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Vectors for moleculars cloning

p align="left">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.

4. 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 .

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.

Single-strain packaging.

Rosenberg have successfully developed a single-strain packaging system by introducing deletion in the cos region of prophage, rendering the prophage DNA unpackagable because cos is the packaging origin. Induction of the lysogen results in the intracellular accumulation of all protein components needed for packaging.

However, packaging of phage DNA is prevented by the lack of cos sites on the prophage DNA. On the other hand, exogenous DNA with cos sites is packaged efficiently to produce an infectious bacteriophage particle. The single-strain system is superior to two-strain system in having a lower background of parental phages. In addition, it uses E. coli C, which lacks the EcoK restriction system, as the host for the lysogen.

7.3 Biological Containment

The biological containment of recombinant phages is an important aspect from the point of view of ethics and eventual biohazards. It is desirable that cloning vectors and recombinants have poor survival in the natural environment and require special laboratory conditions for their replication and survival. According to Blattner, the lytic phages offer a natural advantage in this respect since the phage and the sensitive bacteria coexist only briefly. A newly inserted segment may not be compatible with E. coli metabolism for extended periods. To make the phage vectors more safe, three amber mutations were introduced in its genome. The new vector Xgt WES XC is safer because an amber suppressor host strain is a very rare occurrence in the natural environment. Many vectors carry one of the amber mutations on the genome so that they can be propagated only on an appropriate suppressor host.

8. Phage vectors

Many phage vectors have been constructed in the recent past, each with its own special features. There is no universal lambda vector which can fulfill all the desired objectives of the cloning experiments.

The choice of a vector depends mainly on

§ the size of a DNA fragment to be inserted,

§ the restriction enzymes to be used,

§ the necessity for expression of the cloned fragment,

§ the method of screening to be used to select the desired clones.

Bacteriophage lambda vectors can be broadly classified into two types:

1. replacement vectors ,

2. insertion vectors.

Figure 7. Lambda Phage genome

8.1 Replacement Vectors

Taking advantage of the maximum and minimum genome size essential for efficient packaging and the presence of the nonessential central fragment, it is possible to remove the stuffer fragment and replace it with a foreign DNA fragment in the desired size range. This forms the basis of lambdaderived replacement vectors.

Cloning of a foreign DNA in these vectors involves

· preparation of left and right arms by physical elimination of the nonessential region,

· ligation of the foreign DNA fragment between the arms,

· in vitro packaging and infection.

The replacement vectors contain a pair of restriction sites to excise the central stuffer fragments, which can be replaced by a desired DNA sequence with compatible ends. The presence of identical sites within the stuffer fragment but not in the arms facilitates the separation of the arms and the stuffer on density gradient centrifugation. In many vectors, sets of such sites are provided on attached polylinkers so that an insert can be easily excised. Two purified arms cannot be packaged despite their being ligated to each other, because they fall short of the minimum length required for packaging. This provides positive selection of recombinants. The replacement vectors are convenient for cloning of large (in some cases up to 24 kbp) DNA fragments and are useful in the construction of genomic libraries of higher eukaryotes. Charon and EMBL are among the popular replacement vectors.

8.2 Insertion Vectors

Because the maximum packagable size of lambda genome is 53 kb, small DNA fragments can be introduced without removal of the nonessential (stuffer) fragment. These vectors are therefore called insertion vectors. Cloning of foreign DNA in these vectors exploits the insertional inactivation of the biological function, which differentiates recombinants from nonrecombinants. Insertion vectors are particularly useful in cloning of small DNA fragments such as cDNA.

AgtlO and Agtll are examples of this type of vector. In recent years a multitude of lambda vectors have been constructed. Many innovative approaches have been used to introduce desired properties into the vectors. The following section deals with the strategies adopted for the construction of some of the commonly used vectors and their salient features, utilities, and limitations.

8.3 Storage of Lambda Stocks

Most of the lambda strains are stable for several years when stored at 4°C in SM buffer containing 0.3% freshly distilled chloroform (94). The master stocks of bacteriophage lambda are kept in 0.7% (vol/vol) dimethyl sulfoxide at -70°C for long-term storage. Klinman and Cohen have developed a method for storage of a phage library at -70°C by using top agar containing 30% glycerol.


In my work I determined investigations in Molecular cloning, familiarized with vectors for molecular cloning, summarized the received information and made consequences of scientists researches, defined the main tasks of molecular cloning, and made such conclusions:

1. sequences that permit the propagation of itself in bacteria (or in yeast for YACs) .

2. a cloning site to insert foreign DNA; the most versatile vectors contain a site that can be cut by many restriction enzymes .

3. a method of selecting for bacteria (or yeast for YACs) containing a vector with foreign DNA; uually accomplished by selectable markers for drug resistance .

Cloning vector - a DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA .

General Steps of Cloning with Any Vector :

1. prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary ends ;

2. ligate the foreign DNA into the vector with the enzyme DNA ligase;

3. introduce the DNA into bacterial cells (or yeast cells for YACs) by transformation ;

4. select cells containing foreign DNA by screening for selectable markers (usually drug resistance);


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5. Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Bacteriophage л?vectors, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 2.3-2.125.

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