms, the DNA can then be incorporated into a phage
particle in vitro. Extracts prepared from cells that have all the necessary proteins for the
assembly of lambda can then be mixed with the library DNA and ATP and particles will be
assembled! These particles can then be used to infect E coli and each individual plaque is an
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independent clonal population which represents a single cDNA species. This ability can be
used both to amplify the cDNA library (which is somewhat dangerous because repeated
amplification can lead to a loss of some cDNA sequences) and for the screening of the cDNA
library to isolate the cDNA of interest.
13.4. SCREENING
A lambda -gt10 library can be conveniently screened by plating it at relatively high
concentrations on a bacterial lawn of E coli. High density screening allows the
experimentalist to screen between 100,000 and 1,000,000 independent plaques on a single
plate and makes it theoretically possible to screen for a cDNA that is present only at one copy
per cell in a particular tissue. Screening is done by a "replica plating" procedure. After the
phage infect E coli and form individual plaques, a perfect spatial representation of the
infected plaque can be produced by placing a piece of nitrocellulose on top of the lawn of Ecoli. Nitrocellulose binds DNA with great avidity and so some of the DNA of each plaque
can be transferred to nitrocellulose paper or even several different nitrocellulose papers. each
nitrocellulose sheet should have a representation of the original pattern of infected cells on a
petri dish. The DNA from the library can then be cross-linked to the filter and extraneous
protein can be washed off. The plaques of interest can then be screened using a hybridization
assay.
This takes us to the question of how a library can be screened to isolate candidates for the
cDNA of interest. One of the most straight forward ways to do this is to take advantage of
DNA hybridization. If one can design an oligonucleotide that is complementary to the mRNA
of interest this can be used to screen the library. Such an oligonucleotide probe can be
designed by sequence information from the amino acid sequence of a known protein. In the
50s and 60s biochemical methods were developed to produce amino acid sequence of
overlapping fragments of known purified proteins. Our task is much simpler. It is now
necessary only to know the amino acid sequence of a couple of regions of the protein. To do
this, a purified protein is generally digested either with proteases or biochemical method to
produce a series of peptides. Unlike proteins, which must be treated with care to ensure that
they retain their native conformation, peptides can be treated as bio-organic molecules. They
can be fractionated by fairly standard procedures using HPLC (high pressure liquid
chromatography) which is capable of resolving individual peptides. If a series of individual
peptides can be resolved, the sequence of those peptides can be determined, or at least
partially determined, by Edmund degradation. This series of reaction cleaves individual
amino acids one at a time from a peptide and the resultant amino acid derivatives can be
identified. This procedure can produce sequence information on a series of peptides. To do
this intelligently it is essential that each of the peptides is derived from a single protein
molecule, and the criterion for insuring that this is likely to be the case were discussed in the
section on protein purification. Edmund degradation works via removing single amino acids
from the N-terminal end and can in some cases be applied to an intact protein, however,
generally the N-terminal amino group is chemically modified so this approach usually fails.
A probe is an oligonucleotide that is designed to be complementary to the mRNA of
interest so that it can be used to screen a library. Of course, any mRNA produces a unique
polypeptide when it is translated; but the reverse is not true. Because the triplet code is
degenerate, there are many mRNA sequences that might produce the same amino acid
sequences. Because of this the design of an oligonucleotide probe is not straight forward, but
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a clever experimentalist can make good choices in designing a probe. There are basically two
strategies that can be used. Either the experimentalist can choose to design a relatively short
oligonucleotide that hopefully will have a high degree of homology to the mRNA of interest
or the experimentalist can choose to design a longer probe that is more likely to have some
regions that are not complementary to the mRNA of interest but hopefully will have at least
some sequences that can form a stable duplex. In many cases it makes sense to make a
mixture of different probes, which are homologous, but have different bases in positions
where it is not possible to make a good prediction of which one should be present. This is
called degeneracy. A probe can frequently be 64 or 128 fold degenerate; but too much
degeneracy reduces the specific activity of a probe and increases the chance of hybridization
with the 'wrong' cDNA. The choice of which strategy depends on the amino acid sequences
that is available.
There are a number of other factors that should also be taken into consideration. In
many organisms, there is a preference for the use of particular triplets over the use of other
triplets (codon utilization). Designing a probe that has homology to a known mRNA is
generally not recommended since this may lead to the cloning of the wrong cDNA. Testing of
any probe for its correspondence to known sequences in the data base is, thus, essential.
Using amino acids or amino acid combinations that have fewer potential triplet coding
sequences or lower degree of degeneration (i.e., potential sequences) is of great importance.
If multiple related probes are possible, it is often sensible to screen with a degenerate
oligonucleotide. Once a probe or a series of probes are designed they can be synthesized
chemically and labeled to high specific activity with 32 P. The oligonucleotide probes can
then be incubated with the nitrocellulose filters to allow hybridization. Conditions are chosen
to try to maximize the specificity of the hybridization, but allow for some potential mismatch.
Most importantly, conditions should be chosen so that hybridization which is non-specific or
occurs only with high degree of mismatch is not allowed. Thus, filters are washed to remove
unlabeled or non-specifically bound oligonucleotides. The filters can then be
autoradiographed to identify the regions of the filter corresponding to a, hopefully, specific
signal. Since the filter is a replicate of the original plate, the experimentalist can then return to
this plate and isolate the original plaque or group of plaques responsible for the signal. The
plaques can then be re-plated on fresh E coli (remember each plaque contains phage that can
infected and replicate in E coli), and the process is repeated to eventually isolate a single
plaque that is responsible for the signal (i.e., Plaque purify* it).
Test for specificity. While the isolation of a plaque that gives a strong signal is clearly
an exciting step, it is only the first step. The next question must be asked: is the isolated
cDNA really the one of interest? It could certainly be a cDNA for a related protein or a
completely unrelated protein that just happened to have a sequence that would hybridize to
the probe that was chosen. Thus, it is essential to develop some criteria that the right cDNA
has been isolated or eliminated from contention. There are a number of criteria that will fulfill
this need. The simplest takes advantage of sequence information that can be obtained from
the isolated cDNA. In contrast to proteins where getting sequence information is
experimentally difficult, it is relatively straight forward to get sequence information from
DNA. The cDNA can be subcloned into a convenient vector and sequence information can be
obtained. If the sequence of the cDNA that has been isolated also encodes the sequence of
some of the peptides that had been sequenced but not used to design a probe, this is certainly
persuasive evidence that the correct cDNA has been isolated. It would be hard to argue that
the wrong cDNA had been isolated if sequence of several independent peptides were all
predicted by a cDNA.
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Frequently, there are also elements of the structure of the predicted protein that can be
used to help confirm the correctness of the cloning procedure. During the characterization of
the protein, it is frequently known that the protein for example may be a membrane protein in
which case one might predict the existence of transmembrane sequences. Some proteins are
known to be phosphoproteins which suggest the presence of either serines or threonines in
particular context that will allow kinase to phosphorylate them. Likewise, some proteins are
glycosylated and the presence of amino acid sequences that are associated with glycosylation
will also support the correctness of the cloning approach. Again, it must be emphasized that
all of these are simply criteria that the correct cDNA has been isolated. These must be used
by the experimentalist to develop a convincing case, but none are absolutely fool-proof. In
some cases, the pattern of expression of a protein (a tissue-specific manner) or a change in
mRNA in organisms that carry a particular mutation that is known to influence the activity of
the protein of interest can be a powerful criterion that will allow the experimentalist to make
a persuasive case that the correct cDNA has been isolated. One of the most convincing
approaches is to determine if the protein encoded by the isolated DNA has the biolgical
activity of interest, but it sometimes takes time to do this experiment.
13.6. GETTING FULL LENGTH cDNAS
In some cases, indeed in most cases, the cDNA that is isolated will not be full-length,
i.e. it will correspond only to parts of mRNA but not the entire sequence. In this case it is
necessary to re-screen the library, generally using the cDNA that already has been isolated to
identify either a full-length cDNA or a series of partial cDNAs that would encompass the
entire cDNA of interest. This brings up the interesting question of how an experimentalist
knows whether a full-length cDNA has indeed been isolated. Consideration of basic
molecular biology can provide a number of clues in this question. The molecular weight of a
mRNA can be estimated by northern analysis and this can be compared to the size of the
cDNA that has been isolated. Of it is possible that several mRNAs may be generated from a
single gene by alternative splicing and this should be remembered. A mRNA should include
both a coding region which has a long open-reading frame as well as non-coding sequences
(frequently called UTRs, for untranslated regions) at both the 3' and 5' ends. An open reading
frame is simply an un-interrupted series of triplets that does not contain stop codons. Such a
coding sequence should predict a protein of an appropriate molecular weight which can often
be compared to the molecular weight of the known protein. Upstream of the translation start
site are frequently, but not always, found stop codons. The 3' end of the message frequently
has a poly-A tail. There is almost always special interest in clearly identifying the 5' end of
the mRNA. This sequence is often most difficult to obtain from a cDNA library since it
requires effective reverse transcriptase to the extreme end of the mRNA. Often it is necessary
to return to a cDNA library repeatedly or use specialized approaches to isolate an authentic 5'
end. Often, the best way to identify sequences at the 5' end of a cDNA is to use RACE, which
is a PCR based technique to amplify DNA sequences near either the 5' or the 3' end of a
DNA. The authenticity of a particular 5' end can be confirmed by doing 'primer extension*'
experiments. In this technique, reverse transcriptase is used to extend an oligonucleotide
primer which has been designed to hybridize near the predicted 5' end of a mRNA. The
extension of such a primer should produce a polymer of a specific and predicted size.
13.7. OTHER METHODS OF ISOLATING CDNAS.
The choice of how to isolate cDNAs depends on the interest of the investigator and
the tools that are available. Design of an oligonucleotide probe has been used effectively in
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many cases but there are many other additional approaches that can be used. A few of them
will be listed and described in this section.
Cloning from expression libraries. In many cases a vector can be designed so that the cDNA
will be expressed, frequently as a fusion protein. In this case the cDNA has been incorporated
into a vector in a position where it is within a coding sequence of another protein. The vector
also incorporates promoter sequences that allow the protein to be expressed (both transcribed
and translated). When such a vector is used to make a library it is called an expression
library*. Expression libraries have the advantage and disadvantage that the protein is present.
In some cases this may mean that there may be selective pressure against the expression a
cDNA of interest, but in many cases this expression allows for novel screening approaches.
The straightest forward of these is the use of antibodies to screen a library.
Screening with an antibody is quite similar to screening with an oligonucleotide
probe, but in this case an antibody to the protein is the reagent that is available. This antibody
can be generated experimentally, but it can also be available because of interesting
autoimmune response in an animal model or in a human population. For example, some
cancer patients develop an autoimmune disease that leads to neuronal degeneration. The
antisera from these patients can then be used to isolate a gene which produces a protein that is
recognized by this antibody. The antibody can be added to nitrocellulose filters under
conditions where it binds specifically and the antibody can be then detected by a secondary
antibody that is either labeled with an isotope or covalently attached to an enzyme like horse
radish peroxidase that can be detected using standard enzymatic reactions. Here is a good
web site that provides more information on this type of approach.
If a cDNA is thought to encode a soluble factor that has a known biological effect,
and if that effect can be easily assayed, then the assay could be a way to screen the library,
although it may be difficult to screen a large number of independent isolates.
Complementation. Some genes can be isolated by a classic genetic complementation
approach. If there is a method to select for the expression of a particular gene then this
selection can be used to isolate a cDNA that encodes for that gene. For example it is
relatively straight forward to select either for or against the presence of the enzyme
HGPRTase* (hypoxanthine guanine phospho ribosol transferase) in E. coli or in eukaryotic
cells. If HGPRTase-deficient E. coli can be isolated and then transformed with an expression
vector, those cells expressing the appropriate activity would become HGPRTase+. Since it is
possible to select for such colonies, this would be an easy way to isolate a cDNA for
HGPRTase from any organism. Complementation has been used to isolate many types of
cDNAs including some that regulate complex phenomenon like the cell cycle or membrane
trafficking. The power of this approach is that it provides such strong evidence for specific in
vivo function. Of course, it is essential to independently establish that the correct clone has
been isolated.
Expression on the cell surface with antibody screening. Cell surface receptors are special
interest in biology and they can sometimes be isolated using an expression strategy. Cell
surface molecules on lymphocytes for example have been identified by the isolation of
specific monoclonal antibodies. Likewise, the ligand for many receptors has been isolated
before the nature of the receptor is established. In both cases a cDNA for the cell surface
molecule when expressed, will lead to the presence of a binding site on the cell surface. This
binding site can be used to screen a library either by a method analogous to the antibody
screening mentioned above or by using the ligand or antibody as an affinity reagent to "pan"
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for cells that express the binding site.Functional cloning of receptors. One of the more
interesting classes of cell surface molecules are molecules that encode ionic channels.
Because of the tremendous power and sensitivity of the electrophysiology
(electrophysiologists can even measure the function of a single molecule!), the presence of
one or a few mRNA molecules in a single cell can produce enough ion channels to be
detected relatively easily using an electrophysiological approach. Injection of mRNAs in frog
oocytes can lead to the appearance of particular ion channels that can be detected either
because of their responsiveness to electrical signals or the presence of extracellular ligands.
This approach provided a straight forward assay for the cell-surface receptor for glutamic
acid (glutamate), which is the most common neurotransmitter receptor in the central nervous
system. This type of approach can either rely on expression vectors that can produce the
mRNA of interest or it can rely on a negative criterion. Co-injection of cDNAs can squelch a
signal by hybridizing specifically with the mRNA. Of course the difficulty with any of these
approaches is that it becomes more difficult to screen a large number of mRNAs. This
problem has been successfully conquered by using strategies involving 'sib (for sibling)
selection'. In this strategy, thousands of independent clones are screened at once, and, once a
signal is identified in any one pool, the pool itself can then be subdivided until an individual
clone can be isolated.
Homology screening. One of the most productive, although perhaps less creative approaches
to isolating cDNAs is homology screening*. Once an interesting gene has been isolated from
one species, it is relatively straight forward to use a low stringency hybridization strategy to
isolate cDNAs from another species. Likewise, additional family members from the same
species can frequently be identified. The power of this approach should not be
underestimated. Interesting mutants are frequently obtained in Drosophila by using genetic
screens and identifying the existence of corresponding genes in humans can be tremendously
important. Likewise, because of the large population of humans in the careful monitoring of
their medical care, human genetic diseases are proving an abundant source of interesting
genes and eventually interesting cDNAs. Determining the existence of such cDNAs in model
systems can then be extremely valuable. Good examples of this come from the field of
apoptosis. Some of the original genes like the ICE protease were originally identified in
studies of C. elegans and subsequently human homologs of this genes were isolated.
Likewise, the human oncogene, bcl 2, was initially isolated by genetic studies which led to
the isolation of the cDNA and subsequently homologs were identified in model systems.
PCR-based screens. PCR-based screening is also a method to isolate novel cDNAs. After two
or more members of a family have been isolated, regions of homology can be identified.
These regions of homology are conserved within the family, PCR primers can be designed
and used to amplify reverse transcriptase products of mRNAs in an appropriate tissue. The
molecular weight of known members of the family can be predicted and novel mRNAs may
give rise to novel amplification products. See the section on proteins for a good example of
this. These amplification products in turn can be used to screen cDNA libraries. In some
cases even a single region of conserved structure may be sufficient to isolate novel genes
using the following strategy. Reverse transcriptase can be used to extend a primer which has
been made to a conserved sequence. Such products of course could be heterogeneous because
different reverse transcriptase molecules would extend to different degrees. However, some
restriction enzymes are capable of cleaving single stranded DNA and treatment of such a
product with an enzyme of this type would produce a fragment of a unique size. Such a
fragment can then be homo-polymer tailed (i.e. a sequence of Cs can be added to the end of
the molecule) using terminal transferase. This sequence of Cs can then be used as a site to
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anchor an oligonucleotide primer containing a stretch of Gs. If this primer is extended the
resulting product will be suitable for PCR amplification between the two primers that were
used in its creation.
Plus/minus screening and differential display. Another useful approach to isolating cDNAs of
interest relies not on knowledge of their primary structure, but rather on assumptions about
their expression. Both plus/minus screening and differential display rely on strategies that
seek to isolate cDNAs that are expressed in one situation but not another. For example,
growth factors like NGF or PDGF and hormones like estrogen are known to induce the
expression of novel genes. Thus, a population of cells that are cultured or grown in the
presence and absence of such an experimental manipulation (e.g., +/-NGF, +/-estrogen, + /retinoic acid) should express some genes in common, but have some distinct mRNAs.
Likewise, tissues at different developmental stages may have expression patterns that are of
special interest. Tissues that are related but distinct may also express interesting subset of
genes. There are presumably interesting genes that are expressed in cerebellum, but not basal
ganglia; or in T cells, but not B cells. An isolation of those genes may give a clue to the
function of those tissues or the way gene expression is regulated.
In plus/minus screening, mRNA is isolated from two populations of cells and reverse
transcribed to produce a population of cDNAs. Aliquots of these cDNAs can then be
converted to probe by random hexamer priming and used to screen duplicate lifts from a
library (i.e., two nitrocellulose filters produced from the same plate of plaques or cells. Any
plaque or colony that hybridizes duplicate lifts from a library to one probe but not the other is
a potential candidate for interest, and differential expression can be tested by northern
analysis or a related approach.
Differential display is a simple modification of PCR amplification. In this approach,

mRNA is reverse transcribed using a series of primers. Frequently primers are chosen to have
a random set of oligonucleotide and an oligo-dT section that would hybridize to a poly-A tail.
mRNAs that are homologous to the randomly chosen sequence should be reverse transcribed,
producing a single-stranded cDNA. The addition of another primer, again, randomly chosen
will allow amplification of a subset of the reverse transcribed cDNAs. Depending on the
distance between the two primers, fragments of varying molecular weights will be obtained.
By doing this procedure with mRNA that has been isolated from two different cell
populations, the pattern of expression between the two cell types or cell states can be
determined. Again, an amplified product that is thought to be unique to a particular cell type,
can then be used a probe to screen a library or test expression by northern analysis. Both of
these methods have been used to isolate large number of interesting genes using only their
expression pattern.
Two hybrid screening. One of the more active approaches to isolating cDNA are the twohybrid screens. These screens are named because they take advantage of a specific protein protein interaction that occurs between two proteins each of which is itself a hybrid protein.
The entire assay relies on the ability of one part of each of the hybrid protein to form a
specific interaction that is reasonably stable under physiological conditions with the other. A
number of variations of this approach have been developed, but they all rely on the same
feature.
In the most straight forward version a test cell which expresses an easily assayed
gene, like beta-galactosidase, under the control of a well characterized promoter is produced.
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The promoter is chosen so that it has a low basal activity in the absence of stimulation from a
specific regulatory element.
The same cell is then transfected with an expression vector for a hybrid protein. One
part of the hybrid protein is derived from a transcription factor which is designed to recognize
the DNA regulatory element. Binding to the site, however, is not sufficient to induce gene
expression; rather, a specific mechanism to activate transcription is required. A second part of
this hybrid protein includes a sequence isolated from a particular gene of interest. This
protein can be derived from another transcription factor, from a structural protein, or from an
intracellular signaling protein. The only requirement is that the hybrid itself is not sufficient
to activate expression of the reporter gene.
This cell system is then transfected with an expression library that also expresses a
collection of fused protein. In this case the fusion protein consists of two part. One part of the
fusion protein is coded by the collection of the cDNA libraries that the experimentalist hopes
may encode a protein which will interact with target in the hybrid protein already expressed
in the cell. The second part of this fusion protein is an activator of transcription, frequently
the activating region of VP16, a potent transcription factor. If a cell is transfected with a
hybrid protein that does not recognize the hybrid protein already present in the cell (the bate)
nothing should happen.
In the rare case where VP16 is expressed as a hybrid with the protein that interacts
with the hybrid already present in the cell, this should result in activation of betagalactosidase. Thus, the screen serves as an initial assay for protein-protein interaction and is
structured in such a way that a large number of members of a cDNA library can be quickly
screened and selected for testing for specific interaction. Of course, there are always the
possibility that activation can occur by a non-specific mechanism, but this possibility can be
tested for without too much difficulty.
Screening by databases. The rapid accumulation of sequence information and genetic data
often allows scientists to bypass the steps required to isolate cDNAs. For example, if partial
protein sequence or partial cDNA sequence is available, searching data bases may result in
identifying candidate clones that can be ordered and tested to determine if they are the 'right'
clone. Databases include the sequence of entire genomes as well as short sequences from
cDNAs that serve to tag individual clones (ESTs*, or expressed sequence tags).
13.8. APPLICATIONS
1. Isolating cDNAs allows the experimentalist to use the cDNA to develop expression vectors
so proteins of interest can be produced in high quantities, greatly simplifying the task of
protein purification.
2. Knowing the sequence of an amino acid immediately gives access to the sequence of the
protein. By appreciating protein structure and studying the common motifs present in known
proteins, a great deal of information can be deduced about the possible structure and/or
function of the protein encoded by a known cDNA. Presence of sequences can easily suggest
the protein product may be phosphorylated or may bind a particular small biochemical
molecule, like GTP.
3. The availability of a mRNA allows one to quickly design assays for studying the
expression of the mRNA; labeling cDNA can be used to determine the expression of mRNA
using both northern analysis and RNase protection and the subcellular distribution of RNase
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can be determined by in situ hybridization. Each of these approaches provides a specific
value.
Northern analysis can quickly determine whether the level of expression changes with drug
treatment, hormones, or developmental stage. It reveals whether there are several different
mRNAs that are expressed. Northern analysis relies on the fractionation of isolated mRNAs
on agarose (or occasionally, acrylamide) gels which are then transferred to nitrocellulose. The
transferred mRNAs are then detected by hybridization to a labeled cDNA probe.
RNase protection is a more sensitive method of determining mRNA abundance. In contrast to
northern analysis, RNase protection relies on the resistance of a hybrid molecule to digestion.
Whereas mRNA is normally extremely sensitive to ribonuclease, if RNA is isolated and then
hybridized to a labeled probe (either RNA or DNA) then the hybrid molecule or a portion of
a hybrid molecule can be protected from ribonuclease activity. The amount of protected
probe as well as its size can be easily measured.
DNA arrays allow the determination of the expression of huge numbers of mRNAs in a
single experiment. It is based on the hybridization to nucleic acids that are attached to a solid
phase support.
RT-PCR. even in the absence of a labeled cDNA probe, knowledge of the sequence of a
cDNA can allow a quantitation of expression. Knowing a cDNA's sequence, PCR primers
can be designed and used to amplify a reverse transcriptase product although there are
certainly problems in doing this quantitatively, it can also be a useful and powerful technique.
This approach can also be adapted to in situ approaches.
In situ hybridization. The cDNA can also be used to determine which cells, tissues, or
developmental stages produce a particular mRNA using in situ hybridization. Labeled nucleic
acid is incubated with fixed tissue or cells under conditions where only specifically bound
hybrid is stable. Auto radiography reveals the position of endogenous RNA. Controls with
RNase help prove that hybridization is to RNA not genetic material.
13.9. LET US SUM UP
PCR can be performed for any samples whose DNA of interest need to be amplified.
Both steps and components are critical components of PCR
Same PCR can be used for 100s of variations.
Automation in PCR is also a major breakthrough besides thermostable DNA
polymerases, which made PCR as a success.
1.Evaluate a cloning strategy.
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1. What is cDNA Library?
2. Explaint he key words in cDNA cloning.
3. Describe a screening method.
4. Discuss other methods of isolating cDNA.
13.12. REFERENCE
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LESSON 14: MOLECULAR MAPPING OF GENOME
CONTENTS
14.1. INTRODUCTION
14.2. GENETIC MAPPING
14.3. LINKAGE MAPPING
14.4. PHYSICAL MAPPING
14.5. LET US SUM UP
14.8. REFERENCES
In this lesson we will be discussing molecular mapping of in detail
What is genetic mapping
What are the types of mapping
17.1. INTRODUCTION
Genetic mapping is done for identifying of the location of the genes. The information
can be used for finding out the function of candidate genes involved in diseases. The
convention is to divide genome mapping methods into two categories.
Genetic mapping is based on the use of genetic techniques to construct maps showing
the positions of genes and other sequence features on a genome. Genetic techniques
include cross-breeding experiments or, in the case of humans, the examination of
family histories (pedigrees).
Physical mapping uses molecular biology techniques to examine DNA molecules
directly in order to construct maps showing the positions of sequence features,
including
17.2. GENETIC MAPPING
A genetic map must show the positions of distinctive features of the genes. In a
geographic map these markers are recognizable components of the landscape, such as rivers,
roads and buildings. They represent the possible location of the gene on the genome.
I)
GENES WERE THE FIRST MARKERS TO BE USED
The first genetic maps were, constructed for organisms such as the fruit fly, using
genes as markers. It was believed then, that genes were abstract entities responsible for the
transmission of heritable characteristics from parent to offspring and they were then named as
factors.
To be useful in genetic analysis, a heritable characteristic has to exist in at least two
alternative forms or phenotypes, an example being tall or short stems in the pea plants
originally studied by Mendel. The only genes that could be studied were those specifying
phenotypes that were distinguishable by visual examination. So, for example, the first fruitfly maps showed the positions of genes for body color, eye color, and wing shape. The draw
back was that the analysis was complicated because a single phenotype could be affected by
more than one gene.
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As a next step in mapping biochemical characters were used to distinguish phenotypes.

For example biochemical phenotypes scoring for blood typing. These phenotypes include not
only the standard blood groups such as the ABO series, but also variants of blood serum
proteins and of immunological proteins such as the human leukocyte antigens (the HLA
system). A big advantage of these markers is that many of the relevant genes have multiple
alleles.
II) DNA MARKERS FOR GENETIC MAPPING
Only a fraction of the total number of genes exists in allelic forms that can be
distinguished conveniently. Gene maps are therefore not very comprehensive. Hence there is
a need for other type of markers.
Mapped features that are not genes are called DNA markers. As with gene markers, a
DNA marker must have at least two alleles to be useful.
There are three types of DNA sequence feature that satisfy this requirement:
Restriction fragment length polymorphisms (RFLPs),
Simple sequence length polymorphisms (SSLPs),
Single nucleotide polymorphisms (SNPs).

III) RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPS)
RFLPs were the first type of DNA marker to be studied. Recall that restriction
enzymes cut DNA molecules at specific recognition sequences. This sequence specificity
means that treatment of a DNA molecule with a restriction enzyme should always produce
the same set of fragments. This is not always the case with genomic DNA molecules because
some restriction sites are polymorphic, existing as two alleles, one allele displaying the
correct sequence for the restriction site and therefore being cut when the DNA is treated with
the enzyme, and the second allele having a sequence alteration so the restriction site is no
longer recognized. The result of the sequence alteration is that the two adjacent restriction
fragments remain linked together after treatment with the enzyme, leading to a length
polymorphism this is an RFLP and its position on a genome map can be worked out by
following the inheritance of its alleles, just as is done when genes are used as markers.
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A restriction fragment length polymorphism (RFLP). The DNA molecule on the left
has a polymorphic restriction site (marked with the asterisk) that is not present in the
molecule on the right. The RFLP is revealed after treatment with the restriction enzyme
because one of the molecules is cut into four fragments whereas the other is cut into three
fragments.
Two methods for scoring an RFLP. (A) RFLPs can be scored by Southern
hybridization. The DNA is digested with the appropriate restriction enzyme and separated in
an agarose gel. The smear of restriction fragments is transferred to a nylon membrane and
probed with a piece of DNA that spans the polymorphic restriction site. If the site is absent
then a single restriction fragment is detected (lane 2); if the site is present then two fragments
are detected (lane 3). (B) The RFLP can also be typed by PCR, using primers that anneal
either side of the polymorphic restriction site. After the PCR, the products are treated with the
appropriate restriction enzyme and then analyzed by agarose gel electrophoresis. If the site is
absent then one band is seen on the agarose gel; if the site is present then two bands are seen.
IV) SIMPLE SEQUENCE LENGTH POLYMORPHISMS (SSLPS)
SSLPs are arrays of repeat sequences that display length variations, different alleles
containing different numbers of repeat units. Unlike RFLPs, SSLPs can be multi-allelic as
each SSLP can have a number of different length variants.
Minisatellites, also known as variable number of tandem repeats (VNTRs), in which

the repeat unit is up to 25 bp in length;
Microsatellites or simple tandem repeats (STRs), whose repeats are shorter, usually
dinucleotide or tetranucleotide units.
Microsatellites are more popular than minisatellites as DNA markers, because,

Minisatellites are not spread evenly around the genome but tend to be found more frequently
in the telomeric regions at the ends of chromosomes. In geographic terms, this is equivalent
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to trying to use a map of lighthouses to find one's way around the middle of an island.
Microsatellites are more conveniently spaced throughout the genome.
A) Two alleles of a microsatellite SSLP.

