The overall objectives of this proposal are the determination of genetic and biochemical factors that are responsible for the replication and conjugal transfer of extrachromosomal circular DNA elements in bacteria and the development of plasmid systems for the cloning of foreign DNA in bacteria. Particular emphasis is placed on the colicinogenic-colicin system and several antibiotic resistance (R) plasmids in Escherichia coli. During the coming year a program will be initiated to isolate temperature-sensitive replication mutants of the plasmids Co1E1 and R6K using in vitro mutagenesis of these isolated plasmid molecules and the transformation system of E. coli. In addition, utilizing several different restriction endonucleases that cleave DNA molecules into fragments containing cohesive restriction endonucleases that cleave DNA molecules into fragments containing cohesive ends, we will attempt to obtain low molecular weight derivatives of the plasmids Co1E1, R6K, mini-F and RK2. The intact plasmid, R6K, and low molecular weight derivatives of this plasmid that will be obtained will be analyzed for replication properties in an in vitro system. Another major aspect of the proposed work for the current year will be the development of molecular cloning vehicles starting with the plasmids Co1E1 and RK2 that are more effective and potentially safer for cloning foreign DNA in E. coli and other gram-negative bacteria. BIBLIOGRAPHIC REFERENCES: Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F' km plasmid. Michael A. Lovett and D.R. Helinski. J. Bacteriol. 127:982-987 (1976). Plasmids. Introductory remarks. D.R. Helinski. In Federation Proceedings 35:2024-2025 (1976).