Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid.
The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.
Teixeira
A V
AV
Laboratory for Molecular and Cell Biology, Council for Scientific and Industrial Research, Pretoria, South Africa.
Mizrahi
V
V
Thomson
J A
JA
D001407:Bacillus / Q000235:genetics*; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic
D015500:Chloramphenicol O-Acetyltransferase; D012321:DNA-Directed RNA Polymerases