Concepte fundamentale,

metode i tehnici de biologie

molecular n endocrinologie

Corin Badiu, 2006

Metode de biologie molecular n

endocrinologie

Concepte fundamentale
De la clinica la cauza moleculara
Analiza expresiei genelor
Detectarea mutaiilor
Mutageneza intit
Msurarea transcripiei genelor i
determinarea ARN-ului mesager
Animale transgenice n studiul funciei genelor

Concepte fundamentale
Genomul: aspecte structurale i funcionale
Secvenierea: date i consecine
Proteom i transcriptom

Aspecte structurale

From DNA to Chromosome organization

Dogma fundamentala a biologiei

moleculare
DNA to mRNA (transcription)
mRNA to protein (translation)

Gene
Exon (codes for mRNA)
Introns (spliced out during transcription)
Promoters (part of the gene which binds to many
transcription factor proteins that promotes
transcription)

Gene structure
promoter
region

exons (filled and unfilled boxed regions)

+1
introns (between exons)
transcribed region
mRNA structure
5

3
translated region

Structure of mRNA

Structure of tRNA

Translation

Genomul uman

Genomul uman
Structura:

3,2 * 109 BP (23 cr) + 15.000 BP (mitocondrial)

3*104 gene: 100..10.000, (2000), < 2%

Funcia:

meninerea structurii cromozomiale (telomeri,

matrice histonica); replicare celulara (centromer)
Proteine (zona de gene structurale)
Secvene reglatorii (promoter, inhibitor, izolator)

Reglaj:
esut

Abunden a prot., timp, etap de dezvoltare,

Diversitate:

99,9% IDENTIC; 0,1% diferene, particulariti,

susceptibilitate la boli

Genomul uman
Variabilitate: polimorfism mononucleotidic (SNP), 1:1000
1,42 Mil. SNP, 60.000 n exoni (Science, 2001)
Inserii, deleii, polimorfism repetitiv.
Markeri:

Polimorfism fr semnif. funcional.

Boli monogenice f. rare
- SAG, MEN, MODY,rezistena la androgeni

Diversitate:

Boli poligenice DZ, dislipidemii, Cvasc,

cancer, boli infecioase: subsituii aa + F. risc

Genomul uman
Human Genome Project (1990 - 2001)
Secvena:

3,2 Mild. BP 447 vol x 1000 pag x 1000 cuv

Limbajul:

Posibil 30.000 gene funcionale

Semne de carte: Sequence Taged Sites (STS)
1 cM = 1 Mb, 1% recombinare
1996: MEN1,11q13, markeri transmii la bolnavi

Localizare:
locus susceptibil, izolare ADN, identificarea
genei, verificarea mutaiei la bolnavi
Algoritm:

in silico screening pt cadru de citire,

promoteri, secvene omologe cunoscute (1,5%)

Identification of genes - problems

Promoter

GC rich islands

template for primary transcript

Regions in some promoters that are much

higher in GC content than surrounding
DNA sequence
Identify promoter region of many, but not all genes

Pseudogenes

Gene remnants that look like genes but are no

longer active

Genocopies

More than one copy of an active gene, often with

slight, but important, sequence variations

Identification of genes - problems

Absence of co-linearity between genes and proteins
Genomic DNA sequence generally does not map
directly onto codons for amino acids in proteins
Failure of one gene one polypeptide dogma
Gene
mRNA
Protein

Consider genes with introns

nearly all protein-coding genes have introns
exceptions include histone genes
Gene
Primary transcript
exon
intron

transcription

alternate splicing
transcript processing
mRNA(s)

One gene one mRNA does not hold in general

Translational frameshifting
also yields more than one protein per mRNA

mRNA
lys

protein

tyr

gly

phe

AAA UAU GGC UUU

AAA UAU UGG CUU
lys

tyr

trp

leu

Aplicaii
MEN
MEN1: PTR Ad, Enteropancreatic, Pit ad, Adrenal, carcinoid
11q13: 10 exoni, 1830 bp, 610 aa (menina)
MEN1 mutant: n evaluare pt indicaii
MEN2: AD, (ret)
MTC n 90%, Feocromocitom 50%, PTR 30%
Exonii RET 10, 11, 13, 14, 15,16
Tiroidectomie profilactic
Clasa 3- risc maxim: 883, 918, 922 la 6 luni
Clasa 2- risc mare: 611, 618, 620, 634 la 5 ani
Clasa 1- risc mediu: 609, 768, 804, 891 10 ani
Clasa 0 risc mic: 790, 791 calcitonina periodic

