CELL BIOLOGY, GENETICS AND EVOLUTION

BOT-302 4(3-1)
Assignment topic:
Cosmids

Submitted To:
Prof. M. Muhammad WaqarUlArfeen

Presented By:

Rizwan Sikander
Roll no. 34606

BS CHEMISTRY (3rD SEMESTER-MORNING)

GOVT. POSTGRADUATE COLLEGE SAMUNDRI

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 Contents

Headings Page No.

Introduction 2
Consequences 2
Cosmid features 2
Problem 4
Mechanism 5
Genomic library 9

PAC’s 10
BAC’S 10
References 12

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Introduction

A cosmid is a type of hybrid that contains a cos sequence. Cosmids (cos sites +
plasmid = cosmids) DNA sequences are originally from They are often used as a . Cosmids can
be used to bui. They were first described by Collins and Hohn in 1978. Cosmids can contain 37
to 52 (normally 45) of DNA, limits based on the normal bacteriophage packaging size. They
can as plasmids if they have a suitable origin of replication: for example SV40 ori in
mammalian cells, for double-stranded DNA replication or f1 ori for single-stranded DNA
replication in s. They frequently also contain a gene for selection such as antibiotic resistance, so
that the transformed cells can be identified by plating on a medium containing the antibiotic.
Those cells which did not take up the cosmid would be unable to grow.

Unlike plasmids, they can also be packaged in which allows the foreign genes to be transferred
into or between cells by . Plasmids become unstable after a certain amount of DNA has been
inserted into them, because their increased size is more conducive to . To circumvent this, phage
transduction is used instead. This is made possible by the cohesive ends, also known as cos sites.
In this way, they are similar to using the lambda phage as a vector, except all the lambda genes
have been deleted with the exception of the cos sequence

Cos sequences

Cos sequences are ~200 base pairs long and essential for packaging. They contain
a cosN site where DNA is nicked at each strand, 12 bp apart, by terminase. This causes
linearization of the circular cosmid with two "cohesive" or "sticky ends" of 12bp. (The
DNA must be linear to fit into a phage head.) The cosB site holds the terminase while it is
nicking and separating the strands. The cosQ site of next cosmid (as often results in
linear c) is held by the terminase after the previous cosmid has been packaged, to prevent
degradation by cellular .

Cosmid features and uses

Cosmids 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 derivedfrom

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 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. Selection against wildtypecosmid DNA is simply done via size exclusion.
Cosmids, however, always form colonies and not plaques. Also the clone density is much
lower with around 105 – 106 per µg of ligated DNA.

After the construction of recombinant lambda or cosmid libraries the total DNA is
transferred 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 lambda-replacement vectors.
This compensates for their slightly lower loading capacity. Phage libraries are also stored and
screened more easily than cosmid (colonies!) libraries.

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) to avoid chromosome
scrambling, i.e. the ligation of physically unlinked fragments.

Cosmids are plasmid vectors that contain cos sites. The cos site is the only requirement
for DNA to be packaged into a phage particle.Cosmids were developed in light of this
observation. How do you clone into cosmid vectors?

Clone the DNA into the vector as you would with any plasmid.

Introduce the DNA into the bacterial cell via a phage particle.

Propagate as plasmid.

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 Since phage particles can accept between 38 and 53 kb of DNA and since most cosmids
are about 5 kb, between 33 and 48 kb of DNA can cloned in these vectors.

Problems associated with lambda and cosmid cloning.

Since repeats occur in eukaryotic DNA rearrangements can occur via recombination of
the repeats present on the DNA inserted into lambda or cosmid.

Cosmids are difficult to maintain in a bacterial cell because they are somewhat unstable.

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. Due to the requirement for a large DNA molecule for efficient packaging, there is a
direct selection for hybrids carrying large sections of foreign DNA. The small vector
plasmids do not contribute a large background in the transduced population, which is
therefore markedly enriched for large hybrid plasmids (over 90%). The efficiency of the
in vitro packaging system is on the order of 105 hybrid clones per microgram of foreign
DNA for hybrids in the 20-30 million daltonrange.
Mechanism of cosmid process
The mechanism of packaging DNA into the head of Escherichia coli bacteriophage X has
been extensively studied through the development of in vitro packaging systems (1-4; for
review, see ref. 5). These and studies in vivo (6) led to the following findings: monomeric
circular DNA was not packaged; headto-tail polymers (concatemers) of the unit-length X
DNA molecules were efficiently packaged if the cohesive-end site (cos), substrate for the
packaging-dependent cleavage that produces ,the cohesive ends of mature X DNA, was
23-33 megadaltons (MDal) apart; and only a small region in the proximity of the
cleavage site was required for recognition by the packaging system (7, 8). This
information implies that cos-containing plasmids of less than 23 MDal would not be
efficiently packaged due to the circular form of their DNA and their size, but that
concatemeric derivatives with DNA inserts would be a packaging substrate. The latter
DNA structure resembles a ligation mixture between a cleaved cos-containing plasmid
and DNA to be cloned. It was expected, therefore, that cloning in a cos-containing
plasmid in conjunction with in vitro packaging selects against re-ligated vector molecules
but selects for hybrids in the size range of X DNA, molecules that are recovered only
poorly upon transformation. In our present study, experiments are described in which a
cos-containing ColElrpo plasmid (9, 10) was packaged in nitro after restriction and re-
ligation. The results of this-experiment, as well as of RI plasmid and Pseudomonas
cloning experiments, suggest the use of packageable plasmids as a gene cloning system
that is both highly efficient and selective for recovery of large hybrids. Plasmids
containing a cos site, which are useful as vectors for gene cloning in conjunction with the
packaging system, we refer to as "cosmids." MATERIALS AND METHODS Plasmids
and Bacteria. Preparation of plasmids pJC720 and pJC703 (Fig. 1) has been described (9,
10). The detailed mapping of these plasmids with restriction endonucleases is