In allele 1 the motif GA' is repeated
three times, and in allele 2 it is repeated
five times.
(B). The region surrounding the SSLP is
amplified and the products loaded into
lane A of the agarose gel. Lane B
contains DNA markers that show the
sizes of the bands given after PCR of the
two alleles. The band in lane A is the
same size as the larger of the two DNA
markers, showing that the DNA that was
tested contained allele 2.
SSLP TYPING
(B) SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS)
These are positions in a genome where some individuals have one nucleotide (e.g. a
G) and others have a different nucleotide (e.g. a C). There are vast numbers of SNPs in every
genome, some of which also give rise to RFLPs, but many of which do not because the
sequence in which they lie is not recognized by any restriction enzyme. In the human genome
there are at least 1.42 million SNPs, only 100 000 of which result in an RFLP.
The advantages of SNPs are their abundant numbers and the fact that they can be typed by
methods that do not involve gel electrophoresis. SNP detection is more rapid because it is
based on oligonucleotide hybridization analysis.
A single nucleotide polymorphism (SNP).
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OLIGONUCLEOTIDE HYBRIDIZATION:
Oligonucleotide hybridization is very specific. Under highly stringent hybridization
conditions, a stable hybrid occurs only if the oligonucleotide is able to form a completely
base-paired structure with the target DNA. If there is a single mismatch then the hybrid does
not form. To achieve this level of stringency, the incubation temperature must be just below
the melting temperature or Tm of the oligonucleotide. At temperatures above the Tm, even the
fully base-paired hybrid is unstable. At more than 5 C below the Tm, mismatched hybrids
might be stable. The Tm for the oligonucleotide shown in the figure would be about 58 C.
The Tm in C is calculated from the formula Tm= (4 number of G and C nucleotides) + (2
number of A and T nucleotides). This formula gives a rough indication of the Tm for
oligonucleotides of 1530 nucleotides in length.
The oligonucleotide probe has two
end-labels.
One of these is a fluorescent dye
and the other is a quenching
compound.
The two ends of the oligonucleotide
base-pair to one another, so the
fluorescent signal is quenched.
When the probe hybridizes to its
target DNA, the ends of the
molecule become separated,
enabling the fluorescent dye to emit
its signal.
The two labels are called
molecular beacons'.
17.3. LINKAGE MAPPING

Mapping techniques are all based on genetic linkage. Genetic mapping is based on the
principles of inheritance as first described by Gregor Mendel in 1865. From the results of his
breeding experiments with peas, Mendel concluded that each pea plant possesses two alleles
for each gene, but displays only one phenotype. This is the perfectly correct interpretation of
the interaction between the pairs of alleles studied by Mendel, producing the response called
dominant recessive rule. But he did not encounter some of the situations: One of these is
incomplete dominance, where the heterozygous phenotype is intermediate between the two
homozygous forms. Eg. when red carnations are crossed with white ones, the F1
heterozygotes being pink. Another complication is codominance, when both alleles are
detectable in the heterozygote. Co dominance is the typical situation for DNA markers.
LINKAGE ANALYSIS WITH DIFFERENT TYPES OF ORGANISM
To see how linkage analysis is actually carried out, three different situations are considered:
Linkage analysis with species such as fruit flies and mice, with which we can carry
out planned breeding experiments;
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Linkage analysis with humans, with whom we cannot carry out planned experiments
but instead make use of family pedigrees;
Linkage analysis with bacteria, which do not undergo meiosis.
GENETIC MAPPING IN BACTERIA

The final type of genetic mapping that we must consider is the strategy used with
bacteria. The main difficulty that geneticists faced when trying to develop genetic mapping
techniques for bacteria is that these organisms are normally haploid, and so do not undergo
meiosis. Some other way therefore had to be devised to induce crossovers between
homologous segments of bacterial DNA. The answer was to make use of three natural
methods that exist for transferring pieces of DNA from one bacterium to another:
In conjugation two bacteria come into physical contact and one bacterium (the donor)
transfers DNA to the second bacterium (the recipient). The transferred DNA can be a
copy of some or possibly the donor cells entire chromosome, or it could be a segment
of chromosomal DNA - up to 1 Mb in length - integrated in a plasmid. The latter is
called episome transfer.
Transduction involves transfer of a small segment of DNA - up to 50 kb or so - from
donor to recipient via a bacteriophage.
In transformation the recipient cell takes up from its environment a fragment of DNA,
rarely longer than 50 kb, released from a donor cell.
After transfer, a double crossover must occur so that the DNA from the donor
bacterium is integrated into the recipient cell's chromosome. If this does not occur then the
transferred DNA is lost when the recipient cell divides. The only exception is after episome
transfer, plasmids being able to propagate independently of the host chromosome
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THREE WAYS OF ACHIEVING DNA TRANSFER BETWEEN BACTERIA
(A) Conjugation can result in transfer of
chromosomal or plasmid DNA from
the donor bacterium to the recipient.
Conjugation involves physical contact
between the two bacteria, with transfer
thought to occur through a narrow tube
called the pilus.
(B) Transduction is the transfer of a small

segment of the donor cell's DNA via a
bacteriophage.
(C) Transformation is similar to

transduction but naked' DNA is
transferred.
The events illustrated in (B) and (C) are often accompanied by death of the donor
cell. In (B), death occurs when the bacteriophages emerge from the donor cell; in (C), release
of DNA from the donor cell is usually a consequence of the cell's death through natural
causes.
17.4. PHYSICAL MAPPING
A map generated by genetic techniques is rarely sufficient for directing the
sequencing phase of a genome project. This is for two reasons:
The resolution of a genetic map depends on the number of crossovers that have been
scored. This means that genes that are several tens of kb apart may appear at the same
position on the genetic map.
Genetic maps have limited accuracy. The presence of recombination hotspots
meaning that crossovers are more likely to occur at some points rather than at others
give less accuracy to genetic maps.
These two limitations of genetic mapping mean that for most eukaryotes a genetic
map must be checked and supplemented by alternative mapping procedures before going for
large-scale DNA sequencing. A plethora of physical mapping techniques has been developed
to address this problem, the most important being:
117
I)
Restriction mapping, which locates the relative positions on a DNA molecule of the
recognition sequences for restriction endonucleases;
Fluorescent in situhybridization (FISH), in which marker locations are mapped by
hybridizing a probe containing the marker to intact chromosomes;
Sequence tagged site (STS) mapping, in which the positions of short sequences are
mapped by PCR and/or hybridization analysis of genome fragments.
RESTRICTION MAPPING
Genetic mapping using RFLPs as DNA markers can locate the positions of
polymorphic restriction sites within a genome as seen earlier.
Restriction maps are easy to generate if there are relatively few cut sites for the enzymes
being used. Restriction mapping is therefore more applicable to small rather than large
molecules, with the upper limit for the technique depending on the frequency of the
restriction sites in the molecule being mapped. The technique is as follows.
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RESTRICTION MAPPING
The objective is to map the EcoRI (E) and BamHI (B) sites in a linear DNA molecule
of 4.9 kb. The results of single and double restrictions are shown at the top. The sizes of the
fragments given after double restriction enable two alternative maps to be constructed, as
explained in the central panel, the unresolved issue being the position of one of the three
BamHI sites. The two maps are tested by a partial BamHI restriction (bottom), which shows
that Map II is the correct one
II)
FLUORESCENT in situ HYBRIDIZATION (FISH)
FISH enables the position of a marker on a chromosome or extended DNA molecule

to be directly visualized. In optical mapping the marker is a restriction site and it is visualized
as a gap in an extended DNA fiber. In FISH, the marker is a DNA sequence that is visualized
by hybridization with a fluorescent probe.
in situ HYBRIDIZATION WITH RADIOACTIVE OR FLUORESCENT PROBES
In situ hybridization is a version of hybridization analysis in which an intact
chromosome is examined by probing it with a labeled DNA molecule. The position on the
chromosome at which hybridization occurs provides information about the map location of
the DNA sequence used as the probe.
Fluorescent in situ hybridization.
A sample of dividing cells is dried onto

a microscope slide and treated with
formamide so that the chromosomes
become denatured but do not lose their
characteristic metaphase morphologies
The position at which the probe
hybridizes to the chromosomal DNA is
visualized by detecting the fluorescent
signal emitted by the labeled DNA.
FISH TECHNIQUE
FISH is originally used with metaphase chromosomes. A disadvantage is that the
highly condensed nature of metaphase chromosomes means that only low-resolution mapping
is possible, two markers having to be at least 1 Mb apart to be resolved as separate
hybridization signals. This degree of resolution is insufficient. If metaphase chromosomes
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are too condensed for fine-scale mapping then chromosomes that are more extended should
be used. There are two ways of doing this:
Mechanically stretched chromosomes can be obtained by modifying the preparative

method used to isolate chromosomes from metaphase nuclei. The inclusion of a
centrifugation step generates shear forces which can result in the chromosomes
becoming stretched to up to 20 times their normal length. Individual chromosomes are
still recognizable and FISH signals can be mapped in the same way as with normal
metaphase chromosomes. The resolution is significantly improved and markers that
are 200300 kb apart can be distinguished.
Non-metaphase chromosomes can be used because it is only during metaphase that
chromosomes are highly condensed: at other stages of the cell cycle the chromosomes
are naturally unpacked. This technique is therefore used after preliminary map
information has been obtained, usually as a means of determining the order of a series
of markers in a small region of a chromosome.
Interphase chromosomes contain the most unpacked of all cellular DNA molecules.
To improve the resolution of FISH to better than 25 kb it is therefore necessary to abandon
intact chromosomes and instead use purified DNA. This approach, called fiber-FISH, makes
use of DNA prepared by gel stretching or molecular combing and can distinguish markers
that are less than 10 kb apart.
III) SEQUENCE TAGGED SITE (STS) MAPPING
To generate a detailed physical map of a large genome we need, ideally, a highresolution mapping procedure that is rapid and not technically demanding. One such
technique is STS mapping. A sequence tagged site or STS is simply a short DNA sequence,
generally between 100 and 500 bp in length, that is easily recognizable and occurs only once
in the chromosome or genome being studied.
To map a set of STSs, a collection of overlapping DNA fragments from a single
chromosome or from the entire genome is needed.
To qualify as an STS, a DNA sequence must satisfy two criteria.
The first is that its sequence must be known, so that a PCR assay can be set up to test
for the presence or absence of the STS on different DNA fragments.
The second requirement is that the STS must have a unique location in the
chromosome being studied or in the genome as a whole if the DNA fragment set
covers the entire genome. If the STS sequence occurs at more than one position then
the mapping data will be ambiguous. Care must therefore be taken to ensure that STSs
do not include sequences found in repetitive DNA.
These are easy criteria to satisfy STSs can be obtained in many ways, the most common
sources being expressed sequence tags (ESTs), SSLPs, and random genomic sequences.
Expressed sequence tags (ESTs). These are short sequences obtained by analysis of
cDNA clones. Complementary DNA is prepared by converting an mRNA preparation
into double-stranded DNA. Because the mRNA in a cell is derived from proteincoding genes, cDNAs and the ESTs obtained from them represent the genes that were
being expressed in the cell from which the mRNA was prepared. ESTs are looked
upon as a rapid means of gaining access to the sequences of important genes, and they
120
are valuable even if their sequences are incomplete. An EST can also be used as an
STS, assuming that it comes from a unique gene and not from a member of a gene
family in which all the genes have the same or very similar sequences.
SSLPs. Earlier microsatellites and other SSLPs are used in genetic mapping. SSLPs
can also be used as STSs in physical mapping. SSLPs that are polymorphic and have
already been mapped by linkage analysis are particularly valuable as they provide a
direct connection between the genetic and physical maps.
Random genomic sequences. These are obtained by sequencing random pieces of
cloned genomic DNA, or simply by downloading sequences that have been deposited
in the databases.
17.5. LET US SUM UP

Genome mapping is done to identify the locus of the gene; so that the candidate gene
could be used for further analysis like functional cloning and other mutational analysis.
Methodology could be brought under two broad criteria Genetic and physical mapping.
Mapping techniques use one or more of the following characteristics of the Genome
Unique sequences (REN sites)
Repetitive sequences like micro and mini satellites
Recombination (hotspots)
Single nucleotide differences (SSLP and SNP)
Mono or multi allelic expression of the genes.
Hybridization strategies.
Such genetic maps find use in a number of ways:
Gene Functional analysis
Mutational screening for susceptive individuals
Forensic and legal investigation.
Pedigree analysis
Genetic engineering
1. Differentiate between STS and EST.
2. How SSLP and SNPs are dfferent?
17.7 LESSON END ACTIVITIES
1. What do you understand by sequence tagged sites?
100-500 bp short sequences
recognizable unique location on chromosome
Sequence information should be known.
2. How do you use FISH as a tool for physical mapping?
Use of marker DNA element as the key to identify the locus
Involves hybridization with specific probes of known sequence information; extended
chromosomes are hybridized to the probes- in situ FISH
Fiber FISH use of pure DNA instead of going for non metaphase chromosomes
17.8. REFERENCES
1. Genes VII by Lewin.
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UNIT IV: TRANSFORMATION TECHNIQUES

LESSON 18: CLONING IN GRAM POSITIVE BACTERIA
CONTENTS
18.1. INTRODUCTION
18.2. CONSTRUCTION OF AN IMPROVED SHUTTLE VECTOR
18.3. CLONING DNA IN Bacillus subtilis
18.4. CLONING IN Streptomycetes
18.5. LET US SUM UP
18.8. REFERENFES
The unit deals with cloning strategies in Gram positive bacteria.
18.1. INTRODUCTION
In gram positive bacteria, the base composition of different genomes ranges from <
30% GC to > 70% GC. The preferred codons and the regulatory signals used by the
organisms at one end of the %GC spectrum will not be recognized by the organism at the
other end. There are no universal cloning vehicles for use with all gram positive bacteria.
Rather, one set of system has been developed for high GC organisms (E.g., streptomycetes)
and another for low GC organisms (Eg., Bacillus).
18.2. CONSTRUCTION OF AN IMPROVED SHUTTLE VECTOR
The development of an electroporation protocol that appeared to facilitate
transformation of several streptococcal species as well as species of other gram-positive
bacterial genera was followed by the construction of a plasmid vector that should be useful in
the cloning of virtually any Streptococcal or Enterococcal genetic determinant in E. coli and
in the subsequent transfer of the cloned DNA back to an appropriate gram positive host
strain. The construction of the shuttle vector was initiated with a chimeric plasmid, pDL269.
This plasmid consists of pUC19 with a streptococcal spectinomycin resistance gene cloned
into the multiple cloning sequence. This resistance determinant is expressed constitutively in
E. coli as well as in streptococci. Plasmid pDL269 was digested with AvaIl, self-ligated, and
used to transform E. coli with selection for resistance to spectinomycin. A spectinomycinresistant, ampicillin-sensitive clone contained a plasmid, pDL270, with a 220-bp deletion in
the structural gene for P-lactamase. A 1.5-kb ClaI DNA fragment containing a kanamycin
resistance gene of streptococcal origin was treated with the Klenow fragment of DNA
polymerase I to create blunt ends and then inserted into Avall-digested, Klenow-treated,
pDL270 with T4 DNA ligase. This ligation mixture was used to transform E. coli . Selection
for resistance to spectinomycin and kanamycin produced a clone containing plasmid pDL271.
This plasmid was digested with PvuII and NdeI, treated with Klenow fragnment, and ligated
to a gel-purified 3.5-kb fragment from pJDC9 containing the pUC19 multiple cloning
sequence region flanked by strong E. coli transcription terminators as described by Chen and
Morrison.
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The JDC9-derived fragment was obtained by digestion of this plasmid with BalI and
DraI. Transformation of E. coli, with selection for resistance to kanamycin and
ounterselection for spectinomycin sensitivity, yielded a clone containing plasmid pDL272. A
potential E. coli-Streptococcus and Enterococcus shuttle vector was constructed by the
insertion of a streptococcal replication origin into pDL272. The latter was digested with ScaI
and NdeI and ligated to similarly digested pDL273, which contained the replication region of
the streptococcal plasmid pVA380-1 and the spectinomycin resistance determinant described
above. The ligation mixture was used to transform a competent culture of S. sanguis Challis.
A spectinomycin- and kanamycin-resistant clone was obtained that contained the chimeric
plasmid pDL274. The ability ofthis plasmid to replicate in E. coli was then demonstrated by
transformation of strain DH5a. The size of pDL274 was reduced further by digestion with
ClaI and Ball, treatment with Klenow fragment, blunt end ligation, and transformation of E.
coli with selection for resistance to kanamycin. A 6.9-kb plasmid, designated pDL276, was
obtained from one of the transformant clones derived from this experiment. This plasmid
contained the origin region common to the pUC plasmids, the streptococcal kanamycin
resistance gene expressed constitutively in streptococcal and E. coli host strains, the essential
replication functions of pVA380-1, and the multiple cloning sequence region of pUC19
flanked by strong transcriptional terminators. This plasmid was used to transform S. sanguis
Challis by natural transformation and other streptococcal species by electroporation. It was
also used as a vector for the cloning of a number of streptococcal enterococcal genetic
determinants.
18.3. CLONING DNA IN Bacillus subtilis
Bacillus subtilis is a Gram-positive, sporulating, aerobic bacterium phylogenetically
very distant from E. coli. A B. subtilis cloning system parallel to that developed for E. coli
permits studies of the interaction of the same genes with two, possibly very different, cellular
environments, which would offer greater insight into the diverse processes that convert
genetic information to the corresponding phenotype. The development of the B. subtilis
cloning system has been mainly hindered by the absence of suitable vector replicons. Several
plasmids can be introduced and maintained in the highly transformable B. subtilis strain 168;
however, they lacked genetic markers for selection and DNA cloning. More recently,
plasmids originally isolated from Staphylococcus aureus that code for resistance to
tetracycline or chloramphenicol have been transfected into B. subtilis. These can be tested for
their ability to accept inserted DNA segments, while still maintaining the capacity to replicate
and express their genetic information in this host.
18.3.1. DNA CLONING VECTOR FOR B. subtilis
The development of B.subtilis vectors began with the observation that plasmid from S.
aureus can be transformed into B. subtilis, where they replicate and express antibiotic
resistance normally. None of the natural S. aureus plasmids carries more than one selectable
marker and so improved vectors have been constructed bygene manipulation
.e.g.,Phv11.because of difficulties experienced in direct cloning in B.subtilis ,hybrid
plasmids were constructed which can replicate in both E.coli and B. subtilis.originally most
of these where constructed as fusions between pBR322 and pC194.
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18.3.2. VECTORS DERIVED FROM pAM1
Early in the development of B. subtilis cloning vectors, it was noted that only short
DNA fragments could be efficiently cloned and the longer DNA fragments often undergo
rearrangement. This structural instability is independent of the host recombination systems. if
structural instability is a consequence of rolling circle replication, and then vectors which
replicate by the alternative theta mechanism could be more stable. example pAM1 and
pTB19.series of cloning vectors have been constructed from pAM1.all the vectors carry
genes essential for replication repE , and its regulator copF. The later gene can be
inactivated by inserting a linker into its kpn1 site.
18.3.3. TRANSCRIPTION AND TRANSLATION
The composition of the core RNA polymerase in B. subtilis and other low GChost
resembles that of E.coli. In B. subtilis atleast many promoters contain an essential TGTG
motif upsteam of -10 regions. Mutations of these regions significantly reduce promoter
strength. The promoters also have conserved poly A and poly T upstream of -35 region. The
translation apparatus of B. subtilis differs significantly from that of E.coli. The gram positive
bacteria such as Staphylococcus, Streptococcus, Clostridium and Lactobacillus also lack an
S1 equivalent protein and they two exhibit mRNA selectivity.
18.3.4. IN VITRO CONSTRUCTION OF B. subtilis PLASMID RESISTANT TO
TETRACYCLINE AND CHLORAMPHENICOL
pC194 has a single HindHI site and pT127, a B. subtilis plasmid that encodes
resistance to tetracycline, carries three such sites. This prompted the attempt to insert the
HindIl-generatedsegments of pT127 into the HindIll site of pC194. As expected, the HindIll
cleavage destroyed the transforming activity of the plasmid DNAs. This could be restored by
T4 ligase treatment of cleaved pC194, but not of pT127. However, if the two DNAs were
ligated together, some TcR clones could be recovered. These were resistant to
chloramphenicol as well, and contained plasmid DNA (shown by ethidium bromide/CsCl
density gradient centrifugation). A hybrid plasmid, designated pHV11, was characterized by
electrophoresis and transformation.
The size of pHV1I equals the sum of that of pC194 and of the largest of the HindIII
segments of pT127. Hindtreatment of pHV11 DNA gives rise to two DNA segments, which
match the HindIII-cleaved pC194 and the slowest of the pT127 segments in electrophoretic
mobility. pHV1 plasmid DNA transforms B. subtilis, conferring resistance to tetracycline and
chloramphenicol the 1.5 X 106-dalton DNA segment of pT127, carrying the TcR marker, has
been inserted into the HindIII site of pC194.
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18.3.5. CONSTRUCTION OF E. coli- B. subtilis Hybrid Replicons
The results presented in the preceding section indicate that pC194 can be used as a
HindIII cloning vector in B. subtilis. The insertion of DNA segments other than those of the
plasmid pT127 into pC194 should therefore be feasible. This was tested by constructing
hybrid replicons between pC194 and several E. coli plasmids, each containing a single
HindIII cleavage site.For this purpose, pBR313, pBR322 [both carrying ApR TcR or pWL7
(coding for ApR KmR, W. Goebel, personal communication) were cleaved with HindIII and
ligated to HindIII-treated pC194, and the hybrid DNAs were used totransform E. coli. This
host was preferred to B. subtilis because it was possible to screen for the recombinant
genomes by a simple genetic test. DNA insertion at the HindIII site inactivates the TcR
marker of the pBR plasmids or the KmR marker of pWL7. Many colonies that displayed the
expected marker inactivation and also acquired resistance (to 100lg/ml) of chloramphenicol
were isolated.
Plasmid DNA was extracted from representative clones and analyzed by
electrophoretic and genetic tests.. In all cases the hybrid plasmids isolated from E. coli were
of a size equivalent to the sum of the parents (i.e., the E. coli plasmid and pC194), and were
cleaved by HindIII endonuclease into segments having mobilities that matched those of the
cleaved parental DNAs. These results show that hybrids between E. coli plasmids and pC194
were successfully constructed. Their structures are represented schematically in Fig. 2. In all
cases the hybrid DNAs could transform E. coli not only to ampicillin resistance, but also to
chloramphenicol resistance. This confirms the observed phenotype of the colonies selected
originally (see above) and represents another example of functional, heterospecific gene
transfer among prokaryotes. The plasmids pHV12, pHV14, and pHV15 transformed E. coli
for both markers with essentially the same efficiency. pHV16, however, transformed %Aooo
less efficiently for hloramphenicol resistance. This was not due to the absence of the gene
encodingthe resistance in a fraction of the plasmid population, since the 100 ApR clones
tested by replica-plating were CmR as well. It may possibly be due to inefficient expression
of the chloramphenicol resistance gene in pHV16 under the transformation conditions used.
In all cases transformation of B. subtilis with the hybrid DNAs gave rise to chloramphenicolresistant transformants (Fig. 3). The number of these was proportional to the amount of
pHV12 or pHV14 DNA, up to about 10 ng. The efficiencies were close to 106 transformants
per ,ug of DNA, 30-100 times higher than the value observed with pC194 or pHVll DNAs.
pHV16 DNA transformed much less efficiently, and no proportionality between the number
of transformants and the amount of DNA was observed. In all cases the B. subtilis CmR
transformants have acquired plasmids matching the plasmids isolated from E. coli (not
shown) in size and HindIII restriction pattern. This confirms that pC194 is able to serve as a
vector for cloning HindIIIgenerated DNA segments in B. subtilis. The hybrid plasmid pHV14
contains unique sites for EcoRI, Bam, and Pst, pHV12 for EcoRI and Bam. All of these are
located within the part of the hybrid genomes derived from E. coli plasmids. Insertion of
DNA segments at any of these sites does not interfere with the replication of the parental
plasmids in E. coli . It is therefore very likely that pHV14 and pHV12 can be used as cloning
vectors for DNA segments produced with these restriction enzymes in both E. coli and B.
subtilis. This was confirmed for an EcoRI-generated segment introduced in pHV12.
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18.3.6. LACK OF EXPRESSION OF E. coli Genes In B. subtilis

The hybrid plasmids described in the preceding section carry the gene specifying
resistance to ampicillin. It is present either within the intact transposon TnA (pHV16) or as a
deleted derivative of TnA (pHV12 and pHV14). This gene failed to confer resistance to
ampicillin on B. subtilis. Both the parental strain and the CmR transformants that contained
hybrid plasmids were inhibited by 0.5 jig of ampicillin per ml. In the same hybrid plasmid the
failure to change the drug-sensitive phenotype was not due to a structural modification (e.g.,
deletion) within the Ap gene. The plasmid DNAs extracted from the B. subtilis CmR host
transformed E. coli to both chloramphenicol and ampicillin resistance with equal efficiency
while they transformed B. subtilis to the chloramphenicol resistance only. The lack of
expression of the ApR gene is not related to its orientation relative to the pC194 part of the
hybrid plasmid since pHV15, a plasmid in which pBR322 is inserted in the opposite
orientation to that in pHV14, did not confer ampicillin resistance to B. subtilis. These results
prompted further investigation of the expressionof E. coli genes in B. subtilis.
For that purpose the DNA segment that carries the gene specifying resistance to
kanamycin was excised from pSC105 by the action of EcoRI and inserted into the EcoRI site
of pHV12 by standard techniques. The resulting plasmid, pHV18, conferred resistance to
ampicillin, chloramphenicol, and kanamycin on the E. coli host. It rendered B. subtilis
resistant only to chloramphenicol, transforming competent cells at an efficiency essentially
identical to that of the parental pHV12 DNA (about 106 transformants per ,ug of DNA). Both
the CmR transformants and the parental strain were inhibited by concentrations between 0.5
and 1 ,ug of kanamycin per ml.Plasmid DNA isolated from the CmR B. subtilis transformants
matched the parental E. coli plasmid pHV18 in size and HindIII and EcoRl patterns. This
DNA could transform E. coil to ApR, CmR, and KmR with essentially equal efficiency,
demonstrating the presence of the functional KmR gene on the plasmid. These experiments
show that pHV12 can be used as an EcoRl cloning vector in the B. subtilis system and that
the E. coli KmR gene is not expressed in the B. subtilis host.
126
18.4. CLONING IN Streptomycetes
Cloning in streptomycetes has attracted a lot of interest because of the large number
of antibiotics that are made by members of this genus. Streptomycetes DNa has a G+C
content of 70 75% and this affects the frequency of restriction sites. AT rich recognition
sites are rare and this can be useful if large sized fragments of DNA are wanted. In
Steptomycetes promoters may be several hundred basepairs upstream of the start of the gene
and so can be lost during gene cloning. Also many streptomycetes promoters are complex
and may include tandom sites for recognition by different sigma factors. There are several
ways in which DNA can be introduced into Streptomycetes including transformation,
transfection and conjugation. Transformation is achieved by using protoplast rather than
competent cells and high frequencies of plasmid DNA uptake can be achieved in the presence
of polyethylene glycol. Electroporation has been used to transform streptomycetes since it by
passes the need to develop the protoplast regeneration procedures. Intergeneric conjugation of
mobilizable plasmid from E.coli into Streptomycetes is increasingly used because the
required constructs made easily in E.coli and the conjugation protocols are simple.one
particularly useful phenomenon is that clones horbouring conjugative plasmids can be
detected by the visualization of pocks. The property of pock formation is known as lethal
zygosis.
18.4.1. VECTORS FOR Streptomycetes
With the exception of RS F1010 no plasmid from any other organism found to
replicate in Streptomycetes. For this reason all the cloning vectors used in Streptomycetes are
derived from plamids and phages that occur naturally in them. Plasmid SCP2 is the derivative
of the sex plasmid SCP2. Both plasmids have a size of 31.4kb.the temperate phage C31 is
the Streptomycete equivalent of phage and has been designated as vector. In Phage vectors
plaques can be obtained in overnight but plasmid tranformants can take upto one week to
sporilate. Cloning in Streptomycetes is to analyse the genetics and regulation of antibiotic
synthesis.
127
18.5. LET US SUM UP
There is no universal cloning vector for use with all gram positive bacteria due to the
disparity in GC content.
A major contributing factor to structural instability of recombinant DNA in B. subtilis
appears to be the mode of replication of the plasmid vector.
Different vectors have been derived from SCP2 plasmids including cosmids,
expression vectors, and vectors with promoter less reporter genes, positive selection
vectors, temperature sensitive vectors etc.
Cloning in Streptomycetes plays an important role in the analysis of genetics and
regulation of antibiotic synthesis.

