Cosmid vectors

7 Key excerpts on "Cosmid vectors"

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  eBook - ePub

  Nonmammalian Genomic Analysis

  A Practical Guide

  • Bruce Birren, Eric Lai(Authors)
  • 1996(Publication Date)
  • Academic Press

    (Publisher)

  7

  Cosmid Cloning with Small Genomes

  Rainer Wenzel and Richard Herrmann

  I. Introduction

  Cosmid vectors are useful tools for establishing gene libraries, as has been shown for a number of different prokaryotic and eukaryotic species, like the nematode Caenorhabditis elegans (

  Coulson et al. , 1986

  ) or the bacteria Mycoplasma pneumoniae (Wenzel and Herrmann, 1989 ), Haloferax volcanii (

  Charlebois et al. , 1991

  ), Mycobacterium leprae (

  Eiglmeier et al. , 1993

  ), and Helicobacter pylori (Bukanov and Berg, 1994 ). Their cloning capacity of up to 51 kbp keeps the number of clones required for screening at a manageable range, especially when relatively small prokaryotic genomes have to be analyzed. Since, due to their size, transformation efficiencies of cosmids are rather low, introduction of cosmids into bacterial cells is usually done by in vitro packaging into phage λ particles followed by infection of the host cell. Though packaging efficiency of cosmids is not as good as for phage λ-derived vectors, the cosmids do have the advantage of containing high copy number replicons which give high yields of DNA.

  In the following, various strategies for the construction of an ordered set of cosmid clones representing a bacterial genome are discussed in general. As a guideline, the cloning of the entire genome of the bacterium M. pneumoniae in a contiguous set of cosmid clones is described (Wenzel and Herrmann, 1989 ). This small human pathogenic bacterium has a genome size of about 800 kbp (

  Wenzel et al. , 1992

  ) which generally can be considered to be close to the lower limit of the coding capacity of a self-replicating cell (Morowitz, 1984

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  Introduction to Plant Biotechnology (3/e)

  • H S Chawla(Author)
  • 2011(Publication Date)
  • CRC Press

    (Publisher)

  Cleavage at cos by the λ terminase protein during phage packaging produces a 12-nucleotide cohesive end at the termini of the linear λ genome. Recircularization of the λ genome after bacterial infection is facilitated by base pairing between the complementary cohesive ends. Cosmids were developed to overcome the technical problem of introducing large pieces of DNA into E. coli. Thus, bacteriophage cos DNA sequence was introduced that is required for packaging DNA into preformed phage heads. For successful packaging of DNA, there must be two cos sequences separated by a distance of 38–52 kb. Cosmid cloning vectors with DNA inserts of 30–45 kb can be packaged in vitro into λ phage particles, provided that the ligated double-stranded DNA contains λ cos sequences on either side of the insert DNA. Because the λ phage head can hold up to 45 kb of DNA, the optimal ligation reaction in cosmid cloning produces recombinant molecules with cos sequences flanking DNA segments of ~40 kb. Following adsorption of these phage particles on to suitable E. coli host cells, the cosmid vector circularizes via the cohesive ends and replicates as a plasmid. Cosmid vectors possess an origin of replication, a selectable genetic marker (antibiotic resistance), and suitable cloning sites. For this reason, cosmids are ideal vectors for genome mapping. Like plasmids, cosmids can multiply in large copy number using bacterial ori of replication (ColE1) inside the bacterial cell and do not carry the genes for lytic development. The advantages of cosmids are that (a) relatively large size of insert DNA (up to 45 kb) can be cloned; and (b) DNA can be introduced into the host using bacteriophages derived by in vitro packaging. The disadvantages are that (a) it is difficult to store bacterial host as glycerol stock; and (b) in vitro packaging is needed to maintain cosmids inside the viral heads. BACTERIOPHAGE VECTORS Bacteriophages are viruses that infect bacteria. These are usually called phages

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  BIOS Instant Notes in Molecular Biology

  • Alexander McLennan, Andy Bates, Phil Turner, Michael White(Authors)
  • 2012(Publication Date)
  • Taylor & Francis

    (Publisher)

