MOLECULAR-CLONING AND EXPRESSION OF THE BIODEGRADATIVE THREONINE DEHYDRATASE GENE (TDC) OF ESCHERICHIA-COLI-K12
MOLECULAR-CLONING AND EXPRESSION OF THE BIODEGRADATIVE THREONINE DEHYDRATASE GENE (TDC) OF ESCHERICHIA-COLI-K12
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DOI：
10.1007/bf00425676
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发表时间：
1985-01-01
期刊：
影响因子：
--
通讯作者：
DATTA, P
中科院分区：
文献类型：
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作者：
GOSS, TJ;DATTA, P
通过Tn 5诱变后分离出一个苏氨酸脱氢酶阴性突变株，将tdc：：Tn 5 DNA克隆到pBR 322中，然后在体内替换质粒上的Tn 5元件，从而克隆了大肠杆菌的生物降解性苏氨酸脱氢酶基因（tdc）。亚克隆和核苷酸序列数据显示两个不同的原核启动子样元素，每个包含一个潜在的CAP结合位点和AT丰富的地区，和一个Shine-Dalgarno序列。这些推定的启动子之一，P2，位于紧接上游的tdc编码区，和第二个，P1，大约1个酶上游的P2。从P1中删除潜在的CAP结合位点阻止了tdc基因的表达。然而，去除P2和上游DNA的大片段对酶的合成没有明显的影响。在P1和tdc编码区之间发现了一个936碱基对的开放阅读框架，其产生约32千道尔顿的多肽。这些数据表明，P1，而不是P2，是必要的tdc基因的表达，和DNA序列编码的32 KD多肽和苏氨酸转氨酶是一个单一的转录单位的一部分。
The biodegradative threonine dehydratase gene (tdc) of Escherichia coli was cloned by isolating a dehydratase-negative mutant after Tn5 mutagenesis, cloning the tdc::Tn5 DNA into pBR322 and then replacing the Tn5 element on the plasmid in vivo. Subcloning and nucleotide sequence data revealed two distinct procaryotic promoter-like elements each containing a potential CAP-binding site and AT-rich regions, and a Shine-Dalgarno sequence. One of these putative promoters, P2, was located immediately upstream from the tdc coding region, and a second, P1, was approximately 1 kilobase upstream from P2. Deletion of the potential CAP-binding site from P1 prevented tdc gene expression. However, removal of P2 and a large segment of the upstream DNA had no discernible effect on dehydratase synthesis. A 936-base pair open reading frame was found between P1 and the tdc coding region, which produced a polypeptide of about 32 kilodaltons. The data suggest that P1, and not P2, is necessary for tdc gene expression, and that the DNA sequences coding for the 32 KD polupeptide and threonine dehydratase are part of a single transcriptional unit.