1. Justify the need for antibiotic resistance in vectors.
2. Analyse the lack of expression of E. coli genes in B. subtilis.
1.
2.
3.
4.
Explain hybrid plasmids

Explain the modes of DNA introduction into Streptomycetes
Explain the construction of E.coli B.subtilis hybrid relicons
Explain DNA cloning vector for B.subtilis
18.8. REFERENCES
1. Genes to Clones L.W. Winnecker.
128
LESSON 19: CLONING IN GRAM NEGATIVE BACTERIA
CONTENTS
19.1. INTRODUCTION
19.2. CLONING VECTORS BASED ON E.coli PLASMIDS
19.3. CLONING VECTORS BASED ON M13 BACTERIOPHAGE
19.4. HYBRID PLASMID - M13 VECTORS
19.5. CLONING VECTORS BASED ON BACTERIOPHAGE
19.6. COSMID
19.7. LET US SUM UP
19.10. REFERENCES
The unit discusses about the cloning strategies in Gram negative bacteria.
19.1. INTRODUCTION
Cloning serves two main purposes. Firstly, it allows the large number of recombinant
DNA molecules to be produced from a limited number of starting material. at the outset only
a few nanograms of recombinant DNA may be available, but each bacterium that takes up a
plasmid will divide numerous times to produce a colony, each cell of which contains
multiple copies of the molecule. The second important function of cloning can be described
as purification. The manipulations that results in a recombinant DNA molecule can only
rarely be controlled to the extent that no other DNA molecule are present at the end of the
procedure. The ligation mixture may contain in addition to the desire recombinant molecule,
any number of the following:
1. unligated vector molecules,
2. unligated DNA fragments,
3. vector molecules that have recircularised without new DNA been inserted,
4. Recombinant DNA molecules that carry the wrong inserted DNA fragment.
19.2. CLONING VECTORS BASED ON E.coli PLASMIDS
The simplest cloning vectors, and the ones in most widespread use in gene cloning,
are those based on small bacterial plasmids. A large number of different of different plasmid
vectors are available for use with E.coli. One of the first vectors to be developed and still one
of the most popular is pBR322.
The nomenclature of plasmid cloning vectors:
The name pBR322 conforms to the standard rules for vector nomenclature:
p indicates that this is a plasmid
BR indicates the laboratory in which the vector was originally constructed(BR
stands for Bolivar and rodiguez, the two researchers who developed pBR322)
322 distinguishes this plasmid from the others developed in the same laboratory.
129
19.2.1. THE USEFUL PROPERTIES OF pBR322

The first useful feature of pBR322 is its size (4363bp).the second feature is it carries
two sets of antibiotic resistance genes. Either ampicillin or tetracycline resistance can be used
as selectable markers for cells containing the plasmid, and each marker gene includes unique
restriction sites that can be used in cloning experiments. The third advantage of pBR322 is
that it has the reasonably high copy number. Generally there are about 15 molecules present
in a transformed E.coli cell, but this number can be increased, upto 1000 3000 by plasmid
amplification in the presence of a protein synthesis inhibitor such as chloramphenicol.
130
19.2.2. OTHER TYPICAL E.coli PLASMID VECTORS

a) PBR327-a high copy number plasmid. It was constructed by removing a 1089bp segment
from pBR322.this left the ampR and tetR genes intact, but changed the replicative and
conjugative abilities of the resulting plasmid. As a result pBR327 differs from pBR322 in two
important ways.
1. pBR327 has a high copy number than pBR322, being present at about 30 to 40
molecules per E.coli cell.
131
2. the deletion also destroys the conjugative ability of pBR322, making pBR327 a
non-conjugative plasmid that cannot direct its own transfer to other E.coli cells.
b)pUC8 lac selection plasmid. It is derived from pBR322, although only the replication
origin and the ampR remain. the nucleotide sequence of the ampR has been changed so that it
no longer contains the unique restriction sites; all these cloning sites are now clustered into a
short segment of the lacZ gene carried by pUC8. pUC8 has three important advantages
1. the manipulations involved in construction of pUC8 was accompanied by chance
mutation, within the origin of replication, that results in the plasmid having the copy number
of 500 700 even before amplification.
2. the identification of recombinant cells can be achieved by a single step process, by
plating onto agar medium containing ampicillin + X-gal.
3. pUC8 lies with the clustering of the restriction sites, which allows the DNA
fragment with two different sticky ends to be cloned without resorting to additional
manipulations such as linker attachment.
c)pGEM3Z invitro transcription of cloned DNA. pGEM3Z is very similar to pUC vector: it
carries ampR and lacZ genes, the later containing a cluster of restriction sites and it is almost
exactly the same size. The distinction is that pGEM3Z has two additional short pieces of
DNA each of which acts as a recognition sites for attachment of an RNA polymerase enzyme.
These two promoter sequences lie on either side of the cluster of restriction site used for
introduction of new DNA into the pGEM3Z molecule.
132
19.3. CLONING VECTORS BASED ON M13 BACTERIOPHAGE

The most essential requirement for cloning vector is that it has the means of
replicating in the host cell. For plasmid vectors this requirement is easy to satisfy, as
relatively short DNA sequences are able to act as plasmid origin of replication. M13
bacteriophage replication is more complex. Phage DNA molecules generally carry several
genes that are essential for replication, including genes coding for components of the phage
protein coat and phage specific DNA replicative enzymes. The alteration or deletion of any of
these genes will impair or destroy the replicative ability of the resulting molecule. So it is
difficult to modify phage DNA molecules. The normal M13 genome is 6.4kb in length with
ten closely packed genes each essential for the replication of the phage. There is only a single
507 nucleotide intergenic sequence into which new DNA could be inserted without disturbing
one of these genes.
133
19.3.1. DEVELOPMENT OF THE CLONING VECTOR M13mp2
The first step in construction of an M13 cloning vector was to introduce the lacZ
gene into o the intergenic sequence. This give rise to M13mp1 which forms blue plaques on
X-gal agar. M13mp1 does not posses any unique restriction sites in the lacZ gene. It contains
hexanucleotide GGATTC , near the start of the gene. A single nucleotide change would make
GAATTC which is an EcoR1 site. This alteration was carried out using invitro mutagenesis
results in M13mp2.
19.3.2. M13mp7-SYMMENTRICAL CLONING SITES

The next step in the development of M13 vectors was to introduce additional
restriction sites into the lacZ gene. This was achieved by synthesizing in the test tube a short
oligonucleotide, called polylinker that consist of a series of restriction sites and has EcoR1
sticky ends. This polylinker was inserted into EcoR1 site of M13mp2 to give M13mp7 with
more cloning sites.
134
19.3.4. MORE COMPLEX M13 VECTORS

The latest M13 vectors have more complex polylinker inserted into the lacZ gene. An
example is M13mp8. its advantage is its ability to take DNA fragments with two different
sticky ends. The vector M13mp9 has the same polylinker but in the reverse orientation. A
DNA fragment cloned into M13mp8, excised by double restriction and inserted into M13mp9
will now be in the reverse orientation. This is important in DNA sequencing.
135
136
19.4. HYBRID PLASMID - M13 VECTORS
There is a limit to the size of the DNA fragment that can be cloned with an M13
vector, with 1500bp generally being looked on as the maximum capacity, though fragments
upto 3kb have occasionally been cloned. To get around this problem a number of novel
vectors (phagemids) have been developed by combining a part of the M13 genome with
plasmid DNA. E.g., pEMBL8 which was made by transferring into pUC8 a 1300bp fragment
of the M13 genome. this piece of M13 DNA contains the signal sequence recognized by the
enzymes that convert the normal double stranded M13 molecule into single stranded DNA
before secretion of new phage particles.
19.5. CLONING VECTORS BASED ON BACTERIOPHAGE
Two problems had to be solved before based cloning vectors could be developed.
1. The DNA molecule can be increased in size by only about 5%, representing the
addition of only 3kb of new DNA. If t he total size of the molecule is more than 52kb
then it will not packed into the head structure and infective phage particles will not
be formed. This severely limits the size of the DNA fragment that can be inserted into
an unmodified vector.
2. The genome is so large that it has more than one recognition sequence for virtually
every restriction endonuclease. Restriction cannot be used to cleave the normal
molecule in a way that will allow insertion of new DNA, as the molecule would be
cut into several small fragments that would be very unlikely to reform a viable
genome on relegation.
Cloning vectors primary use being to clone large pieces of DNA from 5kb to 25kb, much
too big to be handled by plasmid or M13 vectors.
19.5.1. INVITRO MUTAGENESIS AND NATURAL SELECTION
Even the deleted genome with the non essential region removed has multiple
recognition sites for most restriction enzymes. This is the problem that is often encountered
when a new vector is being developed. If just one or two sites need to be removed then the
technique of invitro mutagenesis can be used. For example an EcoR1 site GAATTC could be
change to GGATTC which is not recognized by the enzyme.
Instead natural selection was used to provide strains of that lack the unwanted
restriction site. Natural selection can be done using E.coli as host strain that produces EcoR1.
Most DNA that invades the cell will be destroyed by the restriction endonuclease but few
will survive and produce plaques which are mutant phages .
19.5.2. INSERTION AND REPLACEMENT VECTORS
With an insertion vector a large segment of the non essential region has been deleted
and the two arms ligated together. An insertion vector possess atleast one unique restriction
site into which new DNA can be inserted.
Examples,
1. gt10 which can carry upto 8kb of new DNA inserted into a unique EcoR1 site
located in the cI gene
137
2. ZAPII which can carry upto 10kb new DNA into any of six restriction sites
within a polylinker,inactivates lacZ carried by the vector.
Replacement vector has two recognition sites for the restriction enzyme used for
cloning. These sites flank a segment of DNA that is replaced by the DNA to be cloned.
Examples,
1. WES. B in which two EcoR1 sites flanks the replacement fragment and
recombinant selection is solely based on the size.
2. EMBL4 which can carry upto 20kb of inserted DNA by replacing a segment
flanked by pairs of EcoR1, BamH1 and Sal1 sites.
138
19.5.3. CLONING EXPERIMENTS WITH INSERTION OR REPLACEMENT

VECTORS
The cloning experiment with a vector can proceed along the same lines as with a
plasmid vector the molecules are restricted, new DNA is added, the mixture is ligated and
the resulting molecules used to transfect a competent E.coli host.
139
19.6. COSMID
Most sophisticated type of based vector is the cosmid. They are hybrids between a
phage DNA molecule and a bacterial plasmid. It is basically a plasmid that carries a cos site.
It also needs a selectable marker such as the ampicillin resistance gene, and a plasmid origin
of replication, as cosmids lacks all the genes and so do not produce plaques. Instead
colonies are formed on selective media just as with a plasmid vector.
140
19.7. LET US SUM UP
E.coli plasmids play an important role as vectors for cloning in gram negative
bacteria.
The first vector to be developed is pBR322 from which pBR327, pUC8, pGEM3Z
was constructed. These vectors show some differences from pBR322.
The identification of recombinants varies according to the plasmid vectors used.
identification of recombinants by insertional inactivation of the -galactosidase gene
was described in pUC8 where as in pBR322 the selection is by either ampicillin or
tetracycline gene inactivation.
The great attraction of M13 vector is, it offers single-stranded versions of cloned
DNA. Other M13 vectors pairs available are M13mp10/11, M13mp18/19.
Phagemids pEMBL8 require normal M13 as helper phage for replication and phage
coat proteins.
141
The main use of all based vectors is to carry DNA fragments that are too large to be
handled by plasmid or M13 vectors.
Replacement vectors such as EMBL4 can carry upto 20kbof new DNA and some
cosmids can manage fragments upto 40kb.

1. Why replacement vectors are necessary?
2. What is meant by orientation of the polylinker?
1.
2.
3.
4.
5.
Describe useful properties of pBR322.

Give examples for insertion and replacement vectors.
Write about cosmids.
Account on M13 vectors.
Write about the cloning strategy in E.coli.
19.10 REFERENCES
1. Genes to Clones - by L. Winnecker.
142
LESSON 20: CLONING IN FUNGI and Saccharomyces cerevisiae

CONTENTS
20.1. INTRODUCTION
20.2. INTRODUCING DNA INTO FUNGI
20.3. PLAMID VECTORS FOR FUNGI
20.4. YEAST VECTORS
20.5. MOLECULAR ANALYSES WITH YEAST SYSTEMS
20.6. EXPRESSION OF HETEROLOGOUS PROTEINS IN YEAST
20.7. YEAST COSMID VECTORS
20.8. LET US SUM UP
20.11. REFERENCES
In detail the chapter discuss about the cloning in fungi and other Saccharomyces
species.
20.1. INTRODUCTION
Some of the functions present in eukaryotes are not present in prokaryotes like
localization of ATP generating systems to mitochondria, association of DNA with histones,
mitosis and meiosis, and obligate differentiation of cells. Yeast cells are much easier to grow
and manipulate than the plant and animal cells
20.2. INTRODUCING DNA INTO FUNGI
Like E.coli, fungi are not naturally transformable and artificial means have to be used
for introduing foreign DNA. one method involves the use of spheroplasts and was first
developed for S. cerevisiae. In this method, cell wall is removed enzymatically and the
resulting spheroplasts are fused with ethylene glycol in the presence of DNA and calcium
chloride.electroporation provides a simpler and more convenient alternative spheroplast. cells
transformed by electroporation can be selected on the surface of solid media, thus facilitating
subsequent manipulation.
20.3. PLAMID VECTORS FOR FUNGI
If the heterologous DNA introduced into fungi is to be maintained in a extra
chromosomal state then plasmid vectors are required which are capable of replicating in the
fungal host. Four types of plasmid vectors have been developed.
1.
2.
3.
4.
yeast episomal plasmids(YEps)

yeast replicating plasmids(YRps)
yeast centromere plasmids(YCps)
yeast artificial chromosomes(YACs)
143
20.4. YEAST VECTORS
A wide range of vectors are available to meet various requirements for insertion,
deletion alteration and expression of genes in yeast. Most plasmids used for yeast studies are
shuttle vectors, which contain sequences permitting them to be selected and propagated in E.
coli, thus allowing for convenient amplification and subsequent alteration in vitro. The most
common yeast vectors originated from pBR322 and contain an origin of replication (ori),
promoting high copy-number maintenance in E. coli, and the selectable antibiotic markers,
the -lactamase gene, bla (or AmpR), and sometime to tetracycline-resistance gene, tet or
(TetR), conferring resistance to, respectively, ampicillin and tetracycline.
In addition, all yeast vectors contain markers that allow selection of transformants
containing the desired plasmid. The most commonly used yeast markers include URA3, HIS3,
LEU2, TRP1 and LYS2, which complement specific auxotrophic mutations in yeast, such as
ura3-52, his3-1, leu2-1, trp1-1 and lys2-201. These complementable yeast mutations
have been chosen because of their low-reversion rate. Also, the URA3, HIS3, LEU2 and
TRP1 yeast markers can complement specific E. coli auxotrophic mutations.
The URA3 and LYS2 yeast genes have an additional advantage because both positive
and negative selections are possible.
Components of common yeast plasmid vectors
YIp YEp YRp YCp
Plasmid
E. coli genes or segments
ori, bla; tet
+
Yeast genes or segments
URA3; HIS3; LEU2; TRP1;
+
LYS2; etc.
leu2-d
0
0
2 m; 2 m-ori REP3;
ARS1; ARS2; ARS3; etc.
0
CEN3; CEN4; CEN11; etc. 0
Host (yeast) markers
ura3-52; his3-1; leu2-1;
+
trp1-1; lys2-201; etc.
Stability
+
+
0
0
+
0
+
0
0
0
+
+
++ +
20.4.1. YIp VECTORS

The YpI integrative vectors do not replicate autonomously, but integrate into the genome at
low frequencies by homologous recombination. Integration of circular plasmid DNA by
144
homologous recombination leads to a copy of the vector sequence flanked by two direct
copies of the yeast sequence. The site of integration can be targeted by cutting the yeast
segment in the YIp plasmid with a restriction endonuclease and transforming the yeast strain
with the linearized plasmid. The linear ends are recombinogenic and direct integration to the
site in the genome that is homologous to these ends. In addition, linearization increases the
efficiency of integrative transformation from 10- to 50-fold.
The YIp vectors typically integrate as a single copy. However multiple integration do
occur at low frequencies, a property that can be used to construct stable strains
overexpressing specific genes. YIp plasmids with two yeast segments, such as YFG1 and
URA3 marker, have the potential to integrate at either of the genomic loci, whereas vectors
containing repetitive DNA sequences, such as Ty elements or rDNA, can integrate at any of
the multiple sites within genome. Strains constructed with YIp plasmids should be examined
by PCR analysis, or other methods, to confirm the site of integration.
20.4.2. YRp VECTORS

The yeast replicative plasmids able to multiply as independent plasmids because they
carry a chromosomal DNA sequence that includes an oringin of replication. Replication
origins are known to be located very close to several yeast genes, including one or two which
can be used as selectable markes. YRp7 is an example of a replicative plasmid.it is made up
of pBR322 plus the yeast gene TRP1.this gene, which is involved in tryptophan biosynthesis,
is located adjacent to a chromosomal origin of replication.the yeast DNA fragment present in
YRp7 contains both TRP1 and the origin.
145
20.4.3. YEp VECTORS

The YEp yeast episomal plasmid vectors replicate autonomously because of the
presence of a segment of the yeast 2 m plasmid that serves as an origin of replication (2 m
ori). The 2 m ori is responsible for the high copy-number and high frequency of
transformation of YEp vectors.
YEp vectors contain either a full copy of the 2 m plasmid, or, as with most of these
kinds of vectors, a region which encompasses the ori and the REP3 gene. The REP3 gene is
required in cis to the ori for mediating the action of the trans-acting REP1 and REP2 genes
which encode products that promote partitioning of the plasmid between cells at division.
Therefore, the YEp plasmids containing the region encompassing only ori and REP3 must be
propagated in cir+ hosts containing the native 2 m plasmid
Most YEp plasmids are relatively unstable, being lost in approximately 10-2 or more
cells after each generation. Even under conditions of selective growth, only 60% to 95% of
the cells retain the YEp plasmid.
The copy number of most YEp plasmids ranges from 10-40 per cell of cir+ hosts.
However, the plasmids are not equally distributed among the cells, and there is a high
variance in the copy number per cell in populations.
Several systems have been developed for producing very high copy-numbers of YEp
plasmids per cell, including the use of the partially defective mutation leu2-d, whose
expression is several orders of magnitude less than the wild-type LEU2 + allele. The copy
number per cell of such YEp leu2-d vectors range from 200-300, and the high copy-number
persists for many generations after growth in leucine-containing media without selective
pressure. The YEp leu2-d vectors are useful in large-scale cultures with complete media
where plasmid selection is not possible. The most common use for YEp plasmid vectors is to
overproduce gene products in yeast.
146
20.4.4. YCp VECTORS

