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首页> 外文期刊>Journal of bacteriology >Probable Identification of a Membrane-Associated Repressor of Bacillus subtilis DNA Replication as the E2 Subunit of the Pyruvate Dehydrogenase Complex
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Probable Identification of a Membrane-Associated Repressor of Bacillus subtilis DNA Replication as the E2 Subunit of the Pyruvate Dehydrogenase Complex

机译：枯草芽孢杆菌DNA复制的膜相关阻遏物可能鉴定为丙酮酸脱氢酶复合物的E2亚基。

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Two Bacillus subtilis lysogenic libraries were probed by an antibody specific for a previously described membrane-associated inhibitor of B. subtilis DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452–7456, 1988). Three clones that reacted strongly with the antibody contained an entire open reading frame. Sequencing identified one of the clones (R1-2) as containing the E2 subunit of the pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase. An AT-rich sequence in the origin region was identified initially as the site to which extracts from the R1-2 clone were bound. This sequence was almost identical to one detected in Bacillus thuringiensis that also bound the E2 subunit but which was involved in activating the Cry1 protoxin gene of the organism, not in inhibiting DNA replication (T. Walter and A. Aronson, J. Biol. Chem., 274:7901–7906, 1999). However, the exact sequence was not as important in B. subtilis as the AT-rich core region. Binding would occur as long as most of the AT character of the core remained. Purified E2 protein obtained by use of PCR and an expression vector reacted strongly with antibody prepared against the repressor protein and the protein in the R1-2 clone, but its specificity for the AT-rich region was altered. The purified E2 protein was capable of inhibiting membrane-associated DNA replication in vitro, but anti-E2 antibody was variable in its ability to rescue repression when added to the assay.
机译：用对先前描述的 B膜相关抑制剂特异的抗体探查两个枯草芽孢杆菌溶原性文库。枯草芽孢杆菌DNA复制（J. Laffan和W. Firshein，美国国家科学院学报85：7452-7456，1988）。与抗体强烈反应的三个克隆包含一个完整的开放阅读框。测序鉴定出克隆（R1-2）之一含有丙酮酸脱氢酶复合物的E2亚基，二氢脂酰胺乙酰转移酶。最初将起源区域中富含AT的序列鉴定为与R1-2克隆的提取物结合的位点。此序列与苏云金芽孢杆菌中检测到的序列几乎相同，苏木芽孢杆菌也结合了E2亚基，但与激活生物体的Cry1毒素基因有关，而不是抑制DNA复制（T.Walter和A. Aronson，J。Biol。Chem。，274：7901-7906，1999）。但是，确切的顺序在 B中并不那么重要。枯草作为富含AT的核心区域。只要保留了核心的大多数AT特性，就会发生绑定。通过使用PCR和表达载体获得的纯化的E2蛋白与针对阻遏蛋白和R1-2克隆中的蛋白制备的抗体强烈反应，但其对富含AT区域的特异性发生了变化。纯化的E2蛋白能够在体外抑制膜相关DNA的复制，但是将抗E2抗体添加到测定中后，其挽救阻遏能力却是可变的。

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