CLONING AND SEQUENCING OF A BILE ACID-INDUCIBLE OPERON FROM EUBACTERIUM SP STRAIN-VPI-12708
CLONING AND SEQUENCING OF A BILE ACID-INDUCIBLE OPERON FROM EUBACTERIUM SP STRAIN-VPI-12708
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DOI：
10.1128/jb.172.12.7011-7019.1990
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发表时间：
1990-12-01
影响因子：
3.2
通讯作者：
HYLEMON, PB
中科院分区：
文献类型：
--
作者：
MALLONEE, DH;WHITE, WB;HYLEMON, PB
来自真杆菌属的两种胆汁酸诱导性多肽。分子量为 27,000 和大约 45,000 的菌株 VPI 12708 先前已被证明是由驻留在 2.9-kb EcoRI 片段上的基因编码的。我们现在报告了该 EcoRI 片段上游另外三个重叠 DNA 片段的克隆和测序。这四个片段一起含有胆汁酸诱导操纵子的大片段，其编码 27,000-和 45,000-Mr（现在显示为 47,500-Mr）多肽和可能编码分子量为 59,500、58,000、19,500 和 9,000 的四种其他多肽的开放阅读框11,500。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，表观Mr为23,500的胆汁酸诱导性多肽被纯化至同质，并且获得的N端氨基酸序列与从编码19,500-Mr多肽的开放阅读框推导的序列相匹配。含有编码 47,500-Mr 多肽的基因 3'' 下游末端的短 DNA 片段未能成功克隆，但通过聚合酶链式反应合成的 DNA 片段直接测序。通过引物延伸显示胆汁酸诱导型操纵子的mRNA起始位点紧邻编码58,000-Mr多肽的基因的上游。 mRNA起始位点上游的潜在启动子区域与先前鉴定的来自真杆菌属的胆汁酸诱导基因的启动子区域表现出显着的同源性。菌株VPI 12708。我们假设该胆汁酸诱导操纵子编码该细菌中参与胆汁酸7α-脱羟基化途径的大多数酶。
Two bile acid-inducible polypeptides from Eubacterium sp. strain VPI 12708 with molecular weights of 27,000 and approximately 45,000 have previously been shown to be encoded by genes residing on a 2.9-kb EcoRI fragment. We now report the cloning and sequencing of three additional overlapping DNA fragments upstream from this EcoRI fragment. Together, these four fragments contain a large segment of a bile acid-inducible operon which encodes the 27,000- and 45,000-Mr (now shown to be 47,500-Mr) polypeptides and open reading frames potentially coding for four additional polypeptides with molecular weights of 59,500, 58,000, 19,500, and 9,000 to 11,500. A bile acid-inducible polypeptide with an apparent Mr of 23,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to homogeneity, and the N-terminal amino acid sequence that was obtained matched the sequence deduced from the open reading frame coding for the 19,500-Mr polypeptide. A short DNA segment containing the 3'' downstream end of the gene coding for the 47,500-Mr polypeptide was not successfully clones but was directly sequenced from DNA fragments synthesized by polymerase chain reaction. The mRNA initiation site for the bile acid-inducible operon was shown by primer extension to be immediately upstream from the gene encoding the 58,000-Mr polypeptide. A potential promoter region upstream from the mRNA initiation site displayed significant homology with the promoter regions of previously identified bile acid-inducible genes from Eubacterium sp. strain VPI 12708. We hypothesize that this bile acid-inducible operon codes for most of the enzymes involved in the bile acid 7.alpha.-dehydroxylation pathway in this bacterium.