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unpublished. E. coli N205, an E. coli K-12 strain (rk+mk+ recA-su-), was from N.
Sternberg; strain 5K (rk-mk+ thr-thi) was from S. Glover; strain HB101 (rk-mk- leupro-
recA-) was from H. Boyer; and strain GL1 (pel2l; W3101) was from S. W. Emmons (11).
Packaging System. Exogenous DNA was packaged in vitro as described (12), with some
slight modifications: single colonies of strains ligation. Cloning of Pseudomonas DNA
pJC720 was used to clone fragments from Pseudomonas AMi chromosomal DNA
partially digested with HindIII (Exp. 4, Table 1). By gel electrophoresis it was estimated
that more than 80% of the Pseudomonas fragments were larger than 16 MDal and
probably too large to be clonable by packaging with this vector. In spite of this, the
efficiency of hybrid formation is about 3 X 104 per ug of foreign DNA. Twenty-seven of
the first 32 clones tested carried new DNA fragments. The average size of the DNA insert
in these 27 was 10 MDal. On this basis, a few hundred of the clones obtained should
constitute a gene bank (18) of Pseudomonas AM1 chromosomal DNA in E. coli.
Biochemistry: Collins and Hohn 4246 Biochemistry: Collins and Hohn DISCUSSION
We have demonstrated that the packaging of plasmid DNA in X bacteriophage particles
can be used as a method for obtaining plasmid hybrids in the 20- to 30-MDal size range
when using plasmid DNA that has been linked in vitro to foreign DNA fragments. The
yield of clones containing these hybrids is of the order of 3 X 105, under optimal
conditions, per tig of foreign DNA. Furthermore, by the use of small plasmids (less than
8 MDal) that are themselves very inefficiently packaged (unpublished results), the
background of nonhybrid clones is effectively eliminated in a single step without resort to
either modification of the DNA (e.g., alkaline phosphatase treatment or polynucleotide
tailing) or to elaborate selection or screening procedures which are usually the most time-
consuming steps in plasmid cloning experiments. In addition, the use of small cosmids
will allow efficient recovery of cloned fragments in the size of up to 25-30 MDal, the
selection being imposed by the requirement for packaging of a full or nearly full head. In
vitro packaging of X cloning vectors can be made independent (12) or dependent (19) of
the size of the DNA in the range of 24-30 MDal, but a lower size limit for the vector is
set by the requirement for the bacteriophage genes for plaque formation. This
requirement is circumvented in the cosmid cloning system, which is independent of
phage genes responsible for lytic growth. The space thus provided can be taken up by

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DNA to be cloned. Because of the small region required for plasmid replication it is to be
expected that new derivatives for use with other restriction enzymes will be rapidly
developed. Cosmid derivatives of pJC720 and pJC703 have been produced in which
cloning with Bgl II or BamHI can be carried out by using rifampicin selection, with Sal I,
EcoRI, Bgl II, or BamHI by using ampicillin selection, or with Xma I, Kpn I, and Pst I by
using selection for colicin immunity (unpublished results). In addition, a series of cosmid
vectors have been developed (unpublished results), including an 8-MDal cosmid (pJC75-
58) for use with EcoRI, BamHI, and Bgl II which is temperature sensitive, ampicillin
resistant, and mobilization-minus. It is hoped that in conjunction with incapacitated host
strains (20) this latter cosmid will provide an EKII host-vector system that will be most
effective in the production of gene banks of eukaryotic DNA. The packaging of
cosmidsin X particles should allow the use of many standard genetic tricks, previously
only applicable to X, for the selection of deletions or insertions in cloned fragments. Such
selection methods are based on the instability of full bacteriophage heads in chelating
agents, the positive selection for large molecules on infection of pel- hosts, or the
physical separations possible on the basis of density differences between full and partially
filled X particles. We thank Hildburg Stephan for her expert technical assistance, Ulla
Hartmann for preparation of the partially HindIII-cleaved Pseudomonas AM1 DNA, and
DietmarBlohm and Renate Bonewald for preparation of the RIdrdl9 plasmid DNA.
Financial support was provided in part by a grant to T. Hohn from the Swiss National
Fonds

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 Ways to get DNA into bacteria cells

Plasmids, BACs &

sometimes PACs
Cosmids&
sometimes PACs

Lambda
Recombinant DNAs

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Recombinant DNA Library

Collection of many clones derived from a single DNA source.

