Two tandemly located promoters, artificially constructed, are active in a Bacillus subtilis alpha-amylase secretion vector.
An 85 bp DNA fragment, the nucleotide sequence of which had 84% homology with the sequence for the promoter, ribosome binding site and NH2-terminal five amino acids of the Bacillus amyloliquefaciens alpha-amylase gene, was chemically synthesized. In order to analyze the promoter activity of a Bacillus subtilis alpha-amylase secretion vector, the fragment was inserted between the promoter and signal peptide-coding region of Bacillus subtilis alpha-amylase gene. Both promoters, tandemly repeated, functioned in transcribing the B. subtilis alpha-amylase signal peptide-coding region followed by the Escherichia coli beta-lactamase structural gene. The transcription initiation sites were determined by the primer extension method. The extracellular production of beta-lactamase was stimulated by two promoters as compared with that by the plasmids containing either promoter region alone. The change of two amino acids in the NH2-terminal region of the B. subtilis alpha-amylase signal peptide had no effect on the secretion of beta-lactamase from B. subtilis cells.
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D003101:Collodion; D004247:DNA / Q000138:chemical synthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D010209:Paper; D010957:Plasmids / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000502:physiology; D000516:alpha-Amylases / Q000096:biosynthesis*; D001618:beta-Lactamases / Q000276:immunology / Q000378:metabolism
D012333:RNA, Messenger; D003101:Collodion; D004247:DNA; D000516:alpha-Amylases; D001618:beta-Lactamases