  The in vitro packaging of DNA into λ particles (see above) requires only the presence of the λ cos sites spaced by the correct distance (37–52 kb) on linear DNA. The intervening DNA can have any sequence at all; it need not contain any λ genes, for example. The simplest cosmid vector is a normal small plasmid containing a plasmid origin of replication (ori) and a selectable marker, and which also contains a cos site and a suitable restriction site for cloning (Figure 4). After cleavage with a restriction enzyme and ligation with target DNA fragments, the DNA is packaged into λ phage particles. The DNA is re-circularized by annealing of the cos sites after infection, and propagates as a normal plasmid, under selection by ampicillin. As in the case of phage λ (see above), more sophisticated methods are used in a real cloning situation to ensure that multiple copies of the vector or the target DNA are not included in the recombinant. Libraries of clones prepared with Cosmid vectors can be screened as described in Section Q3. The genomes of entire bacteria such as E. coli are available as a set of around 100 cosmid clones. YAC vectors The realization that the components of a eukaryotic chromosome required for stable replication and segregation (at least in the budding yeast Saccharomyces cerevisiae) consist of rather small and well-defined sequences (Section C3) has led to the construction of recombinant chromosomes (yeast artificial chromosomes; YACs). These can be used as vectors for carrying very large cloned fragments. The centromere, telomere, and replication origin sequences (Sections C3, D1, and D3) have been isolated and combined on plasmids constructed in E. coli. The structure of a typical pYAC vector is shown in Figure 5. The method of construction of the YAC clone is similar to that for cosmids in that two end fragments are ligated with target DNA to yield the complete chromosome, which is then introduced (transfected) into yeast cells (Section P3)

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  Introduction to Biotechnology and Biostatistics

  • Khushboo Chaudhary(Author)
  • 2019(Publication Date)
  • Delve Publishing

    (Publisher)

  5.2. CLONING VECTOR FOR RECOMBINANT DNA One of the most important use of recombinant DNA technology in the cloning of (i) random DNA or cDNA segments, often use as probe or (ii) specific genes, Introduction to Biotechnology and Biostatistics 132 which may be either isolated from the genome or synthesized organochemically or in the form c DNA from m RNA. This other DNA molecule is often used in the form of a vector, which could be a plasmid, a bacteriophage, a derived cosmid or phagemid, a transposon or even a virus. Techniques should also be available, which will allow selection of chimeric genomes obtained after insertion of foreign DNA from a mixture of chimeric and the original vector. Another critical desired feature of any cloning vector is that it should posses a site at which foreign DNA can be inserted without disrupting any essential function. Therefore, in each case an enzyme will also have to be selected which will cause a single break. Sometimes vectors are modified by inserting a DNA segment to create unique site for one or more enzymes to facilitate its use in gene cloning. This inserted DNA with restriction sites for several enzymes is sometimes called a polylinker. 5.3. PLASMIDS AS VECTORS Plasmids are defined as autonomous elements, whose genomes exist in the cell as extrachromosomal units. They are self-replicating circular duplex DNA molecules, which are maintained in a characteristic number of copies in a bacterial cell, yeast cell or even in organelles found in eukaryotic cells. These plasmids can be single copy plasmids that are maintained as one plasmid DNA per cell or multicopy plasmids, which are maintained as 10–20 genomes per cell. These are also plasmids, which are under relaxed replication control, thus permitting their accumulation in very large numbers. These are the plasmids which are used as cloning vectors, due to their increased yield potential.

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  Insect Molecular Genetics

  An Introduction to Principles and Applications

  • Marjorie A. Hoy(Author)
  • 2013(Publication Date)
  • Academic Press

    (Publisher)

  This vector contains a cos site, a restriction site for inserting exogenous DNA, and a gene for ampicillin resistance. Exogenous DNA is cut with an appropriate restriction enzyme, as is the vector. The vector and exogenous DNA are ligated together, producing a recombl· nant molecule 37-52 kb long, which can be packaged in λ by in vitro packaging. The packaged vector infects E. coli, injecting its DNA into the host, where it circularizes and multiplies. E. coli cells that receive the cosmid are distinguished from cells that are not infected by their ability to survive on media containing ampicillin. 166 Chapter 7 Cloning and Expression Vectors, Libraries, and Their Screening While having a large capacity for DNA fragments is a benefit in cloning with cosmids, it can be a detriment. If, during a partial digestion with restriction enzymes, two or more genomic DNA fragments join together in the ligation reaction, a clone could be created with fragments that were not initially adjacent to each other. This could be a problem if the researcher is interested in the relationship between a gene of interest and the DNA surrounding it on the chromosome. The problem can be overcome by size fractionating the partial digest. However, even then, cosmid clones could be produced that contain noncontiguous DNA fragments ligated to form a single insert. This problem can be solved by dephosphorylating the foreign DNA fragments to prevent them from ligating together, but this makes cosmid cloning very sensitive to the exact ratio of insert and vector DNAs. If the ratio is unbalanced, vector DNAs could ligate together without containing any exogenous DNA insert. This is resolved by treating the vector to two separate digestions, which generate vector ends that are incapable of ligating to each other after phosphatasing. Commonly used Cosmid vectors include the pJB8 and the pcosEMBL family. pJB8 is probably most useful for constructing genomic libraries.