Genomul uman
MEN1
www.ncbi.nlm.nih.gov
MEN1 + links
Chandrasekharappa, Science 1997

Multiple Endocrine
Neo-Plasia Type I (MEN-I)
Genetics

Autosomal dominantly inherited

Tumours

Parathyroid adenoma

95-100%

Endocrine pancreatic tumours

80%

Pituitary tumours (Prolactin, GH)

30-50%

Mutations of the MENIN-gene on Chr 11q13

Carcinoids (lungs, thymus, gastric, duodenal) 20-50%

Lipomas

10-20%

Thyroid Nodules

10-15%

Lymphomas

<5%

Pedigree MEN-I

Aplicaii
Defecte genetice ale axei de cretere

Bermejo et al, TEM 11, 2, 2000

DNA microarray

Genomica funcional

Genomica funcional
Gene:

Determinarea tuturor secvenelor ce se exprim

Mecanismele de reglaj genetic, SNP ca factori
predispozani, genetica populaional

Proteine:

Polimorfism cu semnificaie funcional:

PROTEOM, TRANSCRIPTOM (esut, funcie).
Produi diferii de degradare (POMC)

Diversitate:

Reglarea expresiei, specificitate tisular

Experimental: oareci transgenici, knockout /knockdown

DNA microarray, electroforeza 2D & Mass Spect

Aplicaii

Diagnosticul genetic; screening genetic

-Men1, SAG, DI central

Terapie: tehnologia genic (insulina, rhGH,

IGF1, rhPTH)
terapia genic n neoplazii

Medicina personalizat

Percent hyperchromicity

DNA melting curve

100

50

0
50

70

90

Temperature oC

Tm is the temperature at the midpoint of the transition

Percent hyperchromicity

Tm is dependent on the G-C content of the DNA

E. coli DNA,
which is 50% G-C,
has a Tm of 69 o C.

50

60

70

80

Temperature oC

average base composition (G-C content) can be

determined from the melting temperature of DNA

Mutation
Types and rates of mutation
Type
Genome
mutation

Mechanism
chromosome
missegregation
(e.g., aneuploidy)

Frequency________
10-2 per cell division

Chromosome
mutation

chromosome
rearrangement
(e.g., translocation)

6 X 10-4 per cell division

Gene
mutation

base pair mutation

10-10 per base pair per
(e.g., point mutation,
cell division or
or small deletion or
10-5 - 10-6 per locus
insertion
generation

Mutation rates* of selected genes

Gene

New mutations per 10 6 gametes

Achondroplasia
Aniridia
Duchenne muscular dystrophy
Hemophilia A
Hemophilia B
Neurofibromatosis -1
Polycystic kidney disease
Retinoblastoma

6
to 40
2.5 to
5
43
to 105
32
to
2
to
44
to 100
60
to 120
5
to 12

57
3

*mutation rates (mutations / locus / generation) can vary

from 10-4 to 10-7 depending on gene size and whether
there are hot spots for mutation (the frequency at most
loci is 10-5 to 10-6).

Polymorphisms exist in the genome

the number of existing polymorphisms is ~1 per 500 bp
there are ~5.8 million differences per haploid genome
polymorphisms were caused by mutations
New germline mutations
each sperm contains ~100 new mutations
a normal ejaculate has ~100 million sperm
100 X 100 million = 10 billion new mutations
~1 in 10 sperm carries a new deleterious mutation
at a rate of production of ~8 X 107 sperm per day,
a male will produce a sperm with a new mutation
in the Duchenne muscular dystrophy gene
approximately every 10 seconds.

Types of base pair mutations

normal sequence

CATTCACCTGTACCA
GTAAGTGGACATGGT
transition (T-A to C-G)

CATCCACCTGTACCA
GTAGGTGGACATGGT

transversion (T-A to G-C)

CATGCACCTGTACCA
GTACGTGGACATGGT

base pair substitutions

transition: pyrimidine to pyrimidine
transversion: pyrimidine to purine
deletion

CATCACCTGTACCA
GTAGTGGACATGGT

insertion

CATGTCACCTGTACCA
GTACAGTGGACATGGT

deletions and insertions can involve one

or more base pairs

Mutation is perpetuated by replication

C G

C G

and

C G

replication of C-G should give daughter strands each with C-G

C G

C A

and

C G

tautomer formation C during replication will result in mispairing

and insertion of an improper A in one of the daughter strands

C A

T A

which could result in a C-G to T-A transition mutation in the next

round of replication, or if improperly repaired

Mismatched (post-replication) repair

the parental DNA strands are
methylated on certain
adenine bases
CH3

5
3
CH3

the mutations are repaired

by excision repair mechanisms
after repair, the newly
replicated strand is methylated

