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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 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. Selection against wildtype cosmid 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 CFU 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 library are also stored and screened easier 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.

2. Phagemids

Phagemids combine desirable properties of both plasmids and filamentous phages. They carry

• the ColEl origin of replication,

• a selectable marker such as antibiotic resistance,

• the major intergenic region of a filamentous phage .

The segments of foreign DNA cloned in these vectors can be propagated as plasmids. When cells harboring these plasmids are infected with a suitable helper bacteriophage, the mode of replication of the plasmid changes under the influence of the gene II product of the incoming virus.

Interaction of the intergenic region of the plasmid with the gene II protein initiates the rolling-circle replication to generate copies of one strand of the plasmid DNA, which are packaged into progeny bacteriophage particles. The single-stranded DNA purified from these particles is used as a template to determine the nucleotide sequence of one strand of the foreign DNA segment, for site-directed mutagenesis or as a strand-specific probe. Phagemids provide high yields of double-stranded DNA and render unnecessary the time-consuming process of subcloning DNA fragments from plasmids to filamentous bacteriophages.

2. Bacteriophage Vectors

Both single-stranded (filamentous) and double-stranded E.coli phages have been exploited as cloning vectors.

Frederick Twort (1915) and Felix d’Herelle (1917) were the first to recognize viruses which infect bacteria, which d'Herelle called bacteriophages (eaters of bacteria). [7]

Figure 5. Frederick Twort and Felix d’Herelle

4.1 Filamentous phages

Filamentous phages are not lytic. They coexist with the infected cells for several generations and are convenient for cloning genes which produce toxic products. Among the filamentous phages, fd, fl, and M13 have been well characterized and their genomes have been sequenced [4]. Their gene functions and molecular mode of propagation are very similar. They infect cells via F pili, and the first mature phage appears within 15 min [6].

Phage M13 is widely used in nucleotide sequencing and site-directed mutagenesis since its genome can exist either in a single-stranded form inside a phage coat or as a doublestranded replicative form within the infected cell. During replication, only the plus strand of the replicative form is selectively packaged by the phage proteins [1]. The replicative form is a covalently closed circular molecule and hence can be used as a plasmid vector and transformed into the host by the usual transformation procedures. The vectors derived from M13, have the same polylinker as that of pUC18 and pUC19, respectively [2]. The DNA fragments having noncomplementary ends can be directionally cloned in this pair of vectors, and the two strands of DNA can be sequenced independently.

4.2.Double-stranded phage vectors

Of the double-stranded phages, bacteriophage lambda-derived vectors are the most popular tools for several reasons:

• acceptance by the phage of large foreign DNA fragments, thereby increasing the chances of screening a single clone carrying a DNA sequence corresponding to a complete gene;

• development and availability of refined techniques aimed at minimizing the problems of background due to nonrecombinants;

• the possibility of screening several thousand clones at a time from a single petri plate; and, finally,

• the ease with which the phage library can be stored as a clear lysate at 4°C for months without significant loss in plaque-forming activity [7].

Recently, a bacteriophage P1 cloning system has been developed which permits cloning of DNA fragments as large as 100 kbp with an efficiency that is intermediate between cosmids and yeast artificial chromosomes .

2. Scope of Present Review

The extensive knowledge of the basic biology of lambda has permitted modifications of its genome to suit the given experimental conditions. In the present review we describe how the utility of lambda as a cloning vector rests essentially in its intrinsic molecular organization. The following sections give an account of various problems encountered in constructing lambda vectors and the strategies that have been adopted to overcome them. A few commonly used vectors are described in detail, taking into account their special values and limitations. The different methods for screening and storage of genomic and cDNA libraries in lambda vectors are also discussed.

2. Life cycle and genetics of Lambda

An understanding of the basic biology of lambda, its mode of propagation, and the genetic and molecular mechanisms that control its life cycle is needed before its applications for genetic manipulations are discussed. This section deals with the basic biology of lambda.

