8. Cloning of the Thymidylate Synthetase Gene of the Phage Phi-3-T

Phi-3-T is a Bacillus subtifis temperate bacteriophage, isolated by Tucker (11) from the soil. Tucker has shown that infection by this phage causes, by lysogenic conversion of thyB. subtifis clones, prototrophy. The phage carries genetic information specifying the thymidylate synthetase activity, the thyP gene. Recently, we have reported purification of B. subtilis DNA segments achieved by EcoRI cleavage followed by gel electrophoresis (6). Since a still further purification can be achieved by cloning the segments (8), we have chosen to use that approach for the isolation of the rhyP gene of the Phi-3-T. We describe here the successful cloning of that gene on the Escherichia coli plasmid pSClO1 (3). The gene complements the thymine deficiency in E. coli, indicating its correct transcription and translation in this new host. The promoter of the gene is likely to be contained within the cloned segment. The hybrid plasmid transforms B. subtifis loo-fold less efficiently than the intact phage DNA. The excised segment, however, displays the same transforming activity (1,000 times less than the intact phage DNA) whether it comes from the phage or from the hybrid plasmid. B. subtifis clones transformed with the hybrid plasmid DNA do not contain detectable pSC 101 sequences, but do show sequences homologous to part of the Phi-3-T genome.