CH3

mutations on the newly

replicated strand are
identified by scanning
for mismatches prior to
methylation of the newly
replicated DNA

CH3

Infasurarea ADN

Recombinant DNA DNA from two

different sources joined together.
1. Cut the DNA and the plasmid using the same
restriction enzyme (these enzymes recognize the
same base sequences.
2. Insert the foreign DNA into the plasmid.
3. Replace the plasmid into the bacterium
4. Allow the bacterium to reproduce all future
generations have the new DNA
5. Collect the product it might be insulin or
growth hormone, or some other molecule.

PCRPolymerase Chain
Reaction
Used to amplifymake large amounts of a
specific piece of DNA from a very small
sample.
1. Heat a starting quantity of DNA to separate the
double helix.
2. Add a collection of all four nucleotides, and
DNA polymerase to copy the DNA, and some
primers, and cool the sample.

PCR

3. Primers are short sections of DNA that are

complementary to the region on both
ends of the DNA that you wish to copy.
Primers act as signals to tell DNA
polymerase where to copy. As the solution
cools, they stick to the DNA you wish to
copy and allow polymerase to do its job.
4. Heating the sample again unwinds the
new duplicated strands; cooling again
allows more primers to bind. If you repeat
this as a cycle, you can make millions of
copies of the original DNA.

Visualizing DNA Sequences

A. So many bases, it is best to visualize them all in some
organized fashion.
1. Restriction enzymes can be used to cut the
chromosomes from many cells into manageable
pieces.
2. There will be a collection of copies of fragment 1,
which is a different size than fragment 2, and so on.
3. The pieces can be ordered according to size using gel
electrophoresis (moving the fragments in an electric
field through a gel matrix). Larger pieces are more
easily retarded by holes in the gel, so they travel less
than smaller pieces: Figure 15.8

Sequence

Sequence

Analiza clonalitatii

Western Blot

Secventializare proteica

Secventializare proteica

Mutageneza tintita

Transgenic animals
DNA transformation: in vivo experiment
Mice are injected either with Type R, non-virulent
Streptococcus or with heat-killed, virulent Type S cells.

The mice are healthy.

Transgenic animals
Mice are injected with both Type R, non-virulent and
heat-killed, Type S Streptococcus

X
DNA carrying genes from
the virulent, heat-killed cells
transforms the non-virulent
bacterial cells, making them
lethal to the mice

Transgenic animals
DNA transformation: in vitro experiment

Type R cells

Type R colonies

Type S cells

Type S colonies

Type R cells
+ DNA from
Type S cells

Mixture of
Type R and Type S
colonies

Transgenic animals
Genotype:
An organisms genetic constitution.

Phenotype:
The observed characteristics of an organism,
as determined by the genetic makeup
(and the environment).
DNA from Type S cells (thus conferring the Type S genotype)
transformed Type R cells into cells havingthe Type S phenotype

Transgenic animals
Injected into nucleus
of a fertilized mouse egg

Plasmid DNA carrying the

growth hormone gene
Egg implanted into uterus
of surrogate mother mouse

Mother mouse gives

birth to transgenic mouse

Transgenic animals
Mouse with growth
hormone transgene

Normal mouse

Mutation alters phenotype

Phenotypic differences between individuals

are due to differences between their genes
These differences have arisen by mutation of DNA
over many thousands of years

CloningTo make an exact genetic copy

of; can be a gene, a cell, or an entire
organism.

International Laboratory Directory

~600 Clinical and research laboratories

~1050 Inherited diseases

~700 clinical tests
~350 research only

Genetics in Specialty Care

: Feature Search*
Behavior Disorder (15) Endocrine (91)

Liver (62)

Blood (97)

Eye (259)

Skeletal Bone (216)

Gastrointestinal (90)

Premature Aging
(5)
Pulmonary (49)

Cancer (82)

Genitourinary (99)

Mitochondrial (16)

Connective Tissue
(34)
Craniofacial (184)

Growth (119)

Metabolic (225)

Heart (162)

Neurologic (All) (907)

Deafness (122)

Immune (36)

Skin (210)

Dental (32)

Renal (86)

Vascular (40)

Ear (11)

Limb Malformation
(76)
*Clinical laboratories

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