The YCp yeast centromere plasmid vectors are autonomously replicating vectors
containing centromere sequences, CEN, and autonomously replicating sequences, ARS. The
YCp vectors are typically present at very low copy numbers, from 1 to 3 per cell, and
possibly more, and are lost in approximately 10 -2 cells per generation without selective
pressure. In many instances, the YCp vectors segregate to two of the four ascospore from an
ascus, indicating that they mimic the behavior of chromosomes during meiosis, as well as
during mitosis. The ARS sequences are believed to correspond to the natural replication
origins of yeast chromosomes, and all of them contain a specific consensus sequence. The
CEN function is dependent on three conserved domains, designated I, II, and III; all three of
these elements are required for mitotic stabilization of YCp vectors. YRp vectors, containing
ARS but lacking functional CEN elements, transform yeast at high frequencies, but are lost at
too high a frequency, over 10% per generation, making them undesirable for general vectors.
The stability and low copy-number of YCp vectors make them the ideal choice for
cloning vectors, for construction of yeast genomic DNA libraries, and for investigating the
function of genes altered in vivo. ARS1, which is in close proximity to TRP1, is the most
commonly used ARS element for YCp vectors, although others have been used. CEN3, CEN4
and CEN11 are commonly used centromeres that can be conveniently manipulated. For
example, the vector YCp50 contains CEN4 and ARS1.
20.4.5. YEAST ARTIFICIAL CHROMOSOMES (YACs)
The initial step in the molecular characterization of eukaryotic genomes generally
requires cloning of large chromosomal fragments, which is usually carried out by digestion
with restriction endonucleases and ligation to specially developed cloning vectors. Usually
147
200 to 800 kb fragments are cloned as Yeast Artificial Chromosomes (YACs), and 100-200
kb fragments are cloned as Bacterial Artificial Chromosomes (BACs or PACs). The
importance of YAC technology has been heightened by the recently developed methods for
transferring YACs to cultured cells and to the germline of experimental animals.
YAC cloning systems are based on yeast linear plasmids, denoted YLp, containing
homologous or heterologous DNA sequences that function as telomeres (TEL) in vivo, as well
as containing yeast ARS (origins of replication) and CEN (centromeres) segments.
Manipulating YLp linear plasmids in vitro is complicated by their inability to be propagated
in E. coli. However, specially developed circular YAC vectors have been developed for
amplification in E. coli. For example, a circular YCp vector, containing a head-to-head dimer
of Tetrahymena or yeast telomeres, is resolved in vivo after yeast transformation into linear
molecules with the free ends terminated by functional telomeres. One common type of YAC
vector that can be propagated in E. coli, contains telomeric sequences in inverted orientation,
which flank a DNA cassette containing the HIS3 gene (Figure 14.2). After amplification in E.
coli and before transforming yeast the plasmid is digested with a restriction endonuclease,
usually BamHI, which excises the HIS3 cassette and generates a linear form in vitro. Yeast
are transformed by this linear structure at high frequencies, although the transformants are
unstable. Despite the presence of a CEN sequence, the YLp is present at high copy numbers
and is lost at high frequency because of its small size. Increasing the size of the YLp by
homologous integration in vivo or by ligation in vitro increases the stability of the plasmid
and reduces the copy number to
approximately one per cell.
Figure. A yeast artificial
chromosome (YAC) cloning
system. The YAC vector contains
telomeric ends that are denoted by
black arrow heads. The vector also
contains a unique SmaI cloning site
flanked by SfiI and NotI 8-base-pair
restriction sites. The vector can be
used to clone 50 to 500 kb
restriction fragments (see the text).
The developed highly-efficient YAC cloning vectors also contain TRP1 and URA3
markers and a SUP4-o gene flanked by the NotI and SfiI rare restriction sites. The SUP4-o
suppressor also harbors a naturally occurring SmaI site. The host strain contains the ade2-1
UAA mutation, causing the formation of a red pigment, unless the mutation is suppressed by
SUP4-o). The YAC vector is cleaved with BamHI and SmaI, treated with alkaline
phosphatase and the two arms are ligated to the exogenous DNA fragments desired to be
cloned. A ade2-1 ura3-52 trp1 host strain is transformed with the ligated mixture. Both arms
148
are anticipated to be present in Ura+ and Trp+ transformants and inserts should be present in
the Ura+ Trp+ Ade- (red) transformants. YACs present in these transformants are then
subjected to pulse-field electrophoresis in order to estimate the size of the inserts.
Figure Recombinational targeted cloning with YAC vectors. A yeast strain is transformed
with a mixture of the two YAC vector arms and large fragments of DNA. Recombination in
vivo results in the formation of a specific YAC clone. The two YAC vector arms are derived
from linearized plasmids that contain targeting segments that are homologous to the termini
of the DNA segment that is to be cloned.
The potential to use YAC cloning technology has been enhanced by the ability to use
homologous recombination for manipulating exogenous DNA in the yeast host. In
recombinationally-targeted YAC cloning, YACs are assembled in vivo, by recombination,
and not by ligation in vitro. Recombination takes place between a target segment of the
exogenous DNA, and the YAC vector that contain sequences homologous to these targets.
Transformation of the two YAC vectors arms and the exogenous segment, flanked by the
target segments, followed by recombination, results in the formation of the desired stable
YACs. The specific target DNA segments for the YAC vector can be obtained from the
exogenous DNA as restriction fragments or PCR products.
Also YACs can be modified after cloning by "retrofitting", using homologous
recombination with yeast plasmids having targeting sequences. For example, a neomycin
resistant gene has to be incorporated into a YAC that will be transferred to mammalian cells
using selection, as was done for transfering the entire human -globin gene in embryonic
stem cells. Also, overlapping YAC clones can be recombined, resulting in larger clones
encompassing more extensive regions. Furthermore, special YAC vectors have designed for
generating terminal and internal deletions of cloned YAC inserts.
YACs have been useful for not only cloning genes but also mammalian telomeric and
centromeric regions, and chromosomal origins of replication.
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20.5. MOLECULAR ANALYSES WITH YEAST SYSTEMS
The accessibility of the yeast genome for genetic manipulation and the available
techniques to introduce exogenous DNA into yeast cells has led to the development of
methods for analyzing and preparing DNA and proteins not only from yeast itself, but also
from other organisms. For example, many mammalian homologs of yeast genes have been
cloned by using heterologous cDNA expression libraries in yeast expression vectors. Also,
yeast is being used to investigate the detailed functions of heterologous proteins, such as
mammalian transcription factors and nuclear
hormone receptor. In fact, like E. coli, yeast has
become a standard microorganism for carrying
out special tasks, some of which are described
in this section.
20.5.1. TWO-HYBRID SYSTEMS
Powerful methods, denoted two-hybrid systems,
have been designed for screening and
investigating interacting proteins. Because of
the ease of the assay, exploratory two-hybrid
screens are usually the first method of choice
when information of interacting proteins are
desired.
Figure. The two-hybrid system. (A) Normally,
the Gal4 transcription activator binds to DNA at
the Gal4p binding sites and activates
transcription of the lacZ reporter gene. (B) A
hybrid of the Gal4 activation domain with the
Yfg2 protein does not activate transcription
because it does not localize at the Gal4 binding site. (C) A hybrid of the Gal4 DNA-binding
site domain with the Yfg1 protein does not activate transcription of the reporter gene because
of the lack of the transcriptional activation domain. (D) Protein-protein interaction between
Yfg1p and Yfg2p reconstitutes Gal4p function and activates transcription of the reporter
gene.
Some of these two-hybrid systems are based on the properties of certain eukaryotic
transcription factors, usually Gal4p, that have two separate domains, one for DNA binding
and the other for transcriptional activation. While the two domains are normally on the same
polypeptide chain, the transcription factor also functions if these two domains are brought
together by noncovalent protein-protein interactions. In practice, gene fusions are constructed
so that the DNA-binding domain is linked to one protein, Yfg1p, and the activation domain is
linked to another protein, Yfg2p, as illustrated in Figure 14.1. Interactions of Yfg1p and
Yfg2p brings the DNA-binding and activation domains close together, leading to the
expression of a reporter gene that is regulated by the transcription factor. Another two-hybrid
system is based on the use of the lexA repressor protein and the lexA operator sequences from
E. coli. These assays are almost always carried out in yeast, although mammalian cells have
been used.
150
Yeast plasmid vectors are available, in which the GAL4 DNA-binding domain and the
GAL4 activation domain are on separate plasmids with convenient restrictions sites and with
selectable yeast markers. These plasmids are used in conjunction with reporter yeast strains,
in which upstream activation sequences from the GAL1-GAL10 region are used to promote
transcription of the E. coli lacZ gene (the PGAL1-lacZ reporter gene) Complete or partial
genes are fused in frame with the GAL4 DNA-binding domain and the GAL4 transcription
activation domains. If these two hybrid proteins interact, then the lacZ reporter gene is
transcribed, leading to the blue color of the strain on medium contain the chromogenic
substrate X-gal. In addition, yeast strains having not only the PGAL1-lacZ but also the PGAL1HIS3 reporter genes are also available. It is advantageous to select directly for expression of
the PGAL1-HIS3 reported gene, followed by a screen for PGAL1-lacZ expression.
Another version of the two-hybrid system uses the lexA operator sequence and the
DNA-binding domain from the E. coli lexA repressor protein. In this system, the activator
domain is a segment of E. coli DNA that expresses an acidic peptide, which acts as a
transcriptional activator in yeast when fused to a DNA-binding domain. As with the GAL4
system, lexA transcriptional activator also contains a nuclear localization signal that directs
the protein into the nucleus. Yeast strains having lexA operators upstream of both the E. coli
lacZ and yeast LEU2 gene have served as reporter genes.
In addition, epitope tags have been built into the constructs of both the GAL4 and lexA
systems, allowing for the detection of expressed hybrid proteins. Although false-positive and
false-negative results can be obtained, a substantial number of protein combinations have
proved to be successfully uncovered with the two-hybrid system and its use has become
widely accepted.
Because of its sensitivity, relatively low-affinity interactions can be detected. Also,
the cloned genes encoding proteins that interact with the target protein becomes immediately
available when the two-hybrid system is used in a screen with libraries of fused genes.
The two hybrid-system has been mainly used for the following three applications:
testing proteins that are believed to interact on the basis of other criteria; defining domains or
amino amino acids critical for interactions of proteins that are already known to interact; and
screening libraries for proteins that interact with a specfic protein. The two hybrid-system has
been successfully used to identify diversed sets of interacting proteins in yeast and
mammalian cells, and it has been particularly successful in studies of oncogenes, tumor
suppressors, protein kinases, and cell-cycle control. Some examples of interacting proteins
uncovered with the two hybrid-system in mammalian cells include Jun and Fos; Ras and the
protein kinase Raf; the retinoblastoma protein or p53 and the SV40 large T antigen; and other
oncoproteins.
20.5.2. THE YEAST ONE-HYBRID (MATCHMAKER) SYSTEM
The yeast one-hybrid system provides the basic tool for conducting a one-hybrid
assay - an in vitro genetic assay used for isolating novel genes encoding proteins that bind to
a target, cis-regulatory element or any other short, DNA binding sequence. The one-hybrid
assay offers maximal sensitivity because detection of the DNA-protein interactions occur
while proteins are in their native configurations in vivo. In addition, the gene encoding the
DNA binding protein of interest is immediately available after a library screening To conduct
a one-hybrid assay, a sequence consisting of tandem copies of a known DNA element is
151
inserted upstream of the HIS3 and lacZ reporter gene promoters (present on separate
vectors).Subsequently, the reporters are integrated site-specifically into the yeast genome to
create the new yeast reporter strains. After construction of the reporter strains, the cDNA
candidates encoding the protein of interest (sometimes from a complete yeast genomic
plasmid library) are expressed as fusion proteins with a target-independent GAL4 activator
domain. A GAL4-AD library can be screened for a cDNA encoding a DNA-binding protein
of interest. After transforming the modified yeast reporter strain with an AD fusion library
that contains candidate cDNA clones and plating, if an AD/library hybrid protein interacts
with the target element, the HIS3 reporter is expressed, allowing colony growth on minimal
medium lacking histidine. If a HIS3/LacZ reporter strain is used, a -galactosidase assay can
be performed to verify the DNA-protein interaction and help eliminate false positives. The gal assay can be conducted as an 'overlay' test.
20.6. EXPRESSION OF HETEROLOGOUS PROTEINS IN YEAST
Although E. coli is still the first choice for the producer of heterologous proteins, the
yeast S. cerevisiae has some attractive features. Proteins produced in yeast, unlike those
produced in E. coli, lack endotoxins. In certain special cases, such as hepatitis B core antigen,
the products produced in yeast have higher activity than those produced in E. coli. In contrast
with using E. coli, several posttranslational processing mechanisms available in yeast have
allowed the expression of several human or human pathogen-associated proteins with
appropriate authentic modifications. Such posttranslational modifications include particle
assembly, amino terminal acetylation, myristylation and proteolytic processing. In addition,
heterologous proteins secreted from specially engineering strains are correctly cleaved and
folding and are easily harvested from yeast culture media. The use of either homologous or
heterologous signal peptides has allowed authentic maturation of secreted products by the
endogenous yeast apparatus.
The importance of yeast for production of protein products by recombinant DNA
methods is illustrated by the fact that the first approved human vaccine, hepatitis B core
antigen, and the first food product, rennin, were produced in yeast.
The cloning of specific cDNAs from other organisms and the study of their function
using yeast as a surrogate does not necessarily require high-level expression of the foreign
protein. In these instances, the aim is just to produce physiological quantities of the protein in
a form that is correctly modified and localized in the cell such that the activity accurately
reflects the activity in the original organism. However, commercial and laboratory
preparations of proteins generally require high expression vectors.
There are numerous varieties of expression vectors currently available for producing
heterologous proteins in yeast, and these are derivatives of the YIp, YEp and YCp plasmids
described above in Section 9. The cDNA, synthetic DNA or genomic DNA lacking introns
are inserted in the vector. Promoters used in expression vectors includes a transcription
initiation site and variable amounts of DNA encoding the 5untranslated region. Because
most of the yeast expression vectors do not contain an ATG in the transcribed region of the
promoter, the heterologous gene must provide an ATG that establishes the correct reading
frame corresponding to the amino-terminus of the protein. It is essential that this ATG
corresponds to the first AUG of the mRNA, because translation almost always initiates at the
first AUG on mRNAs from yeast as well as from other eukaryotes. The 5untranslated region
of the vector also should be similar to the naturally-occurring leader region of abundant
152
mRNAs by lacking secondary structures and being A-rich, and G-deficient, and by having an
A at position -3 relative to the ATG translational initiator codon.
Many of the expression vectors include a known signal for 3 end formation of yeast
mRNA, although vectors lacking such defined signals synthesize transcripts until
encountering a 3-end forming signal from another gene or a fortuitous signal on the plasmid.
Numerous normal and altered yeast promoters have been used, and these are chosen
because of their high activity and some times because of their regulatory properties. Some of
the promoters have been derived from genes encoding alcohol dehydrogenase I, enolase,
glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, triose phosphate
isomerase, galacokinase, repressible acid phosphatase, mating factor, etc. These promoters
almost always produce high levels of transcription of heterologous gene, but there is a wide
variation in the amount of the corresponding proteins that is finally produced in the yeast
strain, depending on the specific heterologous gene.
20.6.1. POST-TRANSLATIONAL PROCESSING AND MODIFICATION OF
HETEROLOGOUS PROTEINS IN YEAST
Another important molecular aspect of recombinant proteins expressed in yeast are
the features of post-translational processing and modification processes specific to yeast,
particularly with attention to therapeutic agents produced in yeast. N-and O-linked
glycosylation patterns in yeast may prove to be different from those in the native host. For
example, yeast adds mannose units to threonine or serine residues, while higher eukaryotes
prefer sialic acid O-linked side chains. Such differences may affect the folding, stability,
activity and immunogenicity of proteins produced in yeast. By contrast, N-linked
glycosylation in yeast largely resembles that of higher eukaryotes. Attention has
also to be paid to possible differences in phosphorylation, acetylation, methylation,
myristylation and isoprenylation of proteins in yeast towards other organisms.
Once synthesized and modified, heterologous proteins produced in yeast may undergo
intracellular proteolytic degradation before they can be purified. In S. cerevisiae, proteolysis
may be unspecific and associated with the vacuole, or specific and coupled to the ubiquitinproteasome system.
20.6.2. GFP FUSION PROTEINS
A relatively recent development of labelling proteins involves the green fluoresent
protein (GFP) as a reporter molecule for intracellular localization and in vivo gene expression
studies. Fusion proteins with the conventional GFP moiety (some 200 amino acids in length)
can be visualized by fluoresecence microscopy at 395 nm (blue light). Interestingly, two
variants of GFP, having particular amino acid replacements, are now available which will
emit fluorescent light of lower (red) or higher (blue) wavelengths. In most cases, the globular
extension in the modified protein will not influence its intracellular localization nor its
function as compared to the native protein, independent of whether the GFP moiety has been
fused to the N-terminus or to the C-terminus. However, this has to be checked for each
protein of interest individually. Variants of the native GFP are available, the genes of which
have been modified such that they are adapted to codon usage in plants, and these have
proven to be advantageous in expression also in the yeast system.
153
20.7. YEAST COSMID VECTORS
Cosmid vectors have proven to be very convenient for cloning and sequencing of
large segments of yeast chromosomal DNA. To construct a library with as complete coverage
as possible with as few clones as possible, the cloned DNA fragments should be randomly
distributed on the DNA. Under these conditions, the number of clones (N) in a library
representing each genomic segment with a given probability (P) is N = ln (1-P)/ln (1-f) where
f is the insert length expressed as fraction of the genome size. For
example, with the size of 12,800 kb for the yeast genome and assuming an average insert
length of 35kb, a cosmid library containing 4600 random clones would represent the yeast
genome at P=99.99%,i.e. about twelve times the genome equivalent. The actual number of
cosmid clones obtained by the usual procedures is very high (>200,000/g DNA). One of the
first yeast cosmid vectors, pHC79, was developed in 1980. In
connection with the yeast genome sequencing programme, two major types of cosmids have
been employed.
(i) pYc3030 generated from pCH79 by adding the yeast 2m plasmid origin of
replication and the yeast HIS3 marker is a shuttle vector that most conveniently allows DNA
to be shuttled between E. coli and yeast cells. It contains a BamH1 cloning sites which is
suitable for accommodating yeast DNA fragments of ca. 30-45 kb in size obtained by partial
digestion of high molecular weight DNA with Sau3A. For cloning, the vector arms
comprising the -phage cos-sites have to be prepared separately and are ligated to a mixture
of partial Sau3A fragments that have been size-fractionated by centrifugation of the digestion
mixture in NaCl gradients. Replica plating which is one of the common procedures used for
the storage and screening of cosmid libraries has been successfully applied to yeast cosmid
libraries. Colonies can be easily purified, and cosmid DNA can be prepared by one of the
'mini-prep' procedures. We found that yeast cosmid can be stored at -20C for several years
without damage. Cosmids have not only been used successfully for chromosomal walking,
but also in complementation analyses; cosmids are maintained in yeast cells in only one or a
few copies.
(ii) pWE15 (and pWE16) are cosmid vectors that have been designed for genomic
walking and rapid restriction mapping [Dujon et al., 1993]. They contain bacteriophage T3
and T7 promoters, respectively, flanking a unique BamH1 cloning site. By using the cosmid
DNA containing a genomic insert as a template for either T3 or T7 polymerase, directional
'walking' probes can be synthesized and used to screen genomic cosmid libraries (or
sublibraries) These vectors contain additional genes (SV2-neo or SV2-dhfr, respectively)
which allow the expression, amplification and rescue of cosmids in mammalian cells. NotI
restriction sites have been placed near the BamH1 site which allows the insert to be removed
as a single large fragment.
20.8. LET US SUM UP
If the heterologous DNA introduced into fungi is to be maintained in a extra

chromosomal state then plasmid vectors are required which are capable of replicating
in the fungal host. Four types of plasmid vectors have been developed.
yeast episomal plasmids(YEps)
yeast replicating plasmids(YRps)
yeast centromere plasmids(YCps)
yeast artificial chromosomes(YACs)
154
The most common yeast vectors originated from pBR322 and contain an origin of
replication (ori), promoting high copy-number maintenance in E. coli, and the
selectable antibiotic markers, the -lactamase gene, bla (or Amp R), and sometime to
tetracycline-resistance gene, tet or (TetR), conferring resistance to, respectively,
ampicillin and tetracycline.
YAC cloning systems are based on yeast linear plasmids, denoted YLp, containing
homologous or heterologous DNA sequences that function as telomeres (TEL) in vivo,
as well as containing yeast ARS (origins of replication) and CEN (centromeres)
segments.
Yeast two-hybrid systems have been designed for screening and investigating
interacting proteins
Cosmid vectors have proven to be very convenient for cloning and sequencing of
large segments of yeast chromosomal DNA.

1. Differentiate between Yeps and YRps.
2. Justify; YAC is preferred for cloning large amounts.
3. Analyse whether YAC will be better tan BAC.
1.
2.
3.
4.
5.
Explain yeast replicative plasmids.

Significance of yeast two hybrid systems.
Explain GFP fusion proteins.
What are the yeast cosmid vectors and explain them.
Explain plasmid vectors for fungi.
20.11. REFERENCES
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LESSON 21: CLONING IN PLANTS
CONTENTS
21.0. AIMS AND OBLECTIVES
21.1. INTRODUCTION
21.2. TRANSFORMATION TECHNIQUES
21.3. BIOLISTICS
21.4. CALCIUM PHOSPHATE PRECIPITATION
21.5. ELECTROPORATION
21.6. GENE SILENCING
21.7. GENE SPLICING
21.8. LIPOFECTION
21.9. MICROINJECTION
21.11. LET US SUM UP
21.14. REFERENCES
21.0. AIMS AND OBLECTIVES
The chapter deals with the cloning strategies in plants.
21.1. INTRODUCTION
The Expression of foreign genenes introduced into plants was first achieved in the
early 1980s. Our ability to manipulate the plant genome has come about through intensive
research into vector systems based on the soil bacterium Agrobacterium tumifaciens and
alternative strategies involving direct DNA transfer. In addition plant viruses have been
developed as vectors for high level transient gene expression.
21.2. TRANSFORMATION TECHNIQUES
The uptake of foreign DNA or transgenes by plant cells is called transformation. A
variety of techniques have been used to introduce transgenes into plant cells; these can be
grouped into the two following broad categories:
1. Agrobacterium mediated and
2. Direct gene transfer.
21.2.1. Agrobacterium MEDIATED GENE TRANSFER
A major method of DNA transfer in plants is Agrobacterium mediated transformation.
The natural living soil bacteria, Agrobacterium tumefaciens is capable of infecting a wide
range of plant species, causing Crown Gall diseases (see image below). It has natural
transformation abilities which can be exploited in plant biotechnology. When A. tumefaciens
infects as a cell, it transfers a copy of its T-DNA, which is a small section of DNA carried on
its Ti (Tumour Inducing) plasmid. The T-DNA is flanked by two (imperfect) 25 base pair
repeats. Any DNA contained within these borders will be transferred to the host cell.
156
The T-DNA section on the Ti plasmid can be replaced by the transgene coding for
the desirable trait attached to the appropriate regulatory sequences. The recombinant bacteria
can then be used to infect both regenerating cell and protoplast cultures.
Marker genes such as those coding for antibiotic resistance are attached to the
transgene so that it is possible to select those cells that have been transformed by the
bacterium. Cell to plant regeneration is carried out on the selected cells and transgene
expression is characterised i.e. it is necessary to check that the gene is expressed at the correct
levels in the correct tissues.
Agrobacterium tumefaciens has been used to transform many dicotyledonous plant
species with relative ease. However, it is, as yet, not possible to transform monocotyledonous
species, which include commercially viable cereal crops such as maize and rice.
A major method of DNA transfer in plants is Agrobacterium mediated transformation.
The natural living soil bacteria, Agrobacterium tumefaciens is capable of infecting a wide
range of plant species, causing Crown Gall diseases (see image below). It has natural
transformation abilities which can be exploited in plant biotechnology. When A. tumefaciens
infects as a cell, it transfers a copy of its T-DNA, which is a small section of DNA carried on
its Ti (Tumour Inducing) plasmid. The T-DNA is flanked by two (imperfect) 25 base pair
repeats. Any DNA contained within these borders will be transferred to the host cell.
The T-DNA section on the Ti plasmid can be replaced by the transgene coding for the
desirable trait attached to the appropriate regulatory sequences. The recombinant bacteria can
then be used to infect both regenerating cell and protoplast cultures.
Marker genes such as those coding for antibiotic resistance are attached to the
transgene so that it is possible to select those cells that have been transformed by the
bacterium. Cell to plant regeneration is carried out on the selected cells and transgene
expression is characterised i.e. it is necessary to check that the gene is expressed at the correct
levels in the correct tissues.
Agrobacterium tumefaciens has been used to transform many dicotyledonous plant
species with relative ease. However, it is, as yet, not possible to transform monocotyledonous
species, which include commercially viable cereal crops such as maize and rice.
157
<
crown gall tumour on tomato.
Plant cells do not have endogenous plasmids. The plasmid vectors used for plant cell
transformation or mostly based on Ti plasmid of A. tumifaciens (tumour inducing plasmids)
and Ri plasmid of A.rhizogenes (root inducing plasmid).These are plant pathogenic gram
negative soil bacteria. A. tumifaciens cause crowngall and A.rhizogenes cause hairy root
diseases to dicot plants. They infect plant cells near wounds, usually at the crown of roots at
the soil surface.
21.2.1.1 MOLECULAR BIOLOGY OF Agrobacterium INFECTION
The process of infection by A. tumifaciens is in the transfer of a small part of Ti
plasmid into the plant cell genome; this DNA sequence is called T-DNA (transferred DNA)
.this infection process is governed by both chromosomal and plasmid borne genes of A.
tumifaciens . infection begins when Agrobacterium cells become attached to plant cells.host
parasite infection is governed by bacterial chromosomal genes generally the chv
genes(chromosomal virulence). chvB , exo genes ,cel genes are concerned with the
biosynthesis of cell attachment polysaccharide due to which the bacterial cells become
firmly adhered to plant cells. chvd, chve are needed for an optimal expression of Ti plasmid
vir genes.
21.2.1.2. PROPERTIES OF CROWN GALL CELLS
Infection by A. tumifaciens produces tumour like growth from which roots and/or
shoots may sometimes be produced.but infection by A.rhizogenes give rise to hairy
roots,which may often show negative geotrophism.
Crown gall and hairy root cells also synthesize unique nitrogenous compounds callad
opines. Agrobacterium cells use opines as their carbon and nitrogen source; the bacteria
usually present in the intercellular spaces of crowngalls. There are different types of opines.
A. tumifaciens generally produce octopine or nopaline, while those as A.rhizogenes produce
either agropine or mannopine.the concerned genes are present in its Ti and Ri plasmid. These
plasmids also carry genes for IAA and cytokinin production.
158
21.2.1.3. THE Ti PLASMID
The Ti plasmid is a large conjugative plasmids or megaplasmid of about 200kb (152
250kb). Ti plasmid is lost when Agrobacterium is grown above 28C; such cured bacteria do
not induce crowngalls, i.e., they become avirulant
The Ti and Ri plasmids are unique bacterial plasmids in the following two respects.
1. They contains some genes (the genes located within their T-DNA), which have regulatory
sequence recognized by plant cells
2. These plasmids naturally transfer their T-DNA into the host plant genome which makes
Agrobacterium a natural genetic engineer
The Ti plasmids are classified into a different type based on the type of opines
produced by their genes. These plasmids contain the following important functional regions.
1. T-DNA contains oncogenes and opines synthesis genes, and is transferred into the host
plant genome.
2. Vir region regulates the transfer of T-DNA into plant cells.
3. Opine catabolism region produce enzymes necessary for the utilization of opines by
Agrobacterium.
4. Conjugative transfer region functions in conjugative transfer of the plasmids; it can also
function in T-DNA transfer when the T-DNA borders are deleted.
5. Origin of replication for propagation in Agrobacterium.
21.2.1.4. ORGANIZATION OF T-DNA

T-DNA (transferred DNA) is 23kb segment of Ti/Ri plasmids wjhich is transferred
into the plant genome during Agrobacterium infection. T-DNA is defined onboth its sides by
a 24bp direct repeat border sequence, contains the genes for tumour/hairyroot induction and
those for opine biosynthesis.
159
All the genes present in the T-DNA contain eukaryotic regulatory sequences. As a result,
these genes are expressed only in plant cells, and they are not expressed in the
Agrobacterium.
21.2.1.5. ORGANIZATION OF VIR REGION
The vir or virulence region of Ti plasmid contains eight operons(virA, virB, virC,
virD. virE, virF, virG, virH) , which together span about 40kb of DNA and has 25 genes.this
region mediates the transfer of T-DNA into plant genome, and hence is essential for
virulence.
21.2.1.6. TRANSFER OF T-DNA
T-DNA transfer is brought about by the vir region, also called vir regulon. A regulon
is a group of operons involved in the same function and subject to some degree of common
regulation. The vir regulon is activated by the phenolics signal molecules acetosyrigone and
-hydroxy syringone, which are produced by wounded tissues of virtually all dicot plant
species, and constitute the wound response.
Integeration of T-DNA into plant genome:
T-DNA enters plant cells in a single stranded form; it is immediately converted into a
double stranded form in the nuclei it has been suggested that the high frequency of
transformation by Agrobacterium is due to the singe stranded T-DNA transfer.
21.2.1.7. VECTORS DERIVED FROM Ti PLASMID
160
The use of wild type Ti plasmid as a vector has the following problems
1. Presence of oncogenes
2. Large size
3. A general lack of unique cloning sites within the T-DNA.
These problems have been resolved by deleting the oncogenes from the T-DNA
(disarming) and developing intermediate vectors and binary vectors to facilitate gene cloning
procedures.
DISARMING
The deletion of genes governing auxin and cytokine production (the onco genes) from
T-DNA of a Ti plasmid is known as disarming. It was a discovered during the early 1980s.
CO-INTEGRATE Ti PLASMID VECTORS
The genes of interest to be transferred into plants are initially cloned in E.coli. Ti
plasmid cannot be used for this purpose due to itsa large size and nonavailbility of unique
restriction sites within DNA. this difficulty is resolved by using a suitable modified E.coli
plasmid for initial cloning of genes. The subsequent gene transfer into plants may utilize one
of the two strategies represented by co-integerate and binary vectors.
Co-integerate vector is produced by integerating the modified E.coli plasmid (used for
cloning) into a disarmed Ti plasmid. The co-integeration of the two plasmids is achieved
within Agrobacterium by homologous recombination.
BINARY VECTOR
The vir region of Ti plasmid need not be present in the same plasmid for an efficient
transfer of T-DNA. Binary vector consist of a pair of plasmids of which one plasmid contains
disarmed T DNA sequence (at least the left and right borders of T DNA must be present),
while the other contains the vir region, and lacks the entire T DNA including the border.
DIRECT GENE TRANSFER METHODS
1.
2.
3.
4.
5.
6.
7.
8.
Biolistics
Calcium phosphate precipitation
Electroporation
Gene silencing
Gene splicing
Lipofection
Microinjection
Viral carriers.
21.3. BIOLISTICS
Biolistics (other wise known as Particle Bombardment) involves directly "shooting" a
piece of DNA into the recipient plant tissue. This is carried out using a gene gun. Tungsten or
gold beads (which are smaller than the plant cells themselves) are coated in the gene of
161
interest and fired through a stopping screen, accelerated by Helium, into the plant tissue. The
particles pass through the plant cells, leaving the DNA inside.
This method can be used on both monocotyledonous and dicotyledonous species
successfully. It is again a relatively simple laboratory procedure. The transformed tissue is
selected using marker genes such as those that code for antibiotic resistance. Whole plants are
then regenerated from the totipotent transformed cells in culture, containing a copy of the
transgene in every single cell
21.4. CALCIUM PHOSPHATE PRECIPITATION

The selected DNA is exposed to calcium phosphate. This mixture creates tiny
granules. Target cells respond to these granules by surrounding and ingesting them
(endoocytosis), allowing the granules to release the DNA and deliver it to the host nuclei and
chromosome(s).
21.5.ELECTROPORATION
The prepared target cells are immersed in a special solution with the selected DNA. A
short but intense electric shock is then passed through the solution. The result is small tears in
the cell walls, which allow the new genetic material access to the nuclei. Then, the cells are
placed into another solution and encouraged to repair their breached walls, locking the
162
donor DNA inside the cell. The selected DNA is incorporated into the host chromosomes to
provide the host with a new gene.
21.6. GENE SILENCING
The gene responsible for the organisms undesirable trait is identified. One method of
silencing that particular gene is to attach a second copy of the gene the wrong way around.
This technique is used to prevent plants like peanuts and wheat from producing the proteins
(allergens) commonly responsible for human allergies.
21.7. GENE SPLICING
Bacteria contain restriction enzymes that form part of the bacteriums immune
system against invasion by another organism or bacteriophage (a bacterial virus). The
restriction enzymes attack the foreign DNA by cutting it into precise sections and preventing
it from being inserted into the bacteriums chromosome. Different bacteria produce different
restriction enzymes that cut any DNA at different places, making the DNA sticky in some
cases, which means they can be pasted directly onto the target organisms prepared DNA.
Using these restriction enzymes from bacteria, molecular biologists can genetically
engineer the DNA for insertion into target (host) cells to modify gene traits. The molecular
biologist then uses another enzyme (ligase) to fuse the new gene into the chromosome.
Alternatively, instead of pasting, the new gene may be inserted into a bacteriums extra
chromosomal DNA molecule (a plasmid), which carries invasion genes that allow it to in
vade the target cell and deliver the gene.
21.8. LIPOFECTION
Small bubbles of fat called liposomes are used as the carriers of selected DNA. The
target cells and the liposomes are placed into a special solution. The liposomes merge with
the cell membrane, allowing the DNA into the cells for inclusion in the chromosome.
21.9. MICROINJECTION
During microinjection, DNA is injected directly into the cell, or even into the cell
nucleus via an inserted cannula. The process is observed and controlled under the
microscope. The DNA is then integrated into the plant genome probably during the cells
own DNA repair processes.
The advantage of microinjection is that with this method, genetic modification in theory no
longer requires marker genes. .
The target gene, which confers a new trait, is introduced directly into a single cell.
The cells transformed in this way are easy to identify if a dye is injected along with
the DNA.
If the process works, it will no longer be necessary to select the transformed cells
using antibiotic resistance or herbicide resistance markers.
So far this method has been used primarily with animal cells. One of the ways in
which plant cells differ from animal cells is that they have a stable cell wall. To use
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microinjection with plants the method has to be adapted to their special
characteristics.
The most important difference is the cell wall and the internal cell pressure of up to 30
bar. If the kind of cannula used for animal cells is used on plant cells it causes a
drastic loss of pressure and the cell dies. To solve the problem, even thinner cannula
tips are being developed and used in combination with a finely adjustable pressure
generator. In this way the cannula enters the plant cell without destroying it.
Another approach to improving microinjection as a transformation method for plant

cells focuses on the efficiency with which the injected genes are integrated into the
plant genome. One way of improving this is to inject special proteins along with the
target gene sequences which support the stable integration of foreign genes into the
plant genome and are produced by A. tumifaciens (agroinjection). This soil bacterium
is naturally capable of inserting its own genes into the genome of plants.
Microinjection
Viruses have the following features as vectors:
1. Virus infects the cells of adult plants
2. They produce large number of copies per cell leading to gene amplification, and
production of the recombinant proteins in large quantities.
3. Some viruses are systemic in nature.
Most plant viruses have RNA genomes; two such viruses that have great potential as
vectors are brome mosaic virus (BMV) and tobacco mosaic virus (TMV). But the greatest
progress has been made with two groups of viruses having DNA genomes, Caulimovirus and
Gemini virus.
21.10.1. CAULIFLOWER MOSAIC VIRUS (CaMV)
It contains about 8 kb double stranded DNA and produces spherical particles and
infects a wide range of dicot crops. The infection are systemic, even its DNA is infectious. It
has eight tightly packed genes of which two small genes gene 2 and 7 are non essential so
only these two genes can be replaced without a loss of infectivity.
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21.10.2. GEMINI VIRUS
These virus infect a variety of monocot and dicot plants and have circular single
stranded DNA. Eg., maize streak virus causes yellow streaks on maize and is able to produce
infection only by natural insect vector (leaf hopper). It multiplies to a high copy number in
the nuclei of dividing cells. MSV genome has been successfully transferred into plants cells
with the help of Agrobacterium (agroinfection). Another Gemini virus, wheat dwarf virus has
been used for successful agroinfection of wheat seedlings.
21.10.3. TOBACCO MOSAIC VIRUS (TMV)
TMV has an RNA genome, which also serves as mRNA. It has four genes of which
core protein genes was non essential. The use of RNA viruses as vectors consists of two
steps.
1.
Use of cDNA copy of the viral genome
2.
in vitro transcription of the recombinant viral genome cDNA to
produce infectious RNA copies
21.10.4. BROME MOSAIC VIRUS (BMV)
It infects several species of graminae, including barley. It has three genomic segments
each packed into a separate particle. The core protein gene is located on the RNA segment 3,
and is the one target site for DNA insertion, although this prevents the formation of virus
particles.
In clonig three types of vector system have been used with varying degree of success
with higher plants- vectors based on naturally ocuring plasmids of Agrobacterium,
direct gene transfer using DNA fragments not attached to a plant cloning vector,
vectors based on plant viruses.
A.tumifaciens is a soil bacterium that causes crown gall disease of dicot plants due to
the present of Ti plasmid.
Ti plasmid have T-DNA region, which gene of intrest can be inserted and is
transferred to plant cells by means of vir genes .
Direct gene transfer methods which are widely used are biolistics,calcium phosphate
precipitation. Electroporation, gene silencing,gene splicing,lipofection and
microinjection.
The two classes of DNA viruses ideally used for gene cloning are Cauliflower mosaic
virus and Gemini virus and RNA viruses used are Tobacco mosaic virus and Brome
mosaic virus.

