Cosmids, Phasmids and Other Advanced Vectors
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POGC05 9/11/2001 10:57 AM Page 64 CHAPTER 5 Cosmids, phasmids and other advanced vectors along the genome to isolate a gene, and this topic is Introduction covered in Chapter 6. In many instances, the desired In the 1970s, when recombinant DNA technology gene will be relatively easy to isolate and a simpler was first being developed, only a limited number cloning vector can be used. Once isolated, the cloned of vectors were available and these were based on gene may be expressed as a probe sequence or as a either high-copy-number plasmids or phage λ. Later, protein, it may be sequenced or it may be mutated phage M13 was developed as a specialist vector to in vitro. For all these applications, small specialist facilitate DNA sequencing. Gradually, a number of vectors are used. purpose-built vectors were developed, of which pBR322 is probably the best example, but the cre- Vectors for cloning large ation of each one was a major task. Over time, a fragments of DNA series of specialist vectors was constructed, each for a particular purpose. During this period, there Cosmid vectors were many arguments about the relative benefits of plasmid and phage vectors. Today, the molecular As we have seen, concatemers of unit-length λ DNA biologist has available an enormous range of vectors molecules can be efficiently packaged if the cos sites, and these are notable for three reasons. First, many substrates for the packaging-dependent cleavage, of them combine elements from both plasmids and are 37–52 kb apart (75–105% the size of λ+ DNA). phages and are known as phasmids or, if they contain In fact, only a small region in the proximity of the an M13 ori region, phagemids. Secondly, many differ- cos site is required for recognition by the packaging ent features that facilitate cloning and expression system (Hohn 1975). can be found combined in a single vector. Thirdly, Plasmids have been constructed which contain a purified vector DNA plus associated reagents can fragment of λ DNA including the cos site (Collins & be purchased from molecular-biology suppliers. Brüning 1978, Collins & Hohn 1979, Wahl et al. The hapless scientist who opens a molecular-biology 1987, Evans et al. 1989). These plasmids have been catalogue is faced with a bewildering selection of termed cosmids and can be used as gene-cloning vectors and each vender promotes different ones. vectors in conjunction with the in vitro packaging Although the benefits of using each vector may be system. Figure 5.1 shows a gene-cloning scheme clear, the disadvantages are seldom obvious. The employing a cosmid. Packaging the cosmid recombin- aim of this chapter is to provide the reader with a ants into phage coats imposes a desirable selection detailed explanation of the biological basis for the upon their size. With a cosmid vector of 5 kb, we different designs of vector. demand the insertion of 32–47 kb of foreign DNA – There are two general uses for cloning vectors: much more than a phage-λ vector can accommod- cloning large pieces of DNA and manipulating ate. Note that, after packaging in vitro, the particle genes. When mapping and sequencing genomes, is used to infect a suitable host. The recombinant the first step is to subdivide the genome into man- cosmid DNA is injected and circularizes like phage ageable pieces. The larger these pieces, the easier it is DNA but replicates as a normal plasmid without to construct the final picture (see Chapter 7); hence the expression of any phage functions. Transfor- the need to clone large fragments of DNA. Large med cells are selected on the basis of a vector drug- fragments are also needed if it is necessary to ‘walk’ resistance marker. POGC05 9/11/2001 10:57 AM Page 65 Cosmids, phasmids and advanced vectors 65 Cosmid ApR Restriction endonuclease target site Foreign DNA restriction fragment cos Restriction endonuclease DNA ligase (high DNA concentration) ++etc. cos+ cos 37–52 kb Packaging in vitro Absorption + injection R Transducing particle Ap containing cosmid recombinant cos Select ApR clone Fig. 5.1 Simple scheme for cloning in a DNA circularizes after cosmid vector. (See text for details.) injection Cosmids provide an efficient means of cloning this way. This is due to the possibility of two or large pieces of foreign DNA. Because of their capa- more genome fragments joining together in the city for large fragments of DNA, cosmids are par- ligation reaction, hence creating a clone containing ticularly attractive vectors for constructing libraries fragments that were not initially adjacent in the of eukaryotic genome fragments. Partial digestion genome. This would give an incorrect picture of with a restriction endonuclease