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

摘要

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 the gene 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 p'age 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.