1. What is meant by Ti Plasmid? Describe advantages and disadvantages.
2. Define Microinjection with plant viral vectors.
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1.
2.
3.
4.
Describe disadvantages involved in vectors derived from Ti plasmids.

Direct gene transfer methods- Discuss.
Write the plant vectors used for cloning.
Explain the cloning strategy in plants.
21.14. REFERENCES
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LESSON 22: CLONING IN ANIMALS
CONTENTS
22.1. INTRODUCTION
22.2. TRANSGENIC ANIMALS
22.3. TRANSGENIC MICE
22.4. KNOCKOUT MICE
22.5. TRANSGENIC SHEEP AND GOATS
22.6. TRANSGENIC CHICKENS
22.7. TRANSGENIC PIGS
22.8. TRANSGENIC FISH
22.13. REFERENCES
The chapter in detail discuss about the transgenic animals and their applications.
22.1. INTRODUCTION
Complex biotechnological procedures have enabled scientists to successfully clone
mice, sheep, cows and other mammals.
The technology is still at early stages and currently, one in three cloned animals is
born abnormally large or with other developmental problems.
Scientists at the Monash Institute of Reproduction and Development in Melbourne
believe these problems could be linked to a process called gene 'imprinting'. Embryos contain
two copies of each gene one from each parent. It is thought that about 60 genes are
imprinted' with instructions to switch one copy on or off to allow for normal growth and
development.
If this doesnt happen correctly and both copies are switched on, or both copies are
switched off, it results in problems in growth and development, both prenatal and postnatal.
We do not understand this imprinting process in cloned embryos.
The closest scientists have come to cloning a non-human primate occurred in October
2004. Biologists successfully transferred cloned monkey embryos into monkey mothers.
None of the resulting pregnancies lasted more than a month.
22.2. TRANSGENIC ANIMALS
A transgenic animal is one that carries a foreign gene that has been deliberately inserted
into its genome. The foreign gene is constructed using recombinant DNA methodology. In
addition to a structural gene, the DNA usually includes other sequences to enable it
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to be incorporated into the DNA of the host and

To be expressed correctly by the cells of the host.
Transgenic sheep and goats have been produced that express foreign proteins in their
milk.
Transgenic chickens are now able to synthesize human proteins in the "white" of the
eggs.
These animals should eventually prove to be valuable sources of proteins for human
therapy.
22.2.1 PRODUCTION OF TRANSGENIC ANIMALS
Since the discovery of the molecular structure of DNA by Watson and Crick in 1953,
molecular biology research has gained momentum. Molecular biology technology combines
techniques and expertise from biochemistry, genetics, cell biology, developmental biology,
and microbiology.
Scientists can now produce transgenic animals because, since Watson and Crick's
discovery, there have been breakthroughs in:
recombinant DNA (artificially-produced DNA)

genetic cloning
analysis of gene expression (the process by which a gene gives rise to a protein)
genomic mapping
The underlying principle in the production of transgenic animals is the introduction of a

foreign gene or genes into an animal (the inserted genes are called transgenes). The foreign
genes "must be transmitted through the germ line, so that every cell, including germ cells, of
the animal contain the same modified genetic material. (Germ cells are cells whose function
is to transmit genes to an organism's offspring.)
To date, there are three basic methods of producing transgenic animals:
1.
2.
3.
DNA microinjection
Retrovirus-mediated gene transfer
Embryonic stem cell-mediated gene transfer
1. Microinjection:
Gene transfer by microinjection is the predominant method used to produce
transgenic farm animals. Since the insertion of DNA results in a random process, transgenic
animals are mated to ensure that their offspring acquire the desired transgene. However, the
success rate of producing transgenic animals individually by these methods is very low and it
may be more efficient to use cloning techniques to increase their numbers. For example, gene
transfer studies revealed that only 0.6% of transgenic pigs were born with a desired gene after
7,000 eggs were injected with a specific transgene.
The mouse was the first animal to undergo successful gene transfer using DNA
microinjection. This method involves:
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transfer of a desired gene construct (of a single gene or a combination of genes that
are recombined and then cloned) from another member of the same species or from a
different species into the pronucleus of a reproductive cell19
the manipulated cell, which first must be cultured in vitro (in a lab, not in a live
animal) to develop to a specific embryonic phase, is then transferred to the recipient
female
2. Retrovirus-Mediated Gene Transfer

A retrovirus is a virus that carries its genetic material in the form of RNA rather than
DNA. This method involves: retroviruses used as vectors to transfer genetic material into the
host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic
constitution
chimeras are inbred for as many as 20 generations until homozygous (carrying the
desired transgene in every cell) transgenic offspring are born
3. Embryonic Stem Cell-Mediated Gene Transfer

The method was successfully used in 1974 when a simian virus was inserted into mice
embryos, resulting in mice carrying this DNA.
This method involves:
isolation of totipotent stem cells (stem cells that can develop into any type of
specialized cell) from embryos
the desired gene is inserted into these cells
cells containing the desired DNA are incorporated into the host's embryo, resulting in
a chimeric animal
Unlike the other two methods, which require live transgenic offspring to test for the
presence of the desired transgene, this method allows testing for transgenes at the cell stage.
22.2.2. SOMATIC CELL NUCLEAR TRANSFER
Dolly, the first animal to be cloned, was created using the technique of somatic cell
nuclear transfer (SCNT). To do this, cells are taken from the animal that is going to be
cloned. In the case of Dolly the sheep, a cell was taken from her udder. These are normal
body cells somatic cells - and the nucleus is removed. The nucleus contains all of the
genetic material to make the animal and so, is termed the donor cell. Egg cells are used for
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cloning because of their ability to grow rapidly. The egg cells nucleus is removed and the
nucleus from the donor cell is inserted in its place.
The egg is then stimulated to grow by numerous stimulants which activates the
reconstructed embryo to divide and grow. The division of the egg cell follows the same
process that would occur if the egg was fertilised by sperm during natural reproduction. The
cell division continues for 5 days until a blastomere forms. A blastomere is a ball of nearly
100 cells all with the same genetic material as the donor. Once a cloned embryo reaches the
blastomere stage of development, it can follow two paths. Either it can be implanted into a
uterus of a female to create a whole organism. This is called reproductive cloning. When
Dolly was born, she was the only lamb born from 277 attempts. She was a clone of the sheep
whose udder cell was used. Blastomeres can also be used as a source of stem cells.
22.2.3. EMBRYO SPLITTING
Another cloning technique is embryo splitting. Using microsurgery (surgery
conducted under a microscope), an embryo is split while it still consists of only a few cells.
Genetically identical individuals develop from each portion in the same way as identical
twins are formed in nature. This technique has been used to successfully create cloned
embryos and cloned animals
22.2.4. CHROMATIN TRANSFER
There are a number of problems associated with nuclear transfer - the method used to
clone Dolly and almost all cloned animals since then. When the nucleus is transferred to a
new egg cell, the egg reprograms the incoming nucleus to allow it to go back to its
undifferentiated state. Because it has come from an adult cell, it no longer needs to produce
the proteins, hormones and other molecules associated with it being an embryo and growing
to produce all the different tissues in a whole body.
Incomplete reprogramming of the donor cell is thought to be a leading factor in the
low success rate of animal cloning. Chromatin transfer is a new cloning technique aimed at
reducing these problems. It involves treating the cell of the animal to be cloned to remove
molecules associated with cell differentiation before the nucleus is removed. This is a very
new method created by Genetic Savings and Clone, a company in the USA that clones pets.
22.3. TRANSGENIC MICE
Normal mice cannot be infected with polio virus. They lack the cell-surface molecule
that, in humans, serves as the receptor for the virus. So normal mice cannot serve as an
inexpensive, easily-manipulated model for studying the disease. However, transgenic mice
expressing the human gene for the polio virus receptor
can be infected by polio virus and even

develop paralysis and other pathological changes characteristic of the disease in
humans.
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Two methods of producing transgenic mice are widely used:
transforming embryonic stem cells (ES cells) growing in tissue culture with the
desired DNA;
injecting the desired gene into the pronucleus of a fertilized mouse egg.
The Embryonic Stem Cell Method (Method "1")

Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of
mouse blastocysts. They can be grown in culture and retain their full potential to produce all
the cells of the mature animal, including its gametes.
1. Make your DNA

Using recombinant DNA methods, build molecules of DNA containing
the structural gene you desire (e.g., the insulin gene)

vector DNA to enable the molecules to be inserted into host DNA molecules
promoter and enhancer sequences to enable the gene to be expressed by host cells
2. Transform ES cells in culture

Expose the cultured cells to the DNA so that some will incorporate it.
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3. Select for successfully transformed cells.
4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts.
5. Embryo transfer
Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized

male). The stimulus of mating elicits the hormonal changes needed to make her uterus
receptive.
Transfer the embryos into her uterus.
Hope that they implant successfully and develop into healthy pups (no more than onethird will).
6. Test her offspring
Remove a small piece of tissue from the tail and examine its DNA for the desired
gene. No more than 1020% will have it, and they will be heterozygous for the gene.
7. Establish a transgenic strain
Mate two heterozygous mice and screen their offspring for the 1:4 that will be
homozygous for the transgene.
Mating these will found the transgenic strain.
The Pronucleus Method (Method "2")
1. Prepare your DNA as in Method 1

2. Transform fertilized eggs
Harvest freshly fertilized eggs before the sperm head has become a pronucleus.
Inject the male pronucleus with your DNA.
When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to
divide by mitosis to form a 2-cell embryo.
3. Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.

An Example
This image (courtesy of R. L. Brinster and R. E. Hammer) shows a transgenic mouse (right)
with a normal littermate (left). The giant mouse developed from a fertilized egg transformed
with a recombinant DNA molecule containing:
the structural gene for human growth hormone

a strong mouse gene promoter
The levels of growth hormone in the serum of some of the transgenic mice were several
hundred times higher than in control mice.
Random vs. Targeted Gene Insertion
The early vectors used for gene insertion could, and did, place the gene (from one to 200
copies of it) anywhere in the genome. However, if you know some of the DNA sequence
172
flanking a particular gene, it is possible
to design vectors that replace that gene.
The replacement gene can be one that
restores function in a mutant animal or

knocks out the function of a
particular locus.
In either case, targeted gene insertion
requires
ired gene
neor, a gene that encodes an enzyme that
inactivates the antibiotic neomycin and its
relatives, like the drug G418, which is lethal
mammalian cells;
tk, a gene that encodes thymidine kinase, an
enzyme that phosphorylates the nucleoside
analog gancyclovir. DNA polymerase fails
discriminate against the resulting nucleotide
and inserts this nonfunctional nucleotide into
freshly-replicating DNA. So ganciclovir kills
cells that contain the tk gene.
Step 1
Treat culture of ES cells with preparation of vector
the
des
to
to
DNA.
Results:
Most cells fail to take up the vector; these cells will be killed if exposed to G418.
In a few cells: the vector is inserted randomly in the genome. In random insertion, the
entire vector, including the tk gene, is inserted into host DNA. These cells are
resistant to G418 but killed by gancyclovir.
In still fewer cells: homologous recombination occurs. Stretches of DNA sequence
in the vector find the homologous sequences in the host genome and the region
between these homologous sequences replaces the equivalent region in the host DNA.
Step 2
Culture the mixture of cells in medium containing both G418 and ganciclovir.
The cells (the majority) that failed to take up the vector are killed by G418.
The cells in which the vector was inserted randomly are killed by gancyclovir
(because they contain the tk gene).
This leaves a population of cells transformed by homologous recombination (enriched
several thousand fold).
173
Step 3
Inject these into the inner cell mass of mouse blastocysts.
22.4. KNOCKOUT MICE
If the replacement gene (A* in the diagram) is nonfunctional (a "null" allele), mating
of the heterozygous transgenic mice will produce a strain of "knockout mice" homozygous
for the nonfunctional gene (both copies of the gene at that locus have been "knocked out").
Knockout mice are valuable tools for discovering the function(s) of genes for which
mutant strains were not previously available. Two generalizations have emerged from
examining knockout mice:
Knockout mice are often surprisingly unaffected by their deficiency. Many genes turn
out not to be indispensable. The mouse genome appears to have sufficient redundancy
to compensate for a single missing pair of alleles.
Most genes are pleiotropic. They are expressed in different tissues in different ways
and at different times in development.
Tissue-Specific Knockout Mice

While "housekeeping" genes are expressed in all types of cells at all stages of
development, other genes are normally expressed in only certain types of cells when turned
on by the appropriate signals (e.g. the arrival of a hormone).
To study such genes, one might expect that the methods described above would work.
However, it turns out that genes that are only expressed in certain adult tissues may
nonetheless be vital during embryonic development. In such cases, the animals do not survive
long enough for their knockout gene to be studied. Fortunately, there are now techniques with
which transgenic mice can be made where a particular gene gets knocked out in only one type
of cell.
The Cre/loxP System
One of the bacteriophages that infects E. coli, called P1, produces an enzyme
designated Cre that cuts its DNA into lengths suitable for packaging into fresh virus
particles. Cre cuts the viral DNA wherever it encounters a pair of sequences designated loxP.
All the DNA between the two loxP sites is removed and the remaining DNA ligated together
again (so the enzyme is a recombinase).
Using "Method 1" (above), mice can be made transgenic for
the gene encoding Cre attached to a promoter that will be activated only when it is
bound by the same transcription factors that turn on the other genes required for the
unique function(s) of that type of cell;
a "target" gene, the one whose function is to be studied, flanked by loxP sequences.
In the adult animal,
those cells that
174
o
o
o
receive signals (e.g., the arrival of a hormone or cytokine)

to turn on production of the transcription factors needed
to activate the promoters of the genes whose products are needed by that
particular kind of cell
Will also turn on transcription of the Cre gene. Its protein will then remove the
"target" gene under study.
All other cells will lack the transcription factors needed to bind to the Cre promoter
(and/or any enhancers) so the target gene remains intact.
The result: a mouse with a particular gene knocked out in only certain cells.
The Cre/loxP system can also be used to remove DNA sequences that block gene
transcription. In such a "knockin" mouse, the "target" gene is turned on in only certain cells.
22.5. TRANSGENIC SHEEP AND GOATS
Until recently, the transgenes introduced into sheep inserted randomly in the genome
and often worked poorly. However, in July 2000, success at inserting a transgene into a
specific gene locus was reported. The gene was the human gene for alpha1-antitrypsin, and
two of the animals expressed large quantities of the human protein in their milk.
This is how it was done.
Sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with a
vector that contained these segments of DNA:
1. 2 regions homologous to the sheep COL1A1 gene. This gene encodes Type 1
collagen. (Its absence in humans causes the inherited disease osteogenesis
imperfecta.)
This locus was chosen because fibroblasts secrete large amounts of collagen and thus
one would expect the gene to be easily accessible in the chromatin.
2. A neomycin-resistance gene to aid in isolating those cells that successfully
incorporated the vector.
3. The human gene encoding alpha1-antitrypsin.
Some people inherit two non- or poorly-functioning genes for this protein. Its
resulting low level or absence produces the disease Alpha1-Antitrypsin Deficiency
(A1AD or Alpha1). The main symptoms are damage to the lungs (and sometimes to
the liver).
4. Promoter sites from the beta-lactoglobulin gene. These promote hormone-driven
gene expression in milk-producing cells.
5. Binding sites for ribosomes for efficient translation of the mRNAs.
Successfully-transformed cells were then
175
fused with enucleated sheep eggs and

Implanted in the uterus of a ewe (female sheep).
Several embryos survived until their birth, and two young lambs have now lived over
a year.
When treated with hormones, these two lambs secreted milk containing large amounts
of alpha1-antitrypsin (650 g/ml; 50 times higher than previous results using random
insertion of the transgene).
On June 18, 2003, the company doing this work abandoned it because of the great
expense of building a facility for purifying the protein from sheep's milk. Purification is
important because even when 99.9% pure, human patients can develop antibodies against the
tiny amounts of sheep proteins that remain.
However, another company, GTC Biotherapeutics, has persevered and in June of 2006 won
preliminary approval to market a human protein, antithrombin, in Europe. Their protein
the first made in a transgenic animal to receive regulatory approval for human therapy was
secreted in the milk of transgenic goats.
22.6. TRANSGENIC CHICKENS
Chickens
grow faster than sheep and goats and large numbers can be grown in close quarters;
synthesize several grams of protein in the "white" of their eggs.
Two methods have succeeded in producing chickens carrying and expressing foreign genes.
Infecting embryos with a viral vector carrying

o the human gene for a therapeutic protein
o promoter sequences that will respond to the signals for making proteins (e.g.
lysozyme) in egg white.
Transforming rooster sperm with a human gene and the appropriate promoters and
checking for any transgenic offspring.
Preliminary results from both methods indicate that it may be possible for chickens to
produce as much as 0.1 g of human protein in each egg that they lay.
Not only should this cost less than producing therapeutic proteins in culture vessels, but
chickens will probably add the correct sugars to glycosylated proteins something that E.
coli cannot do.
22.7. TRANSGENIC PIGS
Transgenic pigs have also been produced by fertilizing normal eggs with sperm cells that
have incorporated foreign DNA. This procedure, called sperm-mediated gene transfer
(SMGT) may someday be able to produce transgenic pigs that can serve as a source of
transplanted organs for humans.
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22.8. TRANSGENIC FISH
Domestic fish production through transgenic techniques offers many potential
economics advantages for commercial aquaculture production, including introduction of new
or novel traits and increased response to selection for faster growth. The traditional method of
producing transgenic fish is still microinjection, however, advancements have been made
using pseudotyped retroviral vectors and electroporation. Some success has also been shown
using particle bombardment, but other methods such as sperm-mediated gene transfer,
liposome-mediated DNA uptake, MPG peptide vector-oligonucleotide complex-mediated
transfer, and nuclear transfer have had limited success in aquaculture. Several factors
influence success of integration and expression, these include: the initial amount of DNA
used in the injection (or electroporation), form of the DNA used (linear versus supercoiled),
site of integration, copy number, and orientation of multiple transgene copies and matrix
attachment regions (MARS).
The species of origin and insertion of the transgene has also been shown to be
important. Most promoters used in aquaculture are either constitutive or inducible, and except
for being able to turn on or off the expression with inducible promoters, the level of
expression remains uncontrolled. Progress has been made toward tissue specific promotion.
The escape or introduction of transgenic fish into natural communities is a major ecological
concern.
Baculovirus infects insects. This virus is rod shaped with a large double stranded
genome. During normal infections, baculovirus produces nuclear inclusion bodies which
consist of virus particles embedded in a protein matrix. This protein matrix is encoded with
the virus and is called polyhedrin and this polyhedrin accounts for 70% of total protein
encoded by the virus as the transcription of the polyhedrin is driven by extremely active
promoters
Baculovirus
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SV40 ; a spherical virus with double stranded circular DNA of size 5.2 kb. The viral
protein contains three viral coded proteins.VP1 is the major protein present in the capsid with
a size of 47000 kDa. Two more proteins VP2 and VP3 are also present. The DNA of virus is
associated with the four histones (H4,H2A,H2B and H3) proteins. The viral DNA can be
segmented into five precise segments coding for five different proteins small T, large T, VP1,
VP2 and VP3. VP1 coding region overlaps VP2 and VP3 in a different translation reading
frame. SV 40 virus infects monkey kidney cell lines
Vectors are often derived from viral components
178
The benefits of these animals to human welfare can be grouped into areas:
Agriculture
Medicine
Industry
The examples below are not intended to be complete but only to provide a sampling of the
benefits
1. Agricultural Applications
a) Breeding
Farmers have always used selective breeding to produce animals that exhibit desired
traits (e.g., increased milk production, high growth rate). Traditional breeding is a timeconsuming, difficult task. When technology using molecular biology was developed, it
became possible to develop traits in animals in a shorter time and with more precision. In
addition, it offers the farmer an easy way to increase yields.
b) Quality
Transgenic cows exist that produce more milk or milk with less lactose or cholesterol,
pigs and cattle that have more meat on them, and sheep that grow more wool. In the past,
farmers used growth hormones to spur the development of animals but this technique was
problematic, especially since residue of the hormones remained in the animal product.
c) Disease resistance
Scientists are attempting to produce disease-resistant animals, such as influenzaresistant pigs, but a very limited number of genes are currently known to be responsible for
resistance to diseases in farm animals.
2. Medical Applications
a) Xenotransplantation
Patients die every year for lack of a replacement heart, liver, or kidney. For example,
about 5,000 organs are needed each year in the United Kingdom alone. Transgenic pigs may
provide the transplant organs needed to alleviate the shortfall. Currently, xenotransplantation
is hampered by a pig protein that can cause donor rejection but research is underway to
remove the pig protein and replace it with a human protein.
b) Nutrition supplements and pharmaceuticals
Products such as insulin, growth hormone, and blood anti-clotting factors may soon
be or have already been obtained from the milk of transgenic cows, sheep, or goats. Research
is also underway to manufacture milk through transgenesis for treatment of debilitating
diseases such as phenylketonuria (PKU), hereditary emphysema, and cystic fibrosis.
179
In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4
grams per litre. This transgenic milk is a more nutritionally balanced product than natural
bovine milk and could be given to babies or the elderly with special nutritional or digestive
needs. Rosie's milk contains the human gene alpha-lactalbumin.
c) Human Gene therapy
Human gene therapy involves adding a normal copy of a gene (transgene) to the
genome of a person carrying defective copies of the gene. The potential for treatments for the
5,000 named genetic diseases is huge and transgenic animals could play a role. For example,
the A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance
that promotes the growth of red cells in humans.
3. Industrial Applications
In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into
the cells of lactating goats. The goats began to manufacture silk along with their milk and
secrete tiny silk strands from their body by the bucketful. By extracting polymer strands from
the milk and weaving them into thread, the scientists can create a light, tough, flexible
material that could be used in such applications as military uniforms, medical microsutures,
and tennis racket strings.
Toxicity-sensitive transgenic animals have been produced for chemical safety testing.
Microorganisms have been engineered to produce a wide variety of proteins, which in turn
can produce enzymes that can speed up industrial chemical reactions.
These ethical issues, better served in their own article, include questions such as:
Should there be universal protocols for transgenesis?

Should such protocols demand that only the most promising research be permitted?
Is human welfare the only consideration? What about the welfare of other life forms?
Should scientists focus on in vitro (cultured in a lab) transgenic methods rather than,
or before, using live animals to alleviate animal suffering?
Will transgenic animals radically change the direction of evolution, which may result
in drastic consequences for nature and humans alike?
Should patents be allowed on transgenic animals, which may hamper the free
exchange of scientific research?
Gene transfer to animal cells can be achieved essentially via three routes.
o The most straight forward is direct DNA transfer, the physical introduction of
foreign DNA into the cell.
o The second route is termed transfection, some physical and chemical
techniques used to persuade cells to take up the DNA from their surroundings
o The third is to package the DNA into animal virus, since virus have evolved
mechanisms to naturally infect cells and to introduce their own nucleic acid
transduction.
The presence of foreign gene is readily identified by southern blot hybridization using
cellular DNA obtained from tail biopsies
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The expression of transferred foreign gene is analysed in S1 nuclease production

assays.

