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Host/vector interactions which affect the viability of recombinant phage lambda clones
Abstract:
A class of recombinant phage lambda clones are recovered from human genomic libraries on Escherichia coli recB21 recC22 sbcB15 cells, which fail to form plaques on wild-type cells. We report experiments which address the mechanism of this inhibition. The introduction of the recombination-stimulating sequence chi into one such clone allows growth of this phage on Rec+ cells. In addition, the insertion of lambda gam+ gene into a rec+-inhibited clone results in the ability of the phage to form plaques on wild-type cells. Since lambda Gam protein is an inhibitor of host RecBC enzyme, we tested a collection of such phage for growth on a variety of hosts altered in RecBC function. Host permissiveness correlated with the inactivation of the RecBC nucleolytic activities and not with the recombinational activities. These observations suggest that the inserted DNA sequences of these phage limit the production of packageable chromosomes. This conclusion is easily reconciled with our current knowledge of the interaction of the host recombination systems with lambda replication and encapsidation. Based on these experiments we have constructed strains, both recombination-proficient and recombination-deficient, which serve as improved hosts for the recovery of genomic sequences which are otherwise inhibitory to the growth of phage lambda.
Insights
Recombinant phage lambda clones inhibited on wild-type Escherichia coli can be rescued by introducing the chi sequence or lambda gam+ gene. This research identifies improved host strains for recovering inhibitory genomic sequences.
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.