Cloning of promoter-active DNA sequences from Chainia (NCL 82.5.1) in Escherichia coli.
The ability of Escherichia coli cells to recognise and use the regulatory signals of genes from Chainia (NCL 82-5-1) was determined in vivo using gene fusions. DNA fragments from Chainia were cloned in E. coli using the promoter probe plasmid pJAC4. Four of the randomly selected recombinants exhibited varying strengths of promoter activity as assessed by the concentration of ampicillin required to kill 50% of the colonies (LD50 values) and by beta-lactamase activity. The origin of the inserts was confirmed by colony hybridization of clones with labelled genomic DNA of Chainia. The beta-lactamase activity of recombinant colonies was substantially higher (> 10-fold) than that of colonies transformed with pJAC4 without any insert. The results show that a few Chainia DNA sequences are recognized by E. coli as transcription initiation signals for the expression of beta-lactamase gene, inspite of the high guanine/cytosine content of the Chainia genome.
Chauthaiwale
V M
VM
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Deshpande
V V
VV
D000192:Actinomycetales / Q000235:genetics*; D000667:Ampicillin / Q000494:pharmacology; D003001:Cloning, Molecular; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007612:Kanamycin; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014169:Transformation, Bacterial; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D015342:DNA Probes; D004269:DNA, Bacterial; D007612:Kanamycin; D000667:Ampicillin; D001618:beta-Lactamases