1. Validate the use of Biolistics method for plants.
2. Validate the use of calcium phosphate method.
1.
2.
3.
4.
Give an account on how Gene knock out mice can be used for functional studies.
Describe the methods involved in cloning animals.
Write the applications of transgenic animals.
Write notes on animal vectors.
22.13. REFERENCES
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UNIT V: RECOMBINANT PRODUCTS IN INDUSTRIES,

BIOTECHNOLOGY AND ETHICS
LESSON 23: BIOTECHNOLOGY IN AGRICULTURE AND ENVIRONMENT
CONTENTS
23.1 INTRODUCTION
23.2 BIOTECHNOLOGY IN AGRICULTURE AND ENVIRONMENT
23.3. LET US SUM UP
The lesson deals with the risks and benefits of recombinant technology in the field of
agricultural and environmental biotechnology.
23.1 INTRODUCTION
Recombinant DNA (rDNA) technology or Genetic Engineering is an umbrella term
for a set of experimental techniques that enable individual genes and DNA sequences to be
manipulated resulting in genetically modified organisms (GMO) and products.
There have been many potential applications of rDNA in medicine, agriculture and
industry. Conventionally proteins and other biological products, processed from human or
animal serum or tissues, often are of low purity. Production of therapeutic products by the
rDNA technology has several advantages such as provision of drugs that could not be
produced by conventional methods, manufacture of sufficient quantities of drugs and
provision for manufacture of safe drugs. Recombinant DNA technology has made a
revolutionary impact in the area of healthcare by enabling mass production of safe, pure and
more effective versions of various biochemical substances used as therapeutics.
It has become possible to introduce genes from higher organisms to microbial cells
such that the recipient cells are capable of synthesizing heterologous proteins. Examples of
such hosts for foreign genes include E.coli, Saccharomyces cerevisiae and other yeasts as
well as filamentous fungi like A.awamori . Products produced in such genetically
manipulated organisms include interferon insulin human serum albumin, factor VIII, factor
IX and epidermal growth factor.
23.2 BIOTECHNOLOGY IN AGRICULTURE AND ENVIRONMENT
Currently Biotechnology techniques are being used in various fields, including
agriculture, veterinary medicine pharmaceutical development, energy conservation and waste
treatment. These techniques increase productivity in crops and livestock; controls pests;
produce new food and fiber crops, and develop effective medicines.
Agricultural biotechnology covers a wide a range of techniques to improve
productivity and quality in crops, livestock and fisheries. Several biotechnological
applications such as plant tissue culture (micro propagation), biopesticides, biofertilizers,
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Diagnostics for diseases of crops ad livestock, embryo transfer in livestock have already been
adopted extensively. New rDNA or genetic engineering technologies are now being used to
transfer genes within and across plant species to generate genetically modified organisms
including transgenic crops.
Transgenic crops incorporate traits such as herbicide tolerance, pest and disease
resistance, product quality improvements and stress tolerance. Several commercially
important crops such as maize, soybean, tomato, cotton potato, mustard and rice are being
targeted for imparting these traits.
i) HERBICIDE TOLERANCE
Good planting conditions for crops also sustain weeds that can reduce crop
productivity as they compete for the same nutrients the desired plant needs. To prevent this,
herbicides are sprayed over crops to eliminate the undesirable weeds. These herbicides are
required to be applied several times during growth cycle leading to not only increased
expenditure to the farmers but also harmful effects to the environment. Many effective broad
spectrum herbicides do not distinguish between weeds and crops, but crop plants can be
modified to make them resistant to herbicides, so as to eliminate weeds more selectively.
When a herbicide is sprayed, it will kill the weeds but have no effect on the crop plants
Farmers can thus reduce the number of times herbicides have to be applied leading to
reduction in the expenditure and damage to the environment.
For eg, the herbicide RoundupTM contains the active ingredient glyphosphate, which
kills plants by binding to the active site of the enzymes enolpyruvalshikimate phosphate
synthase (EPSP synthase). This enzyme is critical for the synthesis of aromatic amino acids.
Roundup is an extremely effective herbicide but it also kills most all species of plants,
including most crop plants. On the other hand its very safe when it comes to humans and
veterinary life because EPSP synthase enzyme is absent in human and animals.
Hence by using rDNA technology it is possible to produce plants that produce
modified EPSP synthase gene which is not inhibited by glyphosphate. Such modifications
have been introduced into crop plants such as cotton and soybean.
ii) INSECT RESISTANCE
Biotechnology has opened up new avenues of natural protection for plants by providing
new biopesticides, such as microorganism, that are toxic to targeted crop pests but do not
harm humans, animals, fish, birds or beneficial insects. As biopesticides act in unique ways,
they can even control pest population that has developed resistance to conventional
pesticides. Using recombinant technology, the gene that makes these microorganisms lethal
to certain insects can be transplanted into the plants on which that insect feeds. The plant that
once was a good source for the insect will now kill it, lessening the need to spray crops with
chemical pesticides to control infestation.
For example, the spores of Bacillus thuringiensis(Bt) contain crystalline (cry) protein
which breaks down to release a toxin, known as delta-endotoxin. This toxin binds to and
creates pores in the intestinal lining, resulting in an ion imbalance, paralysis of the digestive
system and subsequently insect death. Different version of the cry genes, have been identified
against different orders of insect or affect the insect gut in slightly different ways. When the
insect feeds on a leaf or bores into a stem of Bt containing plant, it ingests the toxin and dies.
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The use of Bt varieties has dramatically reduced the amount of chemical pesticides applied to
these crops.
iii) DISEASE RESISTANCE
Plants are susceptible to viral bacterial and fungal diseases. Much progress has been
made in evolving transgenic plants resistant to viruses. Fro example, expression of a gene that
encodes the coat protein of tobacco mosaic virus (TMV) in transgenic tobacco plants has
been shown to cause the plants to resist TMV infection. A number of other viral resistant
plant species have been developed including squash and potatoes. Genetic engineering of
crop plants for resistance to fungal and bacterial infections has been more difficult. However,
by studying the defensive genes that are expressed in naturally disease-resistant plants,
encouraging progress has been made. The proteins encoded by these so called pathogenesis
related proteins (PR proteins) can, in some cases, provide limited disease protection in
transgenic plants.
iv) PRODUCT QUALITY IMPROVEMENT
Product quality improvement includes improving the shelf life of the products. The
most successful research in this aspect is with regard to the tomato plant. Tomatoes need to
be picked while still green so that they are firm enough to withstand mechanical handling and
transport. But they do not develop the same flavor and texture of vine-ripened tomatoes.
Softening of tomatoes and many other fruits is caused y the enzyme pectinase or
polygalacturanase. This enzyme digests the pectin polysaccharide that cements the plant cells
together. Softening of the fruit is caused, in part by this breakdown of pectin. In order to
reduce the levels of PA in ripening tomatoes, researchers placed the PGA gene in reverse
orientation relative to that of the CaMV 35s promoter. This results in transcription of an
antisense RNA that is complementary to the normal sense PGA mRNA, which is able to
arrest the translation of the endogenous PGA mRNA in the tomato fruit. Fruits of these plants
have normal colour and flavor but they soften more slowly and can be picked and processed
after they are ripe. They also have a higher content of soluble solids and are therefore better
than normal tomatoes for processed tomato products.
Transgenic lines of potato having increased levels of starch have also been developed
by introducing a gene construct that expresses a gene from bacteria that produce an enzyme
that enhances starch biosynthesis. A promoter from a potato gene that encodes the major
protein in potato tubers has been used, sop that the expression of the introduced gene is
limited to the tuber. Tubers accumulate approximately 3 to 5 % more starch than normal
potatoes and when they are deep fried absorb less oil and yield chips having fewer calories.
Other plants in which such product
v) RESISTANCE TO ENVIRONMENTAL STRESS
In addition to the biological challenges to plant growth and development, crop plant
need to cope up with abiotic stresses such as drought, cold, heat and soils that are too acidic
or salty to support plant growth. While plant breeders have successfully incorporated genetic
resistance to biotic stresses into many crop plants through cross breeding, their success at
creating crops resistant to abiotic stresses has been more limited, largely because few crops
have close relatives with genes for resistance to those stresses.
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vi) INCREASING YIELD

In addition to increasing crop productivity by using built-in protection against
diseases, pests, environmental stresses and weeds to minimize losses, attempts are being
made to use biotechnology to improve crop yields directly. Crops that are better at accessing
the micronutrients they need are also being developed. Nitrogen is the critical limiting
element for plant growth and step-by-step, researchers from many scientific disciplines are
teasing apart details of the symbiotic relationship that allows nitrogen fixing bacteria to
capture the atmospheric nitrogen and provide it to the plants that harbor them in root nodules.
vi) PLANT BASED PHARMACEUTICALS
Plants are among the most efficient bioreactors which produce quantities of material
with sunlight and soil based nutrients as inputs. Attempts are being made to replace the
traditional fermentation procedure for the production of biopharmaceuticals to plant based
production. The benefits of using plants are the ability to increase production at low cost by
planting more acres, rather than building fermentation capacity, lower capital and operating
cost, simplified downstream processing etc.,
Therapeutic drugs to treat cancer, infectious diseases, autoimmune diseases,
cardiovascular diseases and other conditions and several vaccines can potentially. Plant
transgenic technology is being used to produce a plant that will generate a seed that expresses
a desired therapeutic protein. This seed can propagate under the right growing conditions to
yield plants and seed stock for producing the desired protein. The desired protein can be
extracted from the seed to make a biopharmaceutical. Plant based therapeutics are expected to
be much more cost effective.
23.3. LET US SUM UP
Through recombinant DNA technology protein of interest can be expressed in

convenient host cells and could be easily recovered for use.
Such manipulations are usually done for proteins of clinical, agricultural and
environmental importance.
Genetic modification or Recombinant DNA technology plays a very great role in
various facets of agriculture and environment management.
This includes the production of new plant varieties with improved qualities including
herbicide tolerance, insect resistance and disease resistance. Plant and microbes are
also being modified for the production of pharmaceuticals.

1. Justify the use of biotechnology in agriculture.
2. Analyse whether biotechnology will affect the environment.
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1. Give an account on plant genetic engineering for the production of herbicide tolerant
plants.
2. Why transgenic Bt Cotton was failure in the recent past?
3. What is the scope and use of edible vaccines?
4. Fungicides are used in lesser amount in tropics elaborate
23.6. REFERECNES
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LESSON 24: RISKS AND BENEFITS OF GENETIC ENGINEERING
CONTENTS
24.1. INTRODUCTION
24.2. POTENTIAL BENEFITS OF GENETIC ENGINEERING
24.3. BIOSAFETY REGULATIONS
24.4. GOVERNMENT RULES FOR GMO
24.5. LET US SUM UP
24.8. REFERENCES
The chapter discuss about the merits and demerits of genetic engineering.
24.1. INTRODUCTION
Genetic engineering is widely used field of study for the development of various
products which are of beneficial to human. For all the technique or new finding has its own
pros and cons. In the same way genetic engineering also has its own merits and demerits
which are discussed in the following chapter.
24.2. POTENTIAL BENEFITS OF GENETIC ENGINEERING
The spectrum of potential benefits from the application of genetic engineering and
biotechnology to food crops in developing countries ranges from diagnostic aids, for example
in plant diseases, through to gene mapping, where the genetic characteristics of plants are
visibly cartographed, enabling speedier identification of interesting genetic material for every
kind of plant usable in agriculture. The main objective is to find improved seed varieties, i.e.
varieties with properties such as resistance to or tolerance of plant diseases (fungi, bacteria,
viruses) and animal pests (insects, mites, nematodes) as well as to so-called stress factors
such as climatic variation or aridity, poor soil quality, crop rotation practices, and others. The
idea of genetic engineering, then, is not to invent freakish hybrids but rather to improve
certain properties of important cultivated plants.
An equally important goal of research is the transfer of genes with nitrogen-fixing
capacity onto grain. Ideally, seed varieties which result from such research endeavors should
lead to the cultivation of plants which fit into the concept of "sustainable" agriculture, i.e.
they should not abet erosion or leaching of the soil. To complete the packet of desiderata, a
variety should afford dependable or even high yields at low production costs.
The big edge that recombinant genetics has over conventional breeding is that the
desired properties can be systematically sought, identified, extracted ("snipped") from a plant
or almost any other organism, and within a relatively short time transferred ("spliced") to
another plant. The result is the same as that achieved with conventional methods, but without
the costly and time-consuming cross-breeding they involve.
In addition, gene technology has the capability to provide growers with a greater
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diversity of hardy plant varieties by transposing properties from one species to another - a
further advantage it has over conventional methods. For example, the resistance to such and
such a pest possessed by a variety of bean can be built into maize. To a substantial number of
researchers, biotechnology - especially agricultural biotechnology - presents huge
opportunities for international development. It is obvious that the realization of these
possibilities is expected to be of substantial advantage to the farmers and hence to the rural
communities as a whole
Genetically engineered organisms have many potential applications in agriculture,
including novel foods, pesticides, and animal drugs. Our list of Agricultural Biotechnology
Research Projects suggests the variety of benefits companies have envisioned. These include,
among others, animals engineered for leaner meat, plants engineered for herbicide tolerance
or insect resistance, and bacteria engineered to produce drugs for livestock.
24.2.1. RISKS OF GENETIC ENGINEERING
Many previous Chemical technologies have proved to have adverse effects
unexpected by their developers. DDT, for example, turned out to accumulate in fish and thin
the shells of fish-eating birds like eagles and ospreys. Scientists know of no generic harms
associated with genetically engineered organisms. But specific engineered organisms may be
harmful by virtue of the novel gene combinations they possess. This means that the risks of
genetically engineered organisms must be assessed case by case and that these risks can differ
greatly from one gene-organism combination to another. The answer to this question falls
into the arena of risk assessment.
Risks of Genetic Engineering can be summed up in the following topics:
1.
2.
3.
Potential Harms to Health

Potential Environmental Harms
Risk Assessment
24.2.2. POTENTIAL HARMS TO HEALTH

Some examples of the potential adverse effects of genetically engineered organisms
may have on human health are enlisted below. Most of these examples are associated with the
growth and consumption of genetically engineered crops.
i.)
New Allergens in the Food Supply
Transgenic crops could bring new allergens into foods that sensitive individuals
would not know to avoid. An example is transferring the gene for one of the many allergenic
proteins found in milk into vegetables like carrots. Mothers who know to avoid giving their
sensitive children milk would not know to avoid giving them transgenic carrots containing
milk proteins. The problem is unique to genetic engineering because it alone can transfer
proteins across species boundaries into completely unrelated organisms.
Genetic engineering routinely moves proteins into the food supply from organisms
that have never been consumed as foods. Some of those proteins could be food allergens,
since virtually all known food allergens are proteins. Recent research substantiates concerns
about genetic engineering rendering previously safe foods allergenic. A study by scientists at
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the University of Nebraska shows that soybeans genetically engineered to contain Brazil-nut
proteins cause reactions in individuals allergic to Brazil nuts.
Scientists have limited ability to predict whether a particular protein will be a food
allergen, if consumed by humans. The only sure way to determine whether protein will be an
allergen is through experience. Thus importing proteins, particularly from nonfood sources, is
a gamble with respect to their allergenicity.
ii.)
Antibiotic Resistance
Genetic engineering often uses genes for antibiotic resistance as "selectable markers."
Early in the engineering process, these markers help select cells that have taken up foreign
genes. Although they have no further use, the genes continue to be expressed in plant tissues.
Most genetically engineered plant foods carry fully functioning antibiotic-resistance genes.
The presence of antibiotic-resistance genes in foods could have two harmful effects.
First, eating these foods could reduce the effectiveness of antibiotics to fight disease when
these antibiotics are taken with meals. Antibiotic-resistance genes produce enzymes that can
degrade antibiotics. If a tomato with an antibiotic-resistance gene is eaten at the same time as
an antibiotic, it could destroy the antibiotic in the stomach.
Second, the resistance genes could be transferred to human or animal pathogens,
making them impervious to antibiotics. If transfer were to occur, it could aggravate the
already serious health problem of antibiotic-resistant disease organisms. Although
unmediated transfers of genetic material from plants to bacteria are highly unlikely, any
possibility that they may occur requires careful scrutiny in light of the seriousness of
antibiotic resistance.
In addition, the widespread presence of antibiotic-resistance genes in engineered food
suggests that as the number of genetically engineered products grows, the effects of antibiotic
resistance should be analyzed cumulatively across the food supply.
iii.) Production of New Toxins
Many organisms have the ability to produce toxic substances. For plants, such
substances help to defend stationary organisms from the many predators in their environment.
In some cases, plants contain inactive pathways leading to toxic substances. Addition of new
genetic material through genetic engineering could reactivate these inactive pathways or
otherwise increase the levels of toxic substances within the plants. This could happen, for
example, if the on/off signals associated with the introduced gene were located on the
genome in places where they could turn on the previously inactive genes.
iv.) Concentration of Toxic Metals
Some of the new genes being added to crops can remove heavy metals like mercury
from the soil and concentrate them in the plant tissue. The purpose of creating such crops is
to make possible the use of municipal sludge as fertilizer. Sludge contains useful plant
nutrients, but often cannot be used as fertilizer because it is contaminated with toxic heavy
metals. The idea is to engineer plants to remove and sequester those metals in inedible parts
of plants. In a tomato, for example, the metals would be sequestered in the roots; in potatoes
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in the leaves. Turning on the genes in only some parts of the plants requires the use of genetic
on/off switches that turn on only in specific tissues, like leaves.
Such products pose risks of contaminating foods with high levels of toxic metals if the
on/off switches are not completely turned off in edible tissues. There are also environmental
risks associated with the handling and disposal of the metal-contaminated parts of plants after
harvesting.
v.)
Enhancement of the Environment for Toxic Fungi
Although for the most part health risks are the result of the genetic material newly
added to organisms, it is also possible for the removal of genes and gene products to cause
problems. For example, genetic engineering might be used to produce decaffeinated coffee
beans by deleting or turning off genes associated with caffeine production. But caffeine helps
protect coffee beans against fungi. Beans that are unable to produce caffeine might be coated
with fungi, which can produce toxins. Fungal toxins, such as aflatoxin, are potent human
toxins that can remain active through processes of food preparation.
vi.) Unknown Harms to Health
As with any new technology, the full set of risks associated with genetic engineering
have almost certainly not been identified. The ability to imagine what might go wrong with a
technology is limited by the currently incomplete understanding of physiology, genetics, and
nutrition.
24.2.3. POTENTIAL ENVIRONMENTAL HARMS
a) INCREASED WEEDINESS
One way of thinking generally about the environmental harm that genetically
engineered plants might do is to consider that they might become weeds. Here, weeds mean
all plants in places where humans do not want them. The term covers everything from
Johnson grass choking crops in fields to kudzu blanketing trees to Melaleuca trees invading
the Everglades. In each case, the plants are growing unaided by humans in places where they
are having unwanted effects. In agriculture, weeds can severely inhibit crop yield. In
unmanaged environments, like the Everglades, invading trees can displace natural flora and
upset whole ecosystems.
Some weeds result from the accidental introduction of alien plants, but many were the
result of purposeful introductions for agricultural and horticultural purposes. Some of the
plants intentionally introduced into the United States that have become serious weeds are
Johnson grass, multiflora rose, and kudzu. A new combination of traits produced as a result
of genetic engineering might enable crops to thrive unaided in the environment in
circumstances where they would then be considered new or worse weeds. One example
would be a rice plant engineered to be salt-tolerant that escaped cultivation and invaded
nearby marine estuaries.
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b) GENE TRANSFER TO WILD OR WEEDY RELATIVES
Novel genes placed in crops will not necessarily stay in agricultural fields. If relatives
of the altered crops are growing near the field, the new gene can easily move via pollen into
those plants. The new traits might confer on wild or weedy relatives of crop plants the ability
to thrive in unwanted places, making them weeds as defined above. For example, a gene
changing the oil composition of a crop might move into nearby weedy relatives in which the
new oil composition would enable the seeds to survive the winter. Over wintering might
allow the plant to become a weed or might intensify weedy properties it already possesses.
c) CHANGE IN HERBICIDE USE PATTERNS
Crops genetically engineered to be resistant to chemical herbicides are tightly linked
to the use of particular chemical pesticides. Adoption of these crops could therefore lead to
changes in the mix of chemical herbicides used across the country. To the extent that
chemical herbicides differ in their environmental toxicity, these changing patterns could
result in greater levels of environmental harm overall. In addition, widespread use of
herbicide-tolerant crops could lead to the rapid evolution of resistance to herbicides in weeds,
either as a result of increased exposure to the herbicide or as a result of the transfer of the
herbicide trait to weedy relatives of crops. Again, since herbicides differ in their
environmental harm, loss of some herbicides may be detrimental to the environment overall.
d) SQUANDERING OF VALUABLE PEST SUSCEPTIBILITY GENES
Many insects contain genes that render them susceptible to pesticides. Often these
susceptibility genes predominate in natural populations of insects. These genes are a valuable
natural resource because they allow pesticides to remain as effective pest-control tools. The
more benign, the pesticide, the more valuable the genes that make pests susceptible to it.
Certain genetically engineered crops threaten the continued susceptibility of pests to
one of nature's most valuable pesticides: the Bacillus thuringiensis or Bt toxin. These "Bt
crops" are genetically engineered to contain a gene for the Bt toxin. Because the crops
produce the toxin in most plant tissues throughout the life cycle of the plant, pests are
constantly exposed to it. This continuous exposure selects for the rare resistance genes in the
pest population and in time will render the Bt pesticide useless, unless specific measures are
instituted to avoid the development of such resistance.
e) POISONED WILDLIFE
Addition of foreign genes to plants could also have serious consequences for wildlife
in a number of circumstances. For example, engineering crop plants, such as tobacco or rice,
to produce plastics or pharmaceuticals could endanger mice or deer who consume crop debris
left in the fields after harvesting. Fish that have been engineered to contain metalsequestering proteins (such fish have been suggested as living pollution clean-up devices)
could be harmful if consumed by other fish or raccoons.
f) CREATION OF NEW OR WORSE VIRUSES
One of the most common applications of genetic engineering is the production of
virus-tolerant crops. Such crops are produced by engineering components of viruses into the
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plant genomes. For reasons not well understood, plants producing viral components on their
own are resistant to subsequent infection by those viruses. Such plants, however, pose other
risks of creating new or worse viruses through two mechanisms: recombination and
transcapsidation.
Recombination can occur between the plant-produced viral genes and closely related
genes of incoming viruses. Such recombination may produce viruses that can infect a wider
range of hosts or that may be more virulent than the parent viruses.
Transcapsidation involves the encapsulation of the genetic material of one virus by
the plant-produced viral proteins. Such hybrid viruses could transfer viral genetic material to
a new host plant that it could not otherwise infect. Except in rare circumstances, this would
be a one-time-only effect, because the viral genetic material carries no genes for the foreign
proteins within which it was encapsulated and would not be able to produce a second
generation of hybrid viruses.
g) UNKNOWN HARMS TO THE ENVIRONMENT
As with human health risks, it is unlikely that all potential harms to the environment
have been identified. At this point, biology and ecology are too poorly understood to be
certain that question has been answered comprehensively. This leaves a greater scope for the
risks in environment.
24.2.4. RISK ASSESSMENT
Having identified a list of possible harms that might occur as a result of using or
releasing genetically engineered organisms, the next question is how likely are any of these to
occur? Like the original "brainstorming" of potential harms, the answer to this question
depends greatly on how well the organisms and their interaction in the environment are
understood. Risks must be assessed case by case as new applications of genetic engineering
are introduced. In some circumstances, it is possible to assess risks with great confidence. For
example, it is vanishingly unlikely that genetically engineered palm trees will thrive in the
Arctic regardless of what genes have been added. But for many potential harms, the answers
are far less certain.
Risk assessments can be complicated. Because even rigorous assessments involve
numerous assumptions and judgment calls, they are often controversial when they are used to
support particular government decisions. For example, the approval of the first genetically
engineered squash by the United States Department of Agriculture involved a controversial
risk assessment.
Under the current US regulatory framework for biotechnology, some sort of risk
assessment is routinely produced before decisions to allow commercialization of products
under the Federal Plant Pest Act; the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA); and the Toxic Substances Control Act (TSCA). In the case of the Plant Pest Act,
risk assessments are done according to the procedure specified by the National
Environmental Policy Act (NEPA). Under NEPA, risk assessments could lead to full-blown
environmental impact statements, but so far all evaluations of engineered agricultural
organisms have led to the legal conclusion that no environmental impact statement is needed.
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For the most part, risk assessments are done by scientists and policymakers in the
relevant agencies (USDA or EPA) with information provided by the companies seeking the
approvals. The public often has a brief opportunity to review and comment on the risk
assessments.
There is no standard set of questions that risk assessments must answer because of the
great range of potential impacts of biotechnology products. A risk assessment for a microbial
pesticide, for example, would be substantially different from a risk assessment for genetically
engineered salmon. Like all efforts at risk evaluation, risk assessments done for regulation
depend on the base of scientific knowledge for generation of list of possible harms to be
assessed.
Assessment of these risks and the policies ad procedures adopted to ensure
environmentally safe applications of biotechnology are the concerns biosafety regulations.
Originally biosafety concerns were primarily focused on safety procedures for the rDNA
work within the laboratory but the scope of evaluation of possible risks has further widened
following field releases of these products.
24.3. BIOSAFETY REGULATIONS
The key elements of biosafety regulatory framework includes national policies, a set
of regulations governing risks assessment and risk management, capacity and systems to
monitor, inspect and implement regulations.
24.3.1. BIOSAFETY REGULATIONS IN VARIOUS COUNTRIES
USA: it involves three government agencies
a. Environmental protection agency (EPA)
b. United states department of Agriculture (USDA)
c. Federal drug administration (FDA)
EPA takes lead role in commercialization of the process. If the transgenic process is
herbicide tolerant, then EPA regulates the herbicide but not the transgene and USDA
is the lead agency. USDA grants import permits and field trial authorization. FDA
focus is on whether or not introduced transgene made the new food substantially
different and safe for conception. FDA also regulates approval in case of recombinant
DNA products.
EUROPEAN UNION:
The European Union introduced community biotechnology legislation in the 1990s
as part of an effort to address the issues of GMOs and genetically modified microorganisms
(GMMs). The EU framework is implemented by the EU institutions (the European
commission, the European parliament and council of ministers) and the member countries
(national competent authorities).
Approval for commercialization of products require
i. Case- case environmental and health risk assessment
ii. Step-step authorization procedure after notification has been received.
The EU system requires mandatory labeling of GM foods containing novel DNA/protein.
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24.3.2. REGULATORY FRAMEWORK IN INDIA
India has notified for the manufacture, import, testing after use etc., of GM products
in 1989 under environmental protection act, 1986. Keeping in view the potential risks to
humans and the environment by the indiscriminate use of GMOs and their products, Govt. of
India has also evolved regulatory mechanisms for their development and evaluation.
24.4. GOVERNMENT RULES FOR GMO
The ministry of environment and forests had enacted environment and Protection act
in 1986 to provide the protection and improvement of the environment and related matters.
Under this act, the rules and procedures for the manufacture, import, use, research and release
of GMOs s well as products made by the use of such organisms were notified. These rules
also defined the competent authorities and composition of such authorities for handling of
various aspects of the rules. Presently there are six competent authorities as per the rules,
brief description of their broad responsibilities is given below.
i)
ii)
iii)
iv)
v)
The recombinant DNA advisory committee(RDAC)

The RDAC prepares recommendations, from time to time that are suitable for
implementation/upholding of the safety regulations in research and applications of
GMOs and products thereof. This committee prepared the first Indian recombinant DNA
Biosafety Guideline in 1990, which is adopted by the government for conducting research
and handling of GMOs in India.
Institutional Biosafety committee (IBSC)
IBSC is the nodal point for interaction within the institution for implementation of the
guidelines. As such, in the first instance, it is necessary that the institutions intending to
carry out research activities involving genetic manipulations of micro organisms, plants
and animals should constitute the IBSC. The activities IBSC also include training of
personnel on Biosafety and instituting health monitoring program for laboratory
personnel. The directives are to carry out medical checks including pathological tests
done periodically on persons involved in the work/experiments.
Review committee on genetic manipulation (RCGM)
The RCGM under the Department of Biotechnology has the following functions:
a. To review all approved ongoing research projects involving highg risk category
and controlled experiments.
b. To undertake visits of sites of experimental facilities periodically, where projects
with biohazard potentials are being pursued and also at a time prior to the
commencement of the activity to ensure that adequate safety measures are taken
as per the guidelines.
c. To issue clearance for import of etiological agents and vectors, germ plasm,
organelle etc.., needed for experimental work/training and research.
Genetic engineering Approval committee (GEAC)
GEAC is responsible for approval of activities involving large scale use of hazardous
microorganisms and recombinant products in research and industrial production from
the environment angle.
State Biotechnology Coordination committee (SBCC)
This committee is headed by the chief secretary of the state. The committee
has powers to inspect, investigate and take punitive action in case od violation of the
statuatory provisions. The committee coordinates the activities related to GMOs in the
state with the Central ministries.
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vi)
District level committee (DLC)

This committee is the smallest authoritative unit to monitor the safety regulations
in installations engaged in the use of GMOs in research and applications.
24.5. LET US SUM UP
Despite the benefits of Genetic engineering, there are a number of unfavorable

outcomes which hinder the commercialization of the engineered products.
This includes productions of allergens, newer toxins, mutated viruses, and change in
the usage and prevalence pattern of the organisms due to the introduction of the new
species.
To control such works there are regulatory agencies within a country and all over the
world. They regulate the production and commercialization of transgenic organisms.

