CLONING AND SEQUENCING OF THE GENE WHICH ENCODES THE HIGHLY INDUCIBLE ACETAMIDASE OF MYCOBACTERIUM-SMEGMATIS
CLONING AND SEQUENCING OF THE GENE WHICH ENCODES THE HIGHLY INDUCIBLE ACETAMIDASE OF MYCOBACTERIUM-SMEGMATIS
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DOI：
10.1099/00221287-139-3-575
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发表时间：
1993-03-01
期刊：
影响因子：
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通讯作者：
COLSTON, MJ
中科院分区：
文献类型：
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作者：
MAHENTHIRALINGAM, E;DRAPER, P;COLSTON, MJ
对耻垢分枝杆菌NCTC 8159的乙酰胺酶进行了纯化，并获得了其氨基端序列和通过蛋白水解获得的两个肽的序列。在大肠杆菌中克隆了包括酰胺酶结构基因的DNA片段，使用基于肽序列设计的寡核苷酸探针和从结核分枝杆菌复合体的9个蛋白质抗原编码基因的公开序列计算的密码子使用表。序列分析表明，该基因编码406个氨基酸残基。接近和上游的酰胺酶基因的核苷酸序列含有一个可能的核糖体结合位点，但没有可识别的启动子序列。三个额外的潜在的开放阅读框被发现的上游和非常接近的酰胺酶基因，与共识'-35'和'-10'启动子位点之间的第一和第二个这些。人们希望可以利用乙酰胺酶基因的高度诱导表达来调节分枝杆菌中克隆的其他基因的表达。
The acetamidase of Mycobacterium smegmatis NCTC 8159 was purified, and the sequences of its amino-terminus and of two peptides obtained by proteolysis of the protein were obtained. A DNA fragment including the amidase structural gene was cloned in Escherichia coli, using oligonucleotide probes designed on the basis of the peptide sequences and a codon usage table calculated from published sequences of nine protein-antigen-encoding genes of the Mycobacterium tuberculosis complex. Sequence analysis of the cloned DNA revealed that the amidase gene encoded 406 amino acid residues. The nucleotide sequence close to and upstream of the amidase gene contained a probable ribosome-binding site but no identifiable promoter sequences. Three additional potential open-reading frames were found upstream of and very close to the amidase gene, with consensus '- 35' and '- 10' promoter sites between the first and second of these. It is hoped that the highly inducible expression of the acetamidase gene can be exploited to allow regulated expression of other genes cloned in mycobacteria.