Host/vector interactions which affect the viability of recombinant phage lambda clones.

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Recombinant phage lambda clones derived from human genomic libraries are often inhibited from forming plaques on wild-type Escherichia coli.
• This inhibition poses a challenge for recovering specific genomic sequences using phage lambda cloning systems.

Purpose of the Study:

• To investigate the mechanism underlying the inhibition of specific recombinant phage lambda clones on wild-type E. coli.
• To identify strategies for overcoming this inhibition and developing improved host strains for phage lambda cloning.

Main Methods:

• Recovery of recombinant phage lambda clones from human genomic libraries on defective E. coli strains (recB21 recC22 sbcB15).
• Testing phage growth on wild-type and various mutant E. coli hosts with altered RecBC enzyme function.
• Introduction of the chi sequence and the lambda gam+ gene into inhibited phage clones.

Main Results:

• Introduction of the chi sequence restored plaque formation on Rec+ cells for one inhibited clone.
• Insertion of the lambda gam+ gene (encoding an inhibitor of RecBC enzyme) enabled plaque formation on wild-type cells.
• Host permissiveness correlated with the inactivation of RecBC nucleolytic activities, not recombinational activities.

Conclusions:

• The inserted DNA sequences in these phage clones likely limit the production of packageable chromosomes by interacting with host recombination systems.
• Modified E. coli strains, both recombination-proficient and deficient, were constructed as improved hosts for recovering otherwise inhibitory genomic sequences.
• Understanding the interplay between phage lambda replication, encapsidation, and host RecBC enzyme function is crucial for optimizing cloning strategies.