Furusato T, Takano J, Jigami Y, Tanaka H, Yamane K
J Biochem. 1986 Apr;99(4):1181-90. doi: 10.1093/oxfordjournals.jbchem.a135581.
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.
化学合成了一个85bp的DNA片段，其核苷酸序列与解淀粉芽孢杆菌α-淀粉酶基因的启动子、核糖体结合位点及氨基末端五个氨基酸的序列具有84%的同源性。为了分析枯草芽孢杆菌α-淀粉酶分泌载体的启动子活性，将该片段插入到枯草芽孢杆菌α-淀粉酶基因的启动子与信号肽编码区之间。两个串联重复的启动子在转录枯草芽孢杆菌α-淀粉酶信号肽编码区及随后的大肠杆菌β-内酰胺酶结构基因时发挥作用。通过引物延伸法确定了转录起始位点。与单独含有任一启动子区域的质粒相比，两个启动子刺激了β-内酰胺酶的细胞外产生。枯草芽孢杆菌α-淀粉酶信号肽氨基末端区域两个氨基酸的变化对枯草芽孢杆菌细胞分泌β-内酰胺酶没有影响。
