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Nucleic Acids Research, 1988, Vol. 16, No. 18 9027-9039
© 1988

Articles

A genetic selection for temperature-sensitive variants of the gene V protein of bacteriophage f1

Thomas C. Terwilliger*, Wilder D. Fulford1 and Hal B. Zabin

Department of Biochemistry and Molecular Biology, University of Chicago 920 E. 58th Street, Chicago, IL 60637 1Rockefeller University 1230 York Ave., New York, NY 10021, USA

*To whom correspondence should be addressed

Received April 1, 1988. Revised August 18, 1988. Accepted August 18, 1988.

Complementary negative and positive genetic selections based on the activity of a plasmid-encoded bacteriophage f1 gene V are developed. The negative selection is based on an activity of thegene V protein in E. coli cells which markedly reduces the infection of those cells by f1-related viruses. In order to select against cells expressing active gene V protein, the cells are infected with the phage R386, a derivative of f1 which confers resistance to chloramphenicol, and are plated in the presence of the antibiotic. Those cells which contain gene V protein are infrequently infected with the virus and are unable to grow in the presence of chloramphenicol; those which do not contain the gene V protein are readily infected and can grow in the presence of the antibiotic. The positive genetic selection consists of excising the gene V sequences from the plasmids and using them to replace the gene V of a bacteriophage f1 derivative containing an amber mutation in gene V. Only those genes which encode an active gene V protein can support phage growth and yield plaques. The two genetic selections can be combined in order to yield a substantial enrichment for genes encoding temperature-sensitive gene V proteins.


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