Bacteriophages

Bacteriophages

	Bacteriophages Another suitable cloning vehicle is the DNA of the lambda (λ) phage of E. coli. The lambda phage DNA is linear and double helical for the most part. The ends, however, contain complementary unpaired segments, 12 nucleotides long, on each strand. After injection of the linear viral DNA into E.coli, circularization of the molecule takes place. Pairing of the complementary nucleotides at the ends of linear DNA first occurs. The two remaining nicks are then sealed by the enzyme polynucleotide ligase
Insertion of the lambda phage DNA into the E. coli DNA takes place at a specific site between the gal and the bio operons. The phage DNA circle is opened at another point and spliced to cleaved host DNA with the help of the enzyme integrase. The E. coli DNA now becomes lysogenic and contains phage DNA (provirus). After several cell generations the provirus may be induced to enter the lytic stage by radiation or chemical compounds. Excision of phage DNA from the bacterial DNA requires integrase and another viral gene enzyme called excisionase. Phage DNA is now converted back to the circular form. The circular phage DNA replicates by the rolling circle method, producing several copies. These are cleaved by a specific viral enzyme to form linear DNA with sticky ends
Wild type phage DNA has five EcoRI cleavage sites and a site for Hind III. Derivatives with one, two or three cleavage sites can be obtained by selecting and recombining different mutants. A lambda derivative with two restriction cleavage sites is used for cloning purposes. Cleavage at the two sites permits the removal of a central nonessential segment. Foreign DNA can now replace the excised segment. If the lambda phage is less than 75% of its length, or more than 109%, it does not undergo encapsulation into virus partic1es. This property is utilized to insert small segments of foreign DNA.
Removal of a central fragment between two EcoRI sites results in two fragments with a total length of only 71%. Only small DNA partic1es, up to 17,000 kb length, can be inserted to give DNA which can undergo encapsulation to form viral particles. Lambda dv plasmids with one EcoRI site in the replicating region contain portions of the lambda genome. Simian virus 40 (SV40) has been employed as a cloning vehicle in eukaryotic cells.

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