Genomic Libraries:

Many clones, each of which contains a fragment of chromosomal DNA from a particular
species.

Complete genomic library: Entire genome is represented in at least one clone.

1. Digest chromosomal DNA & vector DNA with restriction enzyme(s).

2. Isolate desired size range of chromosomal DNA fragments.

(Gel Electrophoresis)

3. Ligate chromosomal restriction fragments with vector DNA.

Either package clo Selection:

Only recombinant phage produce plaques.

1. Size limits of DNA that fits into phage particle.

left l arm (15kb) + right l arm (10kb) +

insert (~15kb) = 40kb

2. Requirement for l genetic information:

cos sites & genes in right & left l arms

nes (l and cosmids) or use DNA to transform E. coli (PACs and BACs

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Cosmids:

Plasmids that contain l cos sites.

This allows them to be packaged into l particles.

But don’t have l genes, therefore can clone larger insert fragments; 35-45 kb.

PACs:

Based on P1 bacteriophage.

Replicate as low copy number plasmids inside E. coli.

Can clone inserts in 80-100 kb range.

BACs:

“Bacteria Artificial Chromosomes”

Based on 7 kb F1 plasmid (Ch. 7).

Can clone inserts in 150-300 kb range.

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references
1. Hohn B, Hohn T. Activity of empty, headlike particles for packaging of DNA of
bacteriophage lambda in vitro. ProcNatlAcadSci U S A. 1974 Jun;71(6):2372–2376.
2. Kaiser D, Syvanen M, Masuda T. DNA packaging steps in bacteriophage lambda head
assembly. J Mol Biol. 1975 Jan 15;91(2):175–186.
3. Becker A, Murialdo H, Gold M. Studies on an in vitro system for the packaging and
maturation of phage lambda DNA. Virology. 1977 May 1;78(1):277–290.
4. Becker A, Marko M, Gold M. Early events in the in vitro packaging of bacteriophage
lambda DNA. Virology. 1977 May 1;78(1):291–305.
5. Hohn T, Katsura I. Structure and assembly of bacteriophage lambda. Curr Top
MicrobiolImmunol. 1977;78:69–110.
6. Feiss M, Fisher RA, Crayton MA, Egner C. Packaging of the bacteriophage lambda
chromosome: effect of chromosome length. Virology. 1977 Mar;77(1):281–293.
7. Syvanen M. In vitro genetic recombination of bacteriophage lambda. ProcNatlAcadSci U
S A. 1974 Jun;71(6):2496–2499.
8. Hohn B. DNA as substrate for packaging into bacteriophage lambda, in vitro. J Mol
Biol. 1975 Oct 15;98(1):93–106.
9. Emmons SW, MacCosham V, Baldwin RL. Tandem genetic duplications in phage
lambda. III. The frequency of duplication mutants in two derivatives of phage lambda is
independent of known recombination systems. J Mol Biol. 1975 Jan 15;91(2):133–146.
10. Hohn B, Murray K. Packaging recombinant DNA molecules into bacteriophage particles
in vitro. ProcNatlAcadSci U S A. 1977 Aug;74(8):3259–3263.
11. Morrison DA. Transformation in Escherichia coli: cryogenic preservation of competent
cells. J Bacteriol. 1977 Oct;132(1):349–351.

Collins J. Gene cloning with small plasmids. Curr Top MicrobiolImmunol. 1977;78:122–
170.[P

Emmons SW. Bacteriophage lambda derivatives carrying two copies of the cohesive end
site. J Mol Biol. 1974 Mar 15;83(4):511–525.

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 Umene K, Shimada K, Takagi Y. Packaging of ColE1 DNA having a lambda phage cohesive
end site. Mol Gen Genet. 1978 Feb 7;159(1):39–45. [

Clarke L, Carbon J. A colony bank containing synthetic Col El hybrid plasmids

representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. [

Sternberg N, Tiemeier D, Enquist L. In vitro packaging of a lambda Dam vector containing

EcoRI DNA fragments of Escherichia coli and phage P1. Gene. 1977 May;1(3-4):255–280. . 1.
Hohn, B. &Hohn, T. (1974) Proc. Natl. Acad. Sci. USA 71, 2372-2376. 2. Kaiser, D., Syvanen,
M. & Masuda, T. (1975) J. Mol. Biol. 91, 175-186. 3. Becker, A., Murialdo, H. & Gold, M.
(1977) Virology 78,227- 290. 4.

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