provides suitably their chromosomal organization. The problem can large fragments. However, there is a potential prob- be overcome by size fractionation of the partial lem associated with the use of partial digests in digest. POGC05 9/11/2001 10:57 AM Page 66 66 CHAPTER 5 BamHI HindIII PstI EcoRI EcoRI ApR cos pJB8 (5.4 kb) SalI BamHI HindIII Target genomic DNA cos SalI (1) HindIII (1) SalI (2) Phosphatase (2) Phosphatase H H S S (1) Partial HO HO HO OH Sau3A cos B B cos digestion BamHI BamHI H BBH S BB S (2) Phosphatase HO HO HO OH cos cos Sau3A Sau3A Mix Ligate HO HO 32–47 kb Fig. 5.2 Cosmid cloning scheme of (B) (B) Ish-Horowicz and Burke (1981). (a) H S HO HO Map of cosmid pJB8. (b) Application cos cos to the construction of a genomic library 37–52 kb of fragments obtained by partial Only packageable molecules digestion with Sau3A. This restriction endonuclease has a tetranucleotide recognition site and generates Package fragments with the same cohesive in vitro termini as BamHI (see p. 31). Even with sized foreign DNA, in practice cosmid host by recombination, resulting in the propagation clones may be produced that contain non-contiguous of a non-recombinant cosmid vector. DNA fragments ligated to form a single insert. Such difficulties have been overcome in a cosmid- The problem can be solved by dephosphorylating cloning procedure devised by Ish-Horowicz and the foreign DNA fragments so as to prevent their Burke (1981). By appropriate treatment of the cos- ligation together. This method is very sensitive to mid vector pJB8 (Fig. 5.2), left-hand and right-hand the exact ratio of target-to-vector DNAs (Collins & vector ends are purified which are incapable of self- Brüning 1978) because vector-to-vector ligation ligation but which accept dephosphorylated foreign can occur. Furthermore, recombinants with a dup- DNA. Thus the method eliminates the need to size licated vector are unstable and break down in the the foreign DNA fragments and prevents formation POGC05 9/11/2001 10:57 AM Page 67 Cosmids, phasmids and advanced vectors 67 R Ap cos c2XB KmR (6.8 kb) SmaI cos rep BamHI Target genomic DNA SmaBam Bam Sma (Blunt) (Blunt) R cosAp rep cos (1) Partial Sau3A digestion (2) Phosphatase Sau3A Sau3A Mix Ligate 32–45 kb Fig. 5.3 Cosmid cloning scheme of (B) (B) Bates and Swift (1983). The cosmid c2XB Sma Sma (Blunt) (Blunt) contains two cos sites, separated by a site cos ApR rep cos for the restriction endonuclease SmaI which 37–52 kb creates blunt ends. These blunt ends ligate Only packageable molecules only very inefficiently under the conditions used and effectively prevent the formation of recombinants containing multiple Package copies of the vector. in vitro of clones containing short foreign DNA or multiple promoters flanking the cloning site; and (iii) unique vector sequences. NotI, SacII or Sfil sites (rare cutters, see Chapter 6) An alternative solution to these problems has flanking the cloning site to permit removal of the been devised by Bates and Swift (1983) who have insert from the vector as single fragments. Mam- constructed cosmid c2XB. This cosmid carries a malian expression modules encoding dominant BamHI insertion site and two cos sites separated by a selectable markers (Chapter 10) may also be present, blunt-end restriction site (Fig. 5.3). The creation of for gene transfer to mammalian cells if required. these blunt ends, which ligate only very inefficiently under the conditions used, effectively prevents BACs and PACs as alternatives to cosmids vector self-ligation in the ligation reaction. Modern cosmids of the pWE and sCos series (Wahl Phage P1 is a temperate bacteriophage which has et al. 1987, Evans et al. 1989) contain features such been extensively used for genetic analysis of as: (i) multiple cloning sites (Bates & Swift 1983, Escherichia coli because it can mediate generalized Pirrotta et al. 1983, Breter et al. 1987) for simple transduction. Sternberg and co-workers have devel- cloning using non-size-selected DNA; (ii) phage oped a P1 vector system which has a capacity for POGC05 9/11/2001 10:57 AM Page 68 68 CHAPTER 5 Short arm pac loxP Stuffer + Insert DNA P1 and plasmid r replicons: kan , loxP Ligate Long arm P1 and plasmid pac loxP Insert DNA replicons: kanr, loxP Stuffer 115 kb Pac extract cleaves at pac site and inserts DNA into Fig. 5.4 The phage P1 vector system. P1 heads. Tails are attached P1 heads The P1 vector Ad10 (Sternberg 1990) and tails is digested to generate short and long vector arms. These are dephosphorylated to prevent self-ligation. Size-selected insert DNA + (85–100 kb) is ligated with vector arms, ready for a two-stage processing by packaging extracts. First, the recombinant DNA is cleaved at the pac Allow phage to adsorb site by pacase in the packaging extract.