New pSC101-derivative cloning vectors with elevated copy numbers.
Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (approximately 240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic assay to suppress temperature-sensitive mutants of ffh, encoding the protein component of the Escherichia coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype.
Peterson
James
J
Department of Veterinary Microbiology, Veterinary Medical Research Institute, Iowa State University, 1802 University Boulevard, Building #6, Ames, IA 50011, USA.
Phillips
Gregory J
GJ
D000667:Ampicillin / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D002701:Chloramphenicol / Q000737:chemistry; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics*; D005838:Genotype; D007612:Kanamycin / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074164:RepE protein, E coli; D012097:Repressor Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D007612:Kanamycin; D002701:Chloramphenicol; D000667:Ampicillin; D004265:DNA Helicases