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

• Published in: Gene Vol. 49; no. 2; pp. 253 - 262
• Main Authors: Wertman, Kenneth F., Wyman, Arlene R., Botstein, David
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 1986; Amsterdam Elsevier; New York, NY
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - physiology; Bacteriophage lambda - genetics; Bacteriophage lambda - physiology; Biological and medical sciences; Biotechnology; chi site; cloning vectors; DNA Repair Enzymes; DNA Replication; DNA, Recombinant; Escherichia coli; Escherichia coli - physiology; Escherichia coli Proteins; Exodeoxyribonuclease V; Exodeoxyribonucleases - antagonists & inhibitors; Exodeoxyribonucleases - genetics; Exodeoxyribonucleases - physiology; Fundamental and applied biological sciences. Psychology; gene libraries; Genetic engineering; Genetic technics; genomic libraries; host systems; Lysogeny; Methods. Procedures. Technologies; palindromes; phage lambda; rec+ -inhibited phenotype; RecBC enzyme; recD; Recombinant DNA; recombination; Spi − phenotype; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Viral Plaque Assay; Viral Proteins - genetics; Viral Proteins - physiology; Virus Replication; Spi − phenotype; chi site; recD; Exo I; SDS; chi; Recombinant DNA; RecBC enzyme; bp; genomic libraries; rec + -inhibited phenotype; palindromes; kb; pfu; Virus; Lambda phage group; Bactériophage lambda; Styloviridae; Escherichia coli; Bacteria; Bactériophage; Escherichieae; Enterobacteriaceae
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(86)90286-6

A class of recombinant phage λ 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 λ gam + gene into a rec + -inhibited clone results in the ability of the phage to form plaques on wild-type cells. Since λ 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 λ 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 L