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  Dictionary of DNA and Genome Technology

  • Paul Singleton(Author)
  • 2012(Publication Date)
  • Wiley-Blackwell

    (Publisher)

  inserts will permit ligation to cosmids and subsequent packaging (see the entry SUPERCOS I VECTOR for an alternative approach).

  cosmid walking

  A procedure, similar to CHROMOSOME WALKING (q.v.), in which a clone contig is prepared from a COSMID library. Because the inserts in cosmids are quite short, it is possible to use the entire sequence as a probe – thus avoiding the need to prepare end probes (as is necessary in chromosome walking).

  co-suppression

  See the entry PTGS.

  cotrimoxazole

  See the entry ANTIBIOTIC (synergism).

  counterselection

  Syn. NEGATIVE SELECTION (sense 2).

  coupled transcription–translation(DNA technol.)

  See entry entry CELL-FREE PROTEIN SYNTHESIS.

  coxsackieviruses

  See the entry ENTEROVIRUSES.

  Cosmid: a cosmid cloning vector (diagrammatic). The cosmid (top) is a small plasmid into which has been inserted the cos site (solid black square) of phage λ; cos enables the plasmid, together with its insert, to be packaged (in vitro) in a phage λ head and subsequently to be injected into a bacterial cell for cloning. Cosmids are cut at a single restriction site, linearizing the molecule and leaving the cos site in a non-terminal position. The linearized cosmid molecules are then mixed, in the presence of DNA ligase, with DNA fragments (dashed lines) which have been cut with the same restriction enzyme. Cosmids and fragments bind randomly, via their sticky ends, and in some cases a fragment will be flanked by two cosmids (center); if, in such a molecule, the distance cos-to-cos is about 40–50 kb long, then the cos–cos sequence is the right size for packaging in the phage λ head. Enzymic cleavage at the cos sites (arrowheads) creates sticky ends, and packaging of the recombinant molecule in the phage λ head occurs in vitro in the presence of phage components. After addition of the tail, the phage particle can inject its recombinant DNA into a suitable bacterium (just like an ordinary phage λ). The injected DNA circularizes, via the cos sites, and replicates as a plasmid; its presence in the cell can be detected e.g. by the expression of plasmid-encoded antibiotic-resistance genes. Figure reproduced from Bacteria in Biology, Biotechnology and Medicine

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  Mammalian Cell Biotechnology in Protein Production

  • Hansjörg Hauser, Roland Wagner, Hansjörg Hauser, Roland Wagner(Authors)
  • 2011(Publication Date)
  • De Gruyter

    (Publisher)

  In summary, the origin-vector system in combination with the COS cells is a convenient and versatile expression system for research purposes aimed at short-term overexpression or cloning of particular genes. Since origin-containing plasmid vectors are replicated efficiently, every sue- 1.3 Vectors for Gene Transfer and Expression in Animal Cells 67 cessfully transfected cell will express the gene of interest. There is no recent review of this system. For the reader interested in the history of viral vectors we recommend (Gluzman, 1982; Sambrook, 1987; Strauss et al., 1986). 1.3.3 Vectors Derived from Papilloma Viruses Papilloma viruses were originally classified as members of the papovavirus family (Melnick et al., 1974) because they also have a closed circular double-stranded DNA genome which is complexed with histones, condensed into nucleosomes, and en-capsulated in an icosahedral virion. However, molecular genetics studies have shown that the papillomaviruses constitute a distinct group of viruses. Their genomes are 50 % larger (~ 7900 bp) than SV40 and polyoma virus, and there are virtually no similarities in the sequences. Most papillomaviruses have a single host and grow only in differentiated cutaneous or mucosal epithelium at specific anatomical sites (Broker and Botchan, 1986). There are papillomaviruses of diverse kinds in various vertebrates, including more than forty subtypes in humans (Pfister et al., 1986). They are all known to persist in the form of their episomal DNA in their natural host tissues, the differentiated keratinocytes of epithelia and they are the causative agents of warts (Broker and Botchan, 1986). The only papillomavirus genome which has been used extensively as a vector up to now is the bovine papillomavirus subtype 1 (BPV). The transfor-ming and replication functions of this virus have been studied in great detail.

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