1. Differentiate between the natural breeding and biotechnology based breeding.
2. Analyse the implication of genetic engineering on health.
1. Enlist the potential risk factors involved in the usage of genetically modified organisms.
(GMO)
2. Give an account on the regulatory set up on Biosafety in India.
24.8. REFERENCES
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LESSON 25: ETHICAL ASPECTS OF GENETIC TESTING
CONTENTS
25.1. INTRODUCTION
25.2. GENETIC TESTING
25.3. NON-DNA BASED GENETIC SCREENING
25.4. FAMILY MEDICAL HISTORIES
25.5. APPLICATIONS OF GENETIC TESTING IN THE CONTEXT OF
EMPLOYMENT
25.6. GENES AND DISEASE
25.7. METHODOLOGY FOR GENETIC TESTING
25.8. METHODOLOGY FOR GENETIC MONITORING
25.9. VALIDITY, RELIABILITY AND PREDICTABILITY OF GENETIC TESTS
25.10. POSSIBLE USES OF GENETIC INFORMATION WHICH COULD HARM
INDIVIDUALS
25.11. A POSSIBLE CONSEQUENCE OF CONCERN ABOUT DISCRIMINATION
25.15. REFERENCES
The lesson discusses the ethical issues related to the genetic testing methods available
in the recombinant technology.
25.1. INTRODUCTION
Genetic testing technique plays a vital role in solving many disputes such as it helps
in diagnosing the genetic disorders, parental disputes etc.
25.2. GENETIC TESTING
A persons DNA comprises the genetic information that, within the constraints
imposed by all the environmental influences to which that person is exposed, governs the
growth, development and resulting characteristics of that person. It is becoming increasingly
easy to obtain information regarding particular aspects of someones genetic status by testing
for the presence of particular variant forms of genes or by microscopic examination of their
chromosomes. The information obtained from such genetic tests may sometimes be used to
anticipate the onset of certain genetically determined diseases and to initiate appropriate early
therapy or other anticipatory action.
Genetic testing is also being used at the present time, in the investigation of crime and
in paternity identification. The predictive value of some genetic information means, however,
that some insurance companies would like to access this, in order to obtain what they believe
to be a more accurate assessment of risk for life and health insurance purposes. This Opinion
focuses in particular on employers interest in genetic testing as a way to predict an
employees future health or their susceptibility to particular hazards in the working
environment (genetic screening). They might also wish to monitor the genetic status of an
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employee who is at risk of exposure to agents known to cause genetic damage (genetic
monitoring).
25.3. NON-DNA BASED GENETIC SCREENING
The definition of genetic screening need not be restricted to tests carried out directly
on an individuals DNA. Many genes are coded instructions for making particular proteins.
Others may regulate the timing or quantity of protein synthesised. Consequently it may be
possible in some cases to access indications of genetic status by measuring the activity of a
protein or some of its products. For the purposes of this Opinion, any test that evaluates
specific genes or gene products that may be indicators of a persons genetic status are
considered to be genetic tests.
25.4. FAMILY MEDICAL HISTORIES
Family medical histories can give an indication of possible genetic status with regard
to susceptibility to some diseases and are routinely used by some insurance companies for
persons applying for health insurance. In so far as family medical histories can provide
information on a persons genetic status, which may have a predictive value for future health
as significant as a laboratory performed genetic test, these are included within our definition
of genetic tests.
25.5.
APPLICATIONS OF GENETIC
EMPLOYMENT
TESTING
IN
THE CONTEXT
OF
25.5.1. GENETIC SCREENING AS AN INDICATOR OF FUTURE HEALTH

It is clear that a persons genetic constitution plays a role in their susceptibility to a
variety of diseases. That is not to say that genes are the sole determinant of disease
susceptibility as this may be modulated by environmental, lifestyle, dietary and perhaps other
serendipitous factors. Nevertheless, it is possible that investigation of an individuals genetic
constitution for the presence or absence of particular gene variants might provide some
indication of the likelihood of the individual contracting, in the future, a particular disease.
Employers, either current or prospective, could have an interest in the results of such
genetic screening in so far as these might be a predictor of the future health of an employee,
particularly if they were to imply possible levels of future absenteeism or low work rate
which might impact on profitability. An employee who develops heart disease, for example,
would certainly be likely to require periods of absence from work and, in certain occupations,
might not be able to sustain a normal work rate.
There is also the possibility that sudden onset of a disease condition might result in a
hazard for the employee, other employees or the public. An employer could use the results of
such tests to exclude job applicants on the basis of predicted future health. This type of
genetic testing, where there is no reason to suspect that the employee might possess any
particular genetic constitution, is generally referred to as genetic screening.
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25.5.2. GENETIC SCREENING AS AN INDICATOR OF SUSCEPTIBILITY TO
OCCUPATIONAL HAZARDS
Employers could also have an interest in whether the genetic profile of an employee
might endow them with greater or lesser susceptibility to occupational hazards, such as the
presence of toxic, mutagenic or carcinogenic materials in the workplace environment at
levels below those that are recognized as acceptable. Variants of genes that affect the
metabolism of such genotoxic agents can result in variations in individuals ability to activate
or inactivate these.
Other genes may control the repair of genetic damage. Occupational factors have been
shown to be associated with 10% of cases of adult asthma. Asthma is a polygenic disease in
which many gene environment interactions are involved. Other diseases having an
occupational component and with a known genetic basis include beryllium allergy and
chronic obstructive pulmonary disease resulting from _-1-antitrypsin (AAT) deficiency.
An employer might wish to use such information to deploy workers in areas
appropriate to their particular genetic make up or to exclude them from employment.
Information from this type of genetic screening might also be of value to the employee. Being
aware of the likely level of their own susceptibility to a particular workplace hazard would
permit them to make informed decisions with regard to the type of work they would seek for
their own long-term health and safety.
25.5.3. GENETIC MONITORING
Even in the best regulated employment environment, it may not be possible to ensure
total elimination of the presence of all traces of chemicals or irradiation that could damage a
persons genetic material. In such circumstances it is possible to monitor cells of a potentially
exposed person for genetic damage. Such tests may impact not only on the well-being of the
individual, but also on the next generation. The results of genetic monitoring could reveal a
hitherto unappreciated risk to health and hence is of public health relevance. In this Opinion
this type of genetic testing is referred to as genetic monitoring.
25.6. GENES AND DISEASE
25.6.1. MONOGENIC AND POLYGENIC DISEASES
Although there are many diseases with a recognized genetic component resulting
from a defect in a single gene (monogenic diseases), as a general rule the incidence of such
diseases is low. Monogenic diseases include cystic fibrosis, sickle cell anaemia, Huntingtons
disease and Haemophilia. Cystic fibrosis and sickle cell anaemia are examples of autosomal
recessive diseases, where the relevant genes are carried on one of the 22 pairs of human
chromosomes that are not sex-specific. This means that a copy of the defective gene must be
inherited from both parents if the disease is to be manifest. A person carrying a normal copy
of the gene from one parent and a defective copy from the other are carriers of the disease
gene but do not usually manifest symptoms of the disease.
Huntingtons disease is an example of an autosomal dominant disease. In this case
only a single copy of the defective gene coming from either parent is required for
development of the disease. Haemophilia is an example of a disease resulting from a defect in
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an X-linked gene. The sex specific chromosome pair comprises two X chromosomes (female)
or an X and a Y chromosome (male). As males have only a single X chromosome, they are
more likely to develop such a disease than females with two X chromosomes, as the recessive
gene variant on one of the X chromosomes will not be expressed in the presence of the
normal gene on the other X chromosome.
In contrast to the above examples of diseases resulting from defects in a single gene,
other human diseases with a genetic component are thought to result from interactions
between several genes (polygenic diseases). The incidence of some polygenic diseases is very
high. In most of these cases the genetic basis is incompletely understood and is complicated
by influences of environment, diet and lifestyle. Examples of such polygenic diseases are
heart disease, several cancers and some allergies.
25.6.2. FACTORS AFFECTING DEVELOPMENT OF HEREDITARY DISEASE
The possession of one or more genetic defects does not necessarily dictate that the
person possessing the defect will develop the disease. For example, a woman with the
BRCA1 breast cancer susceptibility gene has an 80% risk of developing breast cancer by age
65. The expressivity of a genetic defect describes the different severities of disease from
which various possessors of the defect may suffer. Penetrance and expressivity, as well as the
timing of disease onset, may be affected by environmental factors, life style or the presence
of a range of other genes.
25.7. METHODOLOGY FOR GENETIC TESTING
Defective versions of a gene may be altered only very slightly in DNA sequence from
the normal version of the gene. Genetic screening aims to identify such small changes in the
gene in question. Only a very small amount of DNA is required for genetic screening. As an
example, the DNA region required is usually amplified thousands of times using the
polymerase chain reaction (PCR). A fluorescent label is then attached to the amplified DNA.
The fluorescence-labelled DNA is then passed through a filter to which is attached a small
length of DNA (the probe) containing a sequence characteristic of the version which is
being screened for. If the version in question is present in the fluorescent amplified DNA then
this will bind to the probe, showing up as fluorescence on the filter. By attaching several
different probes at different points on the filter it is possible to screen for the presence of
several different versions of the gene at the same time. Developments in DNA microarray
technology are likely to make it possible to screen for large numbers of genes with variant
forms simultaneously.
Where a genetic screen involves identification of a protein altered in concentration as a result
of a particular defective gene, the strategy used varies with the protein in question. It may be
recognized using electrophoretic identification, an antibody probe or by an enzyme assay for
example.
25.8. METHODOLOGY FOR GENETIC MONITORING
In contrast to genetic screening, genetic monitoring usually involves microscopic
examination of the karyotypes (chromosome patterns) of white blood cells. Indication of
unacceptable levels of exposure to genotoxic agents comes from observations of changes in
chromosome structures including chromosome breakage, inversion or deletion of sections of
chromosomes or translocation of a part of a chromosome to a different chromosome.
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Recently developed technology permits the observation of quite small alterations in
chromosome structures.
25.9. VALIDITY, RELIABILITY AND PREDICTABILITY OF GENETIC TESTS
25.9.1. VALIDITY OF GENETIC TESTS
At the present time, very few genetic test are available that give information to either
an employer or an employee which could validly be used in the context of decisions
concerning employment. It is likely that this situation may change in the future although it is
difficult to predict the pace of such change. Where employment is linked to health or life
insurance, employers may come under pressure from insurance companies to implement
genetic screening to assess the level of risk to which the employee might be exposed. Validity
of a genetic test would require demonstration of
1) its relevance to health protection of workers,
2) the reliability and reproducibility of the test and
3) the level of predictive value for the test.
In some countries, there are statutory bodies that may be asked to rule on the validity of
genetic tests in particular circumstances.
25.9.2. RELEVANCE OF GENETIC TESTS
At the present time, it is difficult to make a case for any genetic tests to be carried out
as indicators of future health in terms of their relevance to employment. Genetic screening for
susceptibility to workplace environmental hazards clearly has some precautionary relevance
but in many cases the link between a particular genetic status and susceptibility to a particular
hazard has only a theoretical basis at present. In the general debate there have been
exaggerated beliefs about the predictive value of genetic tests, perhaps based on the concept
of genetic determinism, which have been proved to lack foundation. On the other hand,
where there is a possible risk of genetic damage to an employee resulting from exposure to
workplace contaminants, genetic monitoring for chromosomal changes resulting from this
exposure may have a very clear relevance for the health of an employee.
25.9.3. RELIABILITY OF GENETIC TESTS
In such a sensitive area, it is obviously extremely important that procedures for
genetic testing are as reliable as possible, as provision of incorrect information to an
employer or employee could have far reaching consequences. All stages of a scientifically
satisfactory testing procedure should have built in negative and positive controls to ensure the
reliability of the test result. Good laboratory practice would be observed at all times,
including detailed documentation of procedures and results. Even when testing procedures
are optimized, false negatives and false positives will emerge and validation procedures for
the tests may be required.
25.9.4. PREDICTIVE VALUE OF GENETIC TESTS
Even for monogenic diseases, predictive value of genetic testing may be limited.
There is always a possibility that the disease in question might not manifest itself during the
working life of the individual and it is not always possible to predict the severity of the future
disease. The situation is even more complex where diseases with a polygenic basis are
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concerned. At the present time it is virtually impossible accurately to predict, using genetic
tests, either whether the disease will develop at all or, if it does, its timing and severity. Even
if the genetic basis of such diseases becomes fully understood, environmental and lifestyle
factors, which may themselves be unpredictable, will limit the predictability of disease
development. Testing protocols that are less than 100% accurate (most of the cases) will
reduce predictive value still further. False negatives would result in possible exposure of a
susceptible individual to unacceptable levels of occupational hazard whilst false positives
could result in unjustified exclusion of non-susceptible individuals from employment.
25.9.5. STIGMATIZATION AND DISCRIMINATION WITHIN SOCIETY
Stigmatization relates to the perception of an individual by others that may result, for
example, in an individual being spoken of adversely or considered undesirable as a friend or
as a marriage partner by members of a community. The term discrimination can be used to
simply mean the recognition of differences between people, but is more often used to refer to
the unfair treatment of individuals based on such a perceived or actual difference. The
following discussion refers to unfair discrimination rather than discrimination so that the
intended meaning is clear. It also focuses on discrimination rather than stigmatization as the
former represents the mechanism that may result in harm to individuals and families. Genetic
tests can reveal that people have, or have a genetic predisposition to, a disorder.
Stigmatization and discrimination could occur within many aspects of everyday life,
with regard to family relationships, insurance and employment. They already occur for those
who are considered different or disabled. Genetic information, by adding another reason
for such treatment, has the potential to increase the number of people so affected. There are
two basic types of discrimination: direct (where a person with a disability is treated less
favorably than he/she would have been had they not been disabled); and indirect
(requirements or conditions which do not take into account the particular needs a person with
a disability may have in a given situation). There are many situations for potential unfair
discrimination. There are legal assistances that prohibit many forms of discrimination against
people on the grounds of disability. However, for this legislation to protect people against
discrimination on the grounds of genetic condition, the definition of disability would need
to cover genetic pre symptomatic, susceptibility and carrier status.
25.10. POSSIBLE USES OF GENETIC INFORMATION WHICH COULD HARM
INDIVIDUALS
In this section, several situations are mentioned to highlight how genetic information
could potentially be used in ways that would harm individuals. While some are real issues
now, others are more speculative.
1. THE FAMILY
Revealing genetic test results to family members can alter the way in which an
individual is perceived and treated by the family, for better or worse. For example, early-age
onset Alzheimers disease (dementia) is sometimes caused by a mutation in a dominantly
inherited gene. If a man is shown to have inherited such a mutated gene, his siblings could
provide emotional support in the short-term and practical help and care once the dementia
begins to limit their brothers functioning. In contrast, they could begin to withdraw by
reducing contact with their brother and his family. Another sibling who was also tested and
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shown not to have inherited the gene, may feel guilty that he/she has escaped while the
affected brother has not, and may therefore feel an obligation to care for him as his dementia
worsens. There are many uses of genetic testing, discussed in earlier sections of this
document, which can lead to improved health care and provide people with information they
wish to have. The genetic register is a tool for maximizing the health benefits of genetic
information for families.
2. INSURANCE
In the past, life insurance companies obtained genetic information by asking questions
about the health and causes of death of close family members, and such information was used
to determine eligibility for insurance. Recently, as genetic testing for many disorders has
become available, insurance companies have stated that if a person has already had a genetic
test, this fact must be disclosed to the insurer when applying for life insurance. The insurer
may then request the test result if it believes the information is relevant to its decision
whether to provide insurance and if so, whether to do so at standard rates. Insurers will not
ask applicants to undergo genetic tests. In the future, genetic testing could become, in certain
circumstances, a requirement in order to obtain insurance eg for very large policies. These
developments have raised concerns that unfair discrimination could occur if genetic test
information is used inappropriately by insurers. In some instances, the need to disclose that a
genetic test has been performed may cause some people to forego the personal health benefits
of genetic testing in order to access life insurance. On the other hand, those at risk of a
genetic disorder who are tested and shown not to have inherited their familys genetic
predisposition may be keen to reveal that information in order to obtain insurance at standard
rates when they would otherwise have been unable to obtain insurance.
Of particular concern would be the use of genetic information to determine eligibility
for basic health insurance. Current health insurance provisions in Australia rest upon the
notion of community rating and persons cannot be refused private health insurance on the
basis of present or possible future health status, although there are waiting times for preexisting conditions. Social policy related to health insurance may change over time in
Australia such that while genetic information revealed now may not have an adverse impact
on access to health insurance at this point in time, it may do so in the future. To have had
genetic testing may have negative consequences for Australians who move overseas to
countries where genetic test results are used to determine eligibility for health insurance.
3. EMPLOYMENT
Employers may seek genetic information about employees for a variety of reasons.
They may wish to identify existing or potential employees whose genetic make-up places
their health at risk in a particular work environment and to protect them from the potential
harm. The number of known situations in which the work environment is harmful because of
genetic susceptibility is very small but includes the carrier state of sickle cell anaemia and
work at high altitudes, and glucose-6- phosphate dehydrogenase deficiency and work where
environmental oxidants are present. Employers may also decide not to employ people with
certain genetic traits in particular work circumstances. Genetic information about future
health could potentially be used to distinguish between job applicants in terms of their ability
to remain productive, to take a minimum of sick leave, to not require a special work
environment and to not increase the employers superannuation costs. Such use of genetic
information is considered to represent unfair discrimination. Then, an employer would be
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entitled to discriminate against a person who would be unable to carry out the inherent
requirements of the particular employment because of the nature of the disability. Thus, for
example, existing legal provisions may not prevent an employer from discriminating against
a young person who has had a test revealing the presence of a gene for Huntington disease,
Alzheimer disease or other late onset disorders. Although the individual may not develop
symptoms for many years, if at all, the nature of the disorder could prevent the individual
from carrying out the inherent requirements of the employment, thus allowing discrimination
by the employer.
4. FINANCE
The possibility of an indirect use of genetic information to determine access to
finance is highlighted by the situation in the United Kingdom, where many home loans
require a life insurance policy as collateral. It should be noted that United Kingdom insurers
do not require genetic information, at the present time, for mortgages below a certain sum. If
the financial institutions started to require life insurance as collateral for a home loan, and
given that life insurers require disclosure of genetic test results, genetic information could
then become the basis of unfair discrimination.
5. ADOPTION
A childs genetic status may have significant and long-term implications for both a
child and his/her adoptive parents. For the present, adoptive parents receive certain personal
and family health information about a childs biological parents and this is considered
desirable, primarily to assist them in making provision for the future health care of the child.
It is recognized that providing such information could potentially influence whether or not
the adoption proceeds. Some information, such as the result of predictive genetic testing of a
biological parent would not be given to the adoptive parents (unless the biological parent
expressly requested it) as to do so would breach his/her privacy; the fact that a particular
disorder is present in the family would be revealed but not the predictive test result. Some
may argue that when a biological parent has a personal or family history of a heritable genetic
disorder, predictive or susceptibility genetic testing should be performed on a child prior to
adoption in order to determine the childs suitability for adoption. The more generally held
view is that the use of genetic information in such a way would represent disrespect for the
worth of the child as a human person, would fail to respect the childs autonomy to choose
whether or not to be tested on reaching adulthood, and would breach the childs genetic
privacy. It also ignores the fact that the results of genetic testing will, in most cases, reflect
possibilities rather than certainties.
6. HEALTH CARE
Genetic information could potentially be used to determine eligibility for certain
health services. For example, a personal history of a genetic disorder, family history of a
genetic disorder or evidence of predisposition to a genetic disorder could influence eligibility
for assisted reproductive technology and access to donated gametes or organs.
7. FORENSIC AND LEGAL SETTINGS
The sequence of bases in an individuals DNA is unique and as such, provides a
means for unambiguous identification of that individual. DNA fingerprinting/ profiling
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can be used to establish or exclude paternity, and to identify a person who is deceased or who
has committed a crime. Australian governments are moving to establish a national DNA
criminal investigation database system for forensic use.
8. GOVERNMENTS AND GOVERNMENT AGENCIES
Governments could potentially use genetic information to determine eligibility for
health services in certain circumstances. For example, a government could make the
availability of financial support for those with genetic susceptibility contingent on adherence
to prevention strategies, or make those who are unwilling to use prenatal diagnosis bear the
full cost of care of affected children. Governments, as policy makers and sources of funding,
could also require and misuse genetic information as a means of eugenic practice. Individuals
or groups of individuals could be disadvantaged economically, socially and politically
because they have, or do not have, a certain genetic profile. The use of genetic information in
these ways would be ethically unacceptable.
9. PSYCHOLOGICAL DIFFICULTIES
Disclosure of an individuals genetic information to others may cause them to treat
him or her differently. As a direct consequence this may result in psychological problems for
the individual, or there may be an indirect effect if the individuals own perception of
him/herself is changed for the worse.
25.11. A POSSIBLE CONSEQUENCE OF CONCERN ABOUT DISCRIMINATION
Concern about the possibility of stigmatization or discrimination could discourage a
person from taking advantage of the medical benefits of obtaining genetic information, with
consequent adverse effects on his/her health. This could have effects for current and future
family members as well as for the individual. The chance that this outcome will occur can be
minimized if there is legislation to prevent unfair discrimination based on the results of
genetic testing, and if the community is educated about the potential benefits of genetic
testing and has factual information about the circumstances in which it is necessary for test
results to be revealed.
Genetic testing includes investigating ones genetic profile for the presence/absence
of genetic factors that might reveal the susceptibility of identification of a person
against a crime, a disease and certain inherent traits of clinical importance.
Ethical issues relating to use of genetic information includes stigmatization of persons
for the presence of genes that might cause the disease in the latter stages.
It also includes problems at work place that influences the career of the person and
also family related issues.

1. Substantiate the benefits of genetic screening.
2. Establish how discrimination is possible in genetic testing method.
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1. Give an account of genetic testing in individuals.
2. Give the possible benefits of genetic testing.
3. Enlist the possible risks that a person might undergo if his/her genetic data is published.
25.15. REFERENCES
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LESSON 26: ETHICAL ISSUES RELATED TO BIOTECHNOLOGY
CONTENTS
26.1. INTRODUCTION
26.2. USE OF INFORMATION - INTELLECTUAL PROPERTY RIGHTS
26.3. ETHICAL ISSUES RELATING TO BIOTECHNOLOGY
26.4. LET US SUM UP
26.7. REFERENCES
The chapter deals with ethical issues related to biotechnology.
26.1. INTRODUCTION
Biotechnology is a wide field of interest next to the information technology for human
regarding the benefits. Even though there is a broad application for biotechnology in
developing various genetic engineering products, there is both advantages and disadvantages.
In the unit, the ethical issues that are raised against biotechnology are discussed.
26.2. USE OF INFORMATION - INTELLECTUAL PROPERTY RIGHTS
Intellectual property laws confer a bundle of exclusive rights in relation to the
particular form or manner in which ideas or information are expressed or manifested, and not
in relation to the ideas or concepts themselves (see idea-expression divide). The term
"intellectual property" denotes the specific legal rights which authors, inventors and other IP
holders may hold and exercise, and not the intellectual work itself.
Intellectual property laws are designed to protect different forms of subject matter, although
in some cases there is a degree of overlap.
Copyright may subsist in creative and artistic works (e.g. books, movies, music,
paintings, photographs, and software) and give a copyright holder the exclusive right to
control reproduction or adaptation of such works for a certain period of time (historically a
period of between 10 and 30 years depending on jurisdiction, more recently the life of the
author plus several decades).
A patent may be granted for a new, useful, and non-obvious invention, and gives the
patent holder a right to prevent others from practicing the invention without a license from
the inventor for a certain period of time (typically 20 years from the filing date of a patent
application).
A trademark is a distinctive sign which is used to distinguish the products or services

of different businesses.
An Industrial design right protects the form of appearance, style or design of an

industrial object (e.g. spare parts, furniture, or textiles).
A Trade Secret (which is sometimes either equated with, or a subset of, "confidential
information") is secret, non-public information concerning the commercial practices or
proprietary knowledge of a business, public disclosure of which may sometimes be illegal.
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Patents, trademarks, and designs rights are sometimes collectively known as industrial
property, as they are typically created and used for industrial or commercial purposes.
i) COPYRIGHT
Copyright is a set of exclusive rights that regulate the use of a particular expression of
an idea or information. At its most general, it is literally "the rights to copy" an original
creation. In most cases, these rights are of limited duration. The symbol for copyright is "",
and in some jurisdictions may alternatively be written as either (c) or (C).
Copyright may subsist in a wide range of creative, intellectual, or artistic forms or
"works". These include poems, theses, plays, and other literary works, movies, choreographic
works (dances, ballets, etc.), musical compositions, audio recordings, paintings, drawings,
sculptures, photographs, software, radio and television broadcasts of live and other
performances, and, in some jurisdictions, industrial designs. Designs or industrial designs
may have separate or overlapping laws applied to them in some jurisdictions. Copyright is
one of the laws covered by the umbrella term intellectual property.
Copyright law covers only the form or manner in which ideas or information have
been manifested, the "form of material expression". It is not designed or intended to cover the
actual idea, concepts, facts, styles, or techniques which may be embodied in or represented by
the copyright work. For example, the copyright which subsists in relation to a Mickey Mouse
cartoon prohibits unauthorized parties from distributing copies of the cartoon or creating
derivative works which copy or mimic Disney's particular anthropomorphic mouse, but does
not prohibit the creation of artistic works about anthropomorphic mice in general, so long as
they are sufficiently different to not be deemed imitative of the original. In some
jurisdictions, copyright law provides scope for satirical or interpretive works which
themselves may be copyrighted. Other laws may impose legal restrictions on reproduction or
use where copyright does not - such as trademarks and patents.
Copyright laws are standardized through international conventions such as the Berne
Convention in some countries and are required by international organizations such as
European Union or World Trade Organization from their member states.
For eg., books published by the companies.
ii) PATENT
A patent is a right granted for any device, substance, method or process which is new,
inventive and useful.
A patent is legally enforceable and gives the owner the exclusive right to
commercially exploit the invention for the life of the patent.
Patents give effective protection if you have invented new technology that will lead to
a product, composition or process with significant long-term commercial gain.
In return, patent applicants must share their know-how by providing a full description of how
their invention works. This information becomes public and can provide the basis for further
research by others.
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There are four primary incentives embodied in the patent system: the incentive to
invent in the first place; the incentive to disclose the invention once made; the incentive to
invest the sums necessary to experiment, to produce, and finally get the invention on the
market; and the incentive to design around and improve upon earlier patents.
1.
Patents provide incentives for economically efficient research and development
(R&D). Many large modern corporations have annual R&D budgets of hundreds of millions
or even billions of dollars. Without patents, R&D spending would be significantly less or
eliminated altogether, limiting the possibility of technological advances or breakthroughs.
Corporations would be much more conservative about the R&D investments they made, as
third parties would be free to exploit any developments.
2.
In accordance with the original definition of the term "patent," patents facilitate and
encourage disclosure of innovations into the public domain for the common good. If
inventors did not have the legal protection of patents, in many cases, they would prefer or
tend to keep their inventions secret. Awarding patents generally makes the details of new
technology publicly available, for exploitation by anyone after the patent expires, or for
further improvement by other inventors. Furthermore, when a patent's term has expired, the
public record ensures that the patentee's idea is not lost to humanity.
3.
In many industries (especially those with high fixed costs and either low marginal
costs or low reverse engineering costs computer processors, software, and pharmaceuticals
being prototypical examples), once an invention exists, the cost of commercialization
(testing, tooling up a factory, developing a market, etc.) is far more than the initial conception
cost. (For example, the internal "rule of thumb" at several computer companies in the 1980s
was that post-R&D costs were 7-to-1). Unless there is some way to prevent copies from
competing at the marginal cost of production, companies will not make that productization
investment.
4.
Patent rights create an incentive for companies to develop workarounds to patented
inventions, thereby creating improved or alternative technologies that might not otherwise
have been developed.
One interesting side effect of modern day patent usage is that the small-time inventor
can use the exclusive right status to become a licensor. This allows the inventor to
accumulate capital quickly from licensing the invention and may allow rapid innovation to
occur because he or she may choose to not manage a manufacturing buildup for the
invention. Thus the inventor's time and energy can be spent on pure innovation, allowing
others to concentrate on manufacturability.
Patent eg., first patented organism Pseudomonas putida for oil degradation.
iii) TRADEMARK
A trademark or trade mark is a distinctive sign or indicator of some kind which is
used by an individual, business organization or other legal entity to uniquely identify the
source of its products and/or services to consumers, and to distinguish its products or services
from those of other entities. A trademark is a type of intellectual property, and typically
comprises a name, word, phrase, logo, symbol, design, image, or a combination of these
elements. There is also a range of non-conventional trademarks comprising marks which do
not fall into these standard categories.
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The owner of a registered trademark may commence legal proceedings for trademark
infringement to prevent unauthorized use of that trademark. However, registration is not
required. The owner of a common law trademark may also file suit, but an unregistered mark
may be protectable only within the geographical area within which it has been used or in
geographical areas into which it may be reasonably expected to expand.
The term trademark is also used informally to refer to any distinguishing attribute by
which an individual is readily identified, such as the well known characteristics of celebrities.
When a trademark is used in relation to services rather than products, it may
sometimes be called a service mark, particularly in the United States.
iv) INDUSTRIAL DESIGN RIGHTS
Applicant can file for a single international deposit with WIPO or with the
national office in a country party to the treaty. The design will then be protected in as many
member countries of the treaty as desired. Design rights started in the United Kingdom in
1787 with the Designing and Printing of Linen Act and have expanded from there.
Trade secrets
A company can protect its confidential information through non-compete nondisclosure contracts with its employees (within the constraints of employment law, including
only restraint that is reasonable in geographic and time scope). The law of protection of
confidential information effectively allows a perpetual monopoly in secret information - it
does not expire as would a patent. The lack of formal protection, however, means that a third
party is not prevented from independently duplicating and using the secret information once
Industrial design rights are intellectual property rights that protect the visual design of objects
that are not purely utilitarian. An industrial design consists of the creation of a shape,
configuration or composition of pattern or color, or combination of pattern and color in three
dimensional forms containing aesthetic value. An industrial design can be a two- or threedimensional pattern used to produce a product, industrial commodity or handicraft.
Under The Hague Agreement Concerning the International Deposit of Industrial
Designs, a WIPO-administered treaty, a procedure for an international registration exists. An
it is discovered.
The sanctioned protection of such type of information from public disclosure is
viewed as an important legal aspect by which a society protects its overall economic vitality.
A company typically invests time and energy (work) into generating information regarding
refinements of process and operation. If competitors had access to the same knowledge, the
first company's ability to survive or maintain its market dominance would be impaired.
Where trade secrets are recognized, the creator of knowledge regarded as a "trade secret" is
entitled to regard such "special knowledge" as intellectual property.
The precise language by which a trade secret is defined varies by jurisdiction (as do
the particular types of information that are subject to trade secret protection). However, there
are three factors that (though subject to differing interpretations) are common to all such
definitions: a trade secret is some sort of information that:
is not generally known to the relevant portion of the public;
209
confers some sort of economic benefit on its holder (where this benefit must derive
specifically from its not being generally known, not just from the value of the
information itself);
is the subject of reasonable efforts to maintain its secrecy.

