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 .

5. 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.

6. 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.

7. 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

The problem of transfection of recombinant molecules constructed in vitro was overcome by the successful in vitro assembly of viable and infectious phage particles. Two types of in vitro packaging systems have been developed so far, i.e., two-strain packaging and single-strain packaging.

Two-strain packaging.

The basis of the two-strain in vitrop ackaging system is the complementation of two amber mutations. Two lambda lysogens, each carrying a single amber mutation in a distinctly different gene, are induced and grown separately so that they can synthesize the necessary proteins. Neither of the lysogens alone is capable of packaging the phage DNA. The role of various phage products in DNA packaging has been studied in detail[3]. The E protein is the major component of the bacteriophage head, and in its absence all the viral capsid components accumulate. The D protein is involved in the coupled process of insertion of bacteriophage DNA into the prehead precursor and the subsequent maturation of the head. The A protein is required for the cleavage of the concatenated precursor DNA at the cos sites. Two phage lysogens carrying A and E or D and E mutations in the phage genome are induced separately, and cell extracts are prepared. Neither of the extracts can produce infectious phage particles. However, when the extracts are mixed, mature phage particles are produced by complementation.

The major drawback of the two-strain system is the competition of native phage DNA with recombinant molecules. In both the cell extracts, native phage DNA is also present and can be packaged with an efficiency equal to that of the chimeric DNA. This reduces the proportion of recombinants obtained in a library. The problem of regeneration of endogenous phages obtained in the library was partially overcome by the use of b2-deleted prophages, which poorly excise out of the host chromosome or by UV irradiation of packaging extracts.

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