The lambda virus particle contains a linear DNA of 48,502 bp with a single-stranded 5' extension of 12 bases at both ends; these extensions are complementary to each other.

These ends are called cohesive ends or cos. During infection, the right 5' extension (cosR), followed by the entire genome, enters the host cell. Both the cos ends are ligated by E. coli DNA ligase, forming a covalently closed circular DNA which is acted upon by the host DNA gyrase, resulting in a supercoiled structure.

6.1 Development of Lambda

Two Alternative Modes. After infecting the host, the lambda genome may start its replication; this results in the formation of multiple copies of the genome. The protein components necessary for the assembly of mature phage particles are synthesized by the coordinated expression of phage genes. Phage DNA is packaged inside a coat, and the mature phages are released into the environment after cell lysis. This mode of propagation is called the lytic cycle.

Alternatively, the phage genome may enter a dormant stage (prophage) by integrating itself into a bacterial genome by site-specific recombination; during this stage it is propagated along with the host in the subsequent progeny. This stage is termed lysogeny. Changes in environmental and physiological conditions may activate the prophage stage and trigger lytic events.

2. Phage Lambda as a vector

Figure 6. Bacteriophage

The large genome size and complex genetic organization of lambda had posed initial problems with its use as a vector. The problems, however, were surmounted through the sustained efforts of researchers, and lambda has been developed into an efficient vector.

The broad objectives in constructing various phage vectors are

• the presence of cloning sites only in the dispensable fragments,

• the capacity to accommodate foreign DNA fragments of various sizes,

• the presence of multiple cloning sites,

• an indication of incorporation of DNA fragments by a change in the plaque type,

• the ability to control transcription of a cloned fragment from promoters on the vector,

• the possibility of growing vectors and clones to high yield,

• easy and ready recovery of cloned DNA,

• introduction of features contributing to better biological containment.

There are several difficulties in the use of lambda as a vector.

Some of the problems and the general strategies adopted to overcome them are discussed in this section. Manipulation of Restriction Sites The major obstacle to the use of phage lambda as a cloning vector was essentially the presence of multiple recognition sites for a number of restriction enzymes in its genome.

Initially, all attempts were directed toward minimizing the number of EcoRI sites. Murray and Murray in 1974 were able to construct derivatives of lambda with only one or two EcoRI sites. Similarly, Rambach and Toillais constructed lambda derivatives with EcoRI sites only in the nonessential region of the genome by repeated transfer on restrictive and nonrestrictive hosts . After several cycles of digestion, packaging, and growth, phage derivatives with desirable restriction sites and full retention of infectivity were obtained. All but one HindIII sites were removed by recombination of known deletion mutants or substitutions. Recently, oligonucleotides with specific sequences have been synthesized and introduced into the bacteriophage lambda genome. This has provided a variety of cloning sites in the genome [5].

7.1 Size Limitation for Packaging

The second problem was the requirement of a minimum and maximum genome length (38 and 53 kbp, respectively) for the efficient packaging and for the production of viable phage particles. The viability of the bacteriophage decreases when its genome length is greater than 105% or less than 78% of that of wild-type lambda. Genetic studies of specialized transducing bacteriophages showed, however, that the central one-third of the genome, i.e., the region between the J and Ngenes, is not essential for lytic growth. The presence of a nonessential middle fragment of the phage genome was also revealed during construction of viable deletion mutants. These mutants lack most of the two central EcoRI B fragments which are not essential for lytic growth. However, too much DNA cannot be deleted because there is a minimum 38-kbp requirement essential for efficient packaging. The de novo insertion of DNA (even if heterogeneous) is essential for the formation of viable phages. This constitutes a positive selection for recombinant phages carrying insertions. This approach was successfully exploited in constructing recombinant phages carrying E. coli and Drosophila melanogaster DNA [8].

7.2 Transfection of Recombinant Molecules

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