Trade secrets are not protected by law in the same manner as trademarks or patents.
Probably one of the most significant differences is that a trade secret is protected without
disclosure of the secret.
26.3. ETHICAL ISSUES RELATING TO BIOTECHNOLOGY
I)
BIOLOGICAL CONTAINMENT
Biological Containment is an important containment mechanism, especially when

assessing rDNA risks. Based upon existence of natural barriers that limit either Infectivity of
an agent (pathogen, vector) for specific hosts or the Ability of agent to disseminate or survive
in the environment.
There are two levels of biological containment primary and secondary.
Primary containment protects people and the immediate laboratory environment
from exposure to infectious agents. Good microbial techniques and safety equipment provide
sufficient primary containment. Examples of primary barriers include safety equipment such
as biological safety cabinets, enclosed containers, and safety centrifuge cups. Occasionally,
when it is impractical to work in biological safety cabinets, personal protective equipment,
such as lab coats and gloves may act as the primary barrier between personnel and infectious
materials.
Secondary containment protects the environment external to the laboratory from
exposure to infectious materials. Good facility design and operational practices provide
secondary containment. Examples of secondary barriers include work areas that are separate
from public areas, decontamination facilities, handwashing facilities, special ventilation
systems, and airlocks.
II)
PRINCIPLES OF BIOSAFETY
A fundamental objective of any biosafety program is the containment of potentially

harmful biological agents. The term "containment" is used in describing safe methods,
facilities and equipment for managing infectious materials in the laboratory environment
where they are being handled or maintained. The purpose of containment is to reduce or
eliminate exposure of laboratory workers, other persons, and the outside environment to
potentially hazardous agents. The use of vaccines may provide an increased level of personal
protection. The risk assessment of the work to be done with a specific agent will determine
the appropriate combination of these elements.
III) LABORATORY PRACTICE AND TECHNIQUE
The most important element of containment is strict adherence to standard
microbiological practices and techniques. Persons working with infectious agents or
potentially infected materials must be aware of potential hazards, and must be trained and
proficient in the practices and techniques required for handling such material safely. The
210
director or person in charge of the laboratory is responsible for providing or arranging the
appropriate training of personnel.
Each laboratory should develop or adopt a biosafety or operations manual that
identifies the hazards that will or may be encountered, and that specifies practices and
procedures designed to minimize or eliminate exposures to these hazards. Personnel should
be advised of special hazards and should be required to read and follow the required practices
and procedures. A scientist, trained and knowledgeable in appropriate laboratory techniques,
safety procedures, and hazards associated with handling infectious agents must be responsible
for the conduct of work with any infectious agents or materials. This individual should
consult with biosafety or other health and safety professionals with regard to risk assessment.
When standard laboratory practices are not sufficient to control the hazards associated
with a particular agent or laboratory procedure, additional measures may be needed. The
laboratory director is responsible for selecting additional safety practices, which must be in
keeping with the hazards associated with the agent or procedure. Laboratory personnel, safety
practices, and techniques must be supplemented by appropriate facility design and
engineering features, safety equipment, and management practices.
IV) SAFETY EQUIPMENT (PRIMARY
PROTECTIVE EQUIPMENT)
BARRIERS
AND
PERSONAL
Safety equipment includes BSCs, enclosed containers, and other engineering controls
designed to remove or minimize exposures to hazardous biological materials. The BSC is the
principal device used to provide containment of infectious splashes or aerosols generated by
many microbiological procedures. Three types of BSCs (Class I, II, III) Used in
microbiological laboratories are described and illustrated in Appendix A. Open-fronted Class
I and Class II BSCs are primary barriers that offer significant levels of protection to
laboratory personnel and to the environment when used with good microbiological
techniques. The Class II biological safety cabinet also provides protection from external
contamination of the materials (e.g., cell cultures, microbiological stocks) being manipulated
inside the cabinet. The gas-tight Class III biological safety cabinet provides the highest
attainable level of protection to personnel and the environment.
An example of another primary barrier is the safety centrifuge cup, an enclosed
container designed to prevent aerosols from being released during centrifugation. To
minimize aerosol hazards, containment controls such as BSCs or centrifuge cups must be
used when handling infectious agents.
Safety equipment also may include items for personal protection, such as gloves,
coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Personal
protective equipment is often used in combination with BSCs and other devices that contain
the agents, animals, or materials being handled. In some situations in which it is impractical
to work in BSCs, personal protective equipment may form the primary barrier between
personnel and the infectious materials. Examples include certain animal studies, animal
necropsy, agent production activities, and activities relating to maintenance, service, or
support of the laboratory facility.
211
V)
FACILITY DESIGN AND CONSTRUCTION (SECONDARY BARRIERS)
The design and construction of the facility contributes to the laboratory workers'
protection, provides a barrier to protect persons outside the laboratory, and protects persons
or animals in the community from infectious agents that may be accidentally released from
the laboratory. Laboratory directors are responsible for providing facilities commensurate
with the laboratory's function and the recommended biosafety level for the agents being
manipulated.
The recommended secondary barrier(s) will depend on the risk of transmission of
specific agents. For example, the exposure risks for most laboratory work in BSL-1 and BSL2 facilities will be direct contact with the agents, or inadvertent contact exposures through
contaminated work environments. Secondary barriers in these laboratories may include
separation of the laboratory work area from public access, availability of a decontamination
facility (e.g., autoclave), and hand washing facilities. When the risk of infection by exposure
to an infectious aerosol is present, higher levels of primary containment and multiple
secondary barriers may become necessary to prevent infectious agents from escaping into the
environment. Such design features include specialized ventilation systems to ensure
directional air flow, air treatment systems to decontaminate or remove agents from exhaust
air, controlled access zones, airlocks as laboratory entrances, or separate buildings or
modules to isolate the laboratory. Design engineers for laboratories may refer to specific
ventilation Recommendations as found in the ASHRAE Laboratory Design Guide published
by the American Society of Heating, Refrigerating, and Air-Conditioning Engineers.
(ASHRAE)
VI) BIOSAFETY LEVELS
Four BSLs are described, which consist of combinations of laboratory practices and
techniques, safety equipment, and laboratory facilities. Each combination is specifically
appropriate for the operations performed the documented or suspected routes of transmission
of the infectious agents, and the laboratory function or activity. The BSLs described in this
manual should be differentiated from Risk Groups, as described in the NIH Guidelines and
the World Health Organization Laboratory Biosafety Manual.
Risk groups are the result of a classification of microbiological agents based on their
association with, and resulting severity of, disease in humans. The risk group of an agent
should be one factor, to be considered in association with mode of transmission, procedural
protocols, experience of staff, and other factors in determining the BSL in which the work
will be conducted.
The recommended biosafety level(s) for the organisms in represent those conditions
under which the agent ordinarily can be safely handled. The laboratory director is specifically
and primarily responsible for assessing the risks and applying the appropriate biosafety
levels. The institutions Biological Safety Officer (BSO) and IBC can be of great assistance
in performing and reviewing the required risk assessment. At one point in time, under the
NIH Guidelines, BSOs were required only when large scale research or production of
organisms containing recombinant DNA molecules was performed or when work with
recombinant DNA molecules was conducted at BSL-3 or above. IBCs were required only
when an institution was performing non-exempt recombinant DNA experiments. Today,
however, it is strongly suggested that an institution conducting research or otherwise working
212
with pathogenic agents to have a BSO and properly constituted and functioning IBC. The
responsibilities of each now extend beyond those described in the NIH Guidelines and
depend on the size and complexity of the program.
. When information is available to suggest that virulence, pathogenicity, antibiotic
resistance patterns, vaccine and treatment availability, or other factors are significantly
altered, more (or less) stringent practices may be specified. Often an increased volume or a
high concentration of agent may require additional containment practices.
Biosafety Level 1 practices, safety equipment, and facility design and construction
are appropriate for undergraduate and secondary educational training and teaching
laboratories, and for other laboratories in which work is done with defined and characterized
strains of viable microorganisms not known to consistently cause disease in healthy adult
humans. Bacillus subtilis, Naegleria gruberi, infectious canine hepatitis virus, and exempt
organisms under the NIH Guidelines are representative of microorganisms meeting these
criteria. Many agents not ordinarily associated with disease processes in humans are,
however, opportunistic pathogens and may cause infection in the young, the aged, and
immunodeficient or immunosuppressed individuals. Vaccine strains that have undergone
multiple in vivo passages should not be considered avirulent simply because they are vaccine
strains. BSL-1 represents a basic level of scontainment that relies on standard microbiological
practices with no special primary or secondary barriers recommended, other than a sink for
handwashing.
Biosafety Level 2 practices, equipment, and facility design and construction are
applicable to clinical, diagnostic, teaching, and other laboratories in which work is done with
the broad spectrum of indigenous moderate-risk agents that are present in the community and
associated with human disease of varying severity. With good microbiological techniques,
these agents can be used safely in activities conducted on the open bench, provided the
potential for producing splashes or aerosols is low. Hepatitis B virus, HIV, the salmonellae,
and Toxoplasma spp. are representative of microorganisms assigned to this containment
level. BSL-2 is appropriate when work is done with any human-derived blood, body fluids,
tissues, or primary human cell lines where the presence of an infectious agent may be
unknown. (Laboratory personnel working with human-derived materials should refer to the
OSHA Bloodborne Pathogen Standard 2 for specific required precautions).
Primary hazards to personnel working with these agents relate to accidental
percutaneous or mucous membrane exposures, or ingestion of infectious materials. Extreme
caution should be taken with contaminated needles or sharp instruments. Even though
organisms routinely manipulated at BSL-2 are not known to be transmissible by the aerosol
route, procedures with aerosol or high splash potential that may increase the risk of such
personnel exposure must be conducted in primary containment equipment, or in devices such
as a BSC or safety centrifuge cups. Personal protective equipment should be used as
appropriate, such as splash shields, face protection, gowns, and gloves. Secondary barriers
such as hand washing sinks and waste decontamination facilities must be available to reduce
potential environmental contamination.
Biosafety Level 3 practices, safety equipment, and facility design and construction
are applicable to clinical, diagnostic, teaching, research, or production facilities in which
work is done with indigenous or exotic agents with a potential for respiratory transmission,
and which may cause serious and potentially lethal infection. Mycobacterium tuberculosis,
213
St. Louis encephalitis virus, and Coxiella burnetii are representative of the microorganisms
assigned to this level. Primary hazards to personnel working with these agents relate to
autoinoculation, ingestion, and exposure to infectious aerosols.
Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or
production facilities in which work is done with indigenous or exotic agents which may cause
serious or potentially lethal disease as a result of exposure by the inhalation route. Laboratory
personnel have specific training in handling pathogenic and potentially lethal agents, and are
supervised by competent scientists who are experienced in working with these agents. This is
considered a neutral or warm zone.
All procedures involving the manipulation of infectious materials are conducted
within biological safety cabinets or other physical containment devices, or by personnel
wearing appropriate personal protective clothing and equipment. The laboratory has special
engineering and design features.
It is recognized, however, that some existing facilities may not have all the facility
features recommended for Biosafety Level 3 (i.e., double-door access zone and sealed
penetrations). In this circumstance, an acceptable level of safety for the conduct of routine
procedures, (e.g., diagnostic procedures involving the propagation of an agent for
identification, typing, susceptibility testing, etc.), may be achieved in a Biosafety Level 2
facility, providing
1. the exhaust air from the laboratory room is discharged to the outdoors,
2. the ventilation to the laboratory is balanced to provide directional airflow into the
Room,
3. access to the laboratory is restricted when work is in progress, and
4. The recommended Standard Microbiological Practices, Special Practices, and Safety
Equipment for Biosafety Level 3 are rigorously followed.
The decision to implement this modification of Biosafety Level 3 recommendations
should be made only by the laboratory director.
Biosafety level 4 practices, safety equipment, and facility design and construction are
applicable for work with dangerous and exotic agents that pose a high individual risk of lifethreatening disease, which may be transmitted via the aerosol route and for which there is no
available vaccine or therapy. Agents with a close or identical antigenic relationship to BSL-4
agents also should be handled at this level. When sufficient data are obtained, work with
these agents may continue at this level or at a lower level. Viruses such as Marburg or
Congo-Crimean hemorrhagic fever are manipulated at BSL-4. The primary hazards to
personnel working with BSL-4 agents are respiratory exposure to infectious aerosols, mucous
membrane or broken skin exposure to infectious droplets, and autoinoculation.
Biosafety Level 4 is required for work with dangerous and exotic agents that pose a
high individual risk of aerosol-transmitted laboratory infections and life-threatening disease
(e.g. Ebola). Agents with a close or identical antigenic relationship to Biosafety Level 4
agents are handled at this level until sufficient data are obtained either to confirm continued
work at this level, or to work with them at a lower level. This is a hot zone considering that
anybody that contracts a microorganism from a hot zone will most likely die.
214
Members of the laboratory staff have specific and thorough training in handling
extremely hazardous infectious agents and they understand the primary and secondary
containment functions of the standard and special practices, the containment equipment, and
the laboratory design characteristics. They are supervised by competent scientists who are
trained and experienced in working with these agents. Access to the laboratory is strictly
controlled by the laboratory director.
The facility is either in a separate building or in a controlled area within a building,
which is completely isolated from all other areas of the building. A specific facility
operations manual is prepared or adopted. Building protocols for preventing contamination
often uses negatively pressurized facilities, which, if compromised, would severely inhibit an
outbreak of aerosol pathogens.
Within work areas of the facility, all activities are confined to Class III biological
safety cabinets, or Class II biological safety cabinets used with one-piece positive pressure
personnel suits ventilated by a life support system. The Biosafety Level 4 laboratory has
special engineering and design features to prevent microorganisms from being disseminated
into the environment. The laboratory is kept at negative air pressure, so that air flows into the
room if the barrier is penetrated or breached. Furthermore, an airlock is used during personnel
entry and exit.
26.4. LET US SUM UP
Information on the genetic modification and product development has to be protected

in order to prevent from misuse.
There are certain norms to protect such intellectual property which are collectively
called as intellectual property rights (IPR).
This includes patents, trade secrets, copy rights and industrial design rights.
Biological containment plays a very important role in research, production and release
of GMOs.
Containment is implemented by Biosafety cabinets of which there are various levels
each specific for particular functions.

1. Differentiate between trade mark copy right and patent.
2. Justify intelectual property must be protected and revarded.
1. What do you understand by Biological containment?
2. What are intellectual property rights?
3. If you want to work with HIV, which containment facility /safety level is needed?
26.7. REFERENCES
1. Genes to Clones L. Winnecker.
215
LESSON 27: GENETIC ENGINEERING AND BIOWARFARE
CONTENTS
27.1 BIOWARFARE INTRODUCTION
27.2 BIOWEAPONS
27.3 BIOWEAPON CHARACTERISATION
27.4 IDENTIFICATION OF BIOWEAPONS
27.5. LET US SUM UP
27.8. REFERENCES
The lesson discuss about the role of genetic engineering in warfare.
27.1 BIOWARFARE INTRODUCTION
There is an increasing concern within both the scientific and security communities
that the ongoing revolution in biology has great potential to be misused in offensive
biological weapons programs. Biological warfare attacks have often been dismissed as
science fiction or as so immoral as to be beyond imagination.
The history of biological warfare is nearly as old as the history of warfare itself. In
ancient times, warring parties poisoned wells or used arrowheads with natural toxins. Mongol
invaders catapulted plague victims into besieged cities, probably causing the first great
plague epidemic in Europe, and British settlers distributed smallpox-infected blankets to
Native Americans. Indeed, the insights into the nature of infectious diseases gained by Louis
Pasteur and Robert Koch in the nineteenth century did not actually represent a great
breakthrough in the use of infectious organisms as biological weapons.
Similarly, the development of a bioweapon does not necessarily require genetic
engineeringsmallpox, plague and anthrax are deadly enough in their natural states. But the
revolution in biotechnology, namely the new tools for analysing and specifically changing an
organism's genetic material, has led to an increased risk of biowarfare due to several factors.
First, the expansion of modern biotechnology in medical and pharmaceutical research
and production has led to a worldwide availability of knowledge and facilities. Many
countries and regions, where 30 years ago biotechnology merely meant brewing beer and
baking bread, have established high-tech facilities for vaccine or single-cell-protein
production that could be subverted for the production of biological weapons. Today, nearly
all countries have the technological potential to produce large amounts of pathogenic
microorganisms safely.
Second, classical biowarfare agents can be made much more efficiently than their
natural counterparts, with even the simplest genetic techniques.
Third, with modern biotechnology it becomes possible to create completely new
biological weapons. And for technical and/or moral reasons, they might be more likely to be
216
used than classical biowarfare agents. These possibilities have generated new military desires
around the world, including within those countries that have publicly renounced biological
weapons in the past.
27.2 BIOWEAPONS
Biological warfare (BW), also known as a germ warfare, biological weapons,
bioweapons and bioengineering, is the use of any pathogen (bacterium, virus or other
disease-causing organism) as a weapon of war. Note that using nonliving toxic products, even
if produced by living organisms (e.g., toxins), is considered chemical warfare under the
provisions of the Chemical Weapons Convention. A biological weapon may be intended to
kill, incapacitate or seriously impede an adversary. It may also be defined as the material or
defense against such employment.
The creation and stockpiling of biological weapons ("offensive BW") was outlawed
by the 1972 Biological Weapons Convention (BWC), signed by over 100 countries. The
BWC remains in force. The rationale behind the agreement is to avoid the devastating impact
of a successful biological attack which could conceivably result in thousands, possibly even
millions, of deaths and cause severe disruptions to societies and economies. Oddly enough,
the convention prohibits only creation and storage, but not usage, of these weapons.
However, the consensus among military analysts is that, except in the context of
bioterrorism, BW is of little military use. Many countries pursue "defensive BW" research
(defensive or protective applications) which is not prohibited by the BWC. As a tactical
weapon, the main military problem with a BW attack is that it would take days to be
effective, and therefore, unlike a nuclear or chemical attack, would not immediately stop an
opposing force.
As a strategic weapon, BW is again militarily problematic, because it is difficult to
prevent the attack from spreading, either to allies or to the attacker, and while an attack is
taking effect, the opponent can undertake massive retaliation.
For a proliferator, there are several main advantages of biological weapons:
(i)
they are easy to manufacture
(ii)
the starting materials, such as bacterial and viral strains and plasmids, can be
easily obtained by requesting them from the scientists working with them or
from culture repositories
(iii)
The ever-expanding microbial genome databases now provide a parts-list of
all potential genes involved in pathogenicity and virulence, adhesion and
colonization of host cells, immune response evasion and antibiotic resistance
from which to pick and choose the most lethal combinations.
Ongoing efforts in functional genomics using DNA arrays and proteomic analysis
have begun to reveal the subsets of genes in the genome of each pathogen that are required
for infection and that are involved in virulence and antibiotic resistance. One might argue that
this could further accelerate the potential misuse of genomic data through the identification of
the most relevant genes in the pathogen collection.
Fortunately, the same advances in microbial genomics that could be used to produce
bioweapons can also be used to set up countermeasures against them. One of the most
217
important contributions may come from the development of more rapid methods for detecting
biological agents, regardless of whether or not they have been engineered. Comparative
genome hybridization using DNA arrays has already proven useful in the identification of
strain-specific differences related to virulence and antigenicity in Helicobacter pylori and
Streptococcus pneumoniae.
The readout from such a detector could provide information on the full genetic
complement of any biological warfare agent, even if it contained genes or plasmids from
other species, had unusual virulence or antibiotic-resistance properties or was a synthetic
organism built from component genes.
The ability to quickly identify and characterize a potential biowarfare agent in a single
assay will greatly reduce the delays inherent in current detection methods. This future
scenario could serve as a possible deterrent to the deployment of biowarfare agents if the
verification of an attack and the identification of an infectious agent can be accomplished
quickly, allowing for a rapid and highly effective response.
27.3 BIOWEAPON CHARACTERISATION
Ideal characteristics of biological weapons are high infectivity, high potency,
availability of vaccines, and delivery as an aerosol.
Diseases most likely to be considered for use as biological weapons are contenders
because of their lethality (if delivered efficiently), and robustness (making aerosol delivery
feasible).
The biological agents used in biological weapons can often be manufactured quickly
and easily. The primary difficulty is not the production of the biological agent but delivery in
an effective form to a vulnerable target.
For example, anthrax is considered an effective agent for several reasons. First, it
forms hardy spores, perfect for dispersal aerosols. Second, pneumonic (lung) infections of
anthrax usually do not cause secondary infections in other people. Thus, the effect of the
agent is usually confined to the target. A pneumonic anthrax infection starts with ordinary
"cold" symptoms and quickly becomes lethal, with a fatality rate that is 80% or higher.
Finally, friendly personnel can be protected with suitable antibiotics.
A mass attack using anthrax would require the creation of aerosol particles of 1.5 to 5
micrometres. Too large and the aerosol would be filtered out by the respiratory system. Too
small and the aerosol would be inhaled and exhaled. Also, at this size, nonconductive
powders tend to clump and cling because of electrostatic charges. This hinders dispersion. So,
the material must be treated with silica to insulate and discharge the charges. The aerosol
must be delivered so that rain and sun does not rot it, and yet the human lung can be infected.
There are other technological difficulties as well.
Diseases considered for weaponization, or known to be weaponized include anthrax,
ebola, Marburg virus, bubonic plague, cholera, tularemia, brucellosis, Q fever, machupo,
Coccidioides mycosis, Glanders, Melioidosis, Shigella, Rocky Mountain spotted fever,
typhus, Psittacosis, yellow fever, Japanese B encephalitis, Rift Valley fever, and smallpox.
Naturally-occurring toxins that can be used as weapons include ricin, SEB, botulism toxin,
saxitoxin, and many mycotoxins. The organisms causing these diseases are known as select
218
agents. Their possession, use, and transfer are regulated by the centers for disease control and
prevention act.
27.4 IDENTIFICATION OF BIOWEAPONS
Biodefense fully integrates the sustained efforts of the national and homeland
security, medical, public health, intelligence, diplomatic, and law enforcement communities.
Biological weapons agents as part of a long-term campaign of aggression and terror. Health
care providers and public health officers are the first lines of defense. Private, local, and state
capabilities are being augmented by and coordinated with Federal assets, to provide layered
defenses against biological weapons attacks. The traditional approach toward protecting
agriculture, food, and water, focusing on the natural or unintentional introduction of a disease
being strengthened by focused efforts to address current and anticipated future biological
weapons threats that may be deliberate, multiple, and repetitive.
The growing threat of biowarfare agents and bioterrorism has led to the development
of specific field tools that perform on-the-spot analysis and identification of encountered
suspect materials. One such technology, being developed by researchers from the Lawrence
Livermore National Laboratory (LLNL), employs a "sandwich immunoassay", in which
fluorescent dye-labeled antibodies aimed at specific pathogens are attached to silver and gold
nanowires. Researchers at Ben Gurion University in Israel are developing a different device
called the BioPen, essentially a "Lab-in-a-Pen", which can detect known biological agents in
under 20 minutes using an adaptation of the ELISA, a similar widely employed
immunological technique, that in this case incorporates fiber optics.
27.5. LET US SUM UP
Biowarfare or bioweapons include application of living organisms as weapons.

For example B.anthrax was modified for mass infection through spores and caused
devastating results.
The advantages of bioweapons over chemical or other kind of warfare are numerous
such as the productivity, usability, and efficiency.

1. Evaluate the use of anthrox as biological weapon.
2. Discuss the creation of how biological weapon.
1. Give an account of beneficial and harmful effects of Bioweapons.
2. How will you characterise the bioweapons?
27.8. REFERENCES

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UNIT I: NUCLEIC ACID AND PROTEIN SYNTHESIS AND REGULATION

LESSON 1: NUCLEIC ACID - DNA

1.0. AIMS AND OBJECTIVES

Fig. 1 STRUCTURE OF DNA

The pitch or angle between each base pair.

Flattened out on helix

Through minor groov,1

Fig.1 STRUCTURAL COMPARISON OF DNA AND RNA

Fig.2 CLOVER LEAF STRUCTURE OF tRNA

CTT Leu (L)

CCT Pro (P)

CAT His (H)

CGT Arg (R)

ATT Ile (I)

ACT Thr (T)

AAT Asn (N)

AGT Ser (S)

GTT Val (V)

GCT Ala (A)

GAT Asp (D)

GGT Gly (G)

Fig.1 PROTEIN SYNTHESIS

Fig.1 LAC OPERON

lacZ encodes -galactosidase (LacZ), an intracellular enzyme that cleaves the

lacY encodes -galactoside permease (LacY), a membrane bound transport protein

lacA encodes -galactoside transacetylase (LacA), an enzyme that transfers an acetyl

Fig.2 lac OPERON IN DETAIL

Fig.3 TRYPTOPHAN OPERON

Fig.4 TRANSCRIPTIONAL ATTENUATION OF TRYPTOPHAN OPERON

5.2.2. PLASMIDS IN ORGANISMS OTHER THAN BACTERIA

Fig.1 STRUCTURE OF pBLUESCRIPT (PHAGEMID)

Fig.2 STRUCTURE OF pBLUESCRIPT

Fig.1 STRATEGY FOR CONSTRUCTION OF CLONINNG VECTOR

The genomic map of CaMV

7.2.1. CLONING STRATEGY

First, it is necessary to produce or obtain a library including the sequence of interest.

7.2.2. cDNA LIBRARIES

What are the usual cloning strategies?

8.10. PLUS/MINUS SCREENING AND DIFFERENTIAL DISPLAY

8.11. TWO HYBRID SCREENING

8.12. SCREENING BY DATABASES

UNIT III: RECOMBINANT TECHNIQUES

Fig. BLOTTING TECHNIQUE

Blotting of from gels (nucleic acid/protein)

Highly purified mRNA (naturally single stranded) can be used as probe;

Labeling of probes: Preparation of the probes essentially needs a label to be incorporated

Nick translation. Pancreatic DNase I introduces single-stranded nicks by cleaving internal

(A) Kinase end-labeling of oligonucleotides. The 5 -terminal phosphate of the oligonucleotide

Identification of the recombinant clone carrying the desired DNA insert

9.2.2. SOUTHERN BLOTTING

Fig. SOUTHERN BLOTTING

8.Prehybridization: Generally a prehybridization step is required before

9. Development: The radioactive labeled target sequence can be visualized after

Fig. NORTHERN BLOTTING

The first step in transformation is to select a piece of DNA to be inserted

Figure. A PCR cycle.

EXPONENTIAL TEMPLATE DUPLICATION

In this example, only 2 RAPD PCR products are formed:

The procedure used during the development of SSCP was as follows:

Single-stranded DNA mobilities are dependent on temperature. For best results,

Sensitivity of SSCP is affected by pH. Double-stranded DNA fragments are

Under optimal conditions, approximately 80 to 90% of the potential base exchanges

Optimization of SSCP analysis to detect the maximum number of mutations requires

A single nucleotide polymorphism, or SNP is a DNA sequence variation occurring