Peoples O P, Sinskey A J
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Mol Microbiol. 1989 Mar;3(3):349-57. doi: 10.1111/j.1365-2958.1989.tb00180.x.
A series of expression plasmids containing either the complete insert from plasmid pUCDBK1 (Peoples et al., 1987) or sub-fragments thereof were constructed in a tac promoter vector. Analysis of protein lysates of induced cultures of these clones identified the gene encoding NADPH-specific acetoacetyl-CoA reductase in the 2.3kb of sequence located downstream from the beta-ketothiolase gene in plasmid pUCDBK1. The complete nucleotide sequence (2.1kb) of this region was determined. An open reading frame was located 88bp downstream from the stop codon of the thiolase gene encoding a potential polypeptide of Mr 25,000, which is in good agreement with that observed for the overexpressed protein on SDS-PAGE. N-terminal protein sequence data obtained by Edman degradation of the purified Mr = 25,000 polypeptide were used to identify the correct start of the NADPH-specific acetoacetyl-CoA reductase gene. Hence in Z. ramigera, the genes encoding beta-ketothiolase (phbA) and NADPH-specific acetoacetyl-CoA reductase (phbB) are organized as phbA-phbB. S1-nuclease analysis of Z. ramigera RNA identified a transcription start site 85 bp upstream from the phbA structural gene locating the promoter region.
一系列表达质粒在tac启动子载体中构建而成，这些质粒包含质粒pUCDBK1（Peoples等人，1987年）的完整插入片段或其亚片段。对这些克隆诱导培养物的蛋白质裂解物进行分析，确定了位于质粒pUCDBK1中β-酮硫解酶基因下游2.3kb序列中编码NADPH特异性乙酰乙酰辅酶A还原酶的基因。测定了该区域的完整核苷酸序列（2.1kb）。在硫解酶基因的终止密码子下游88bp处发现了一个开放阅读框，编码一个潜在的25000 Mr多肽，这与SDS-PAGE上观察到的过表达蛋白一致。通过对纯化的25000 Mr多肽进行Edman降解获得的N端蛋白质序列数据用于确定NADPH特异性乙酰乙酰辅酶A还原酶基因的正确起始位置。因此，在枝动杆菌中，编码β-酮硫解酶（phbA）和NADPH特异性乙酰乙酰辅酶A还原酶（phbB）的基因组织形式为phbA-phbB。对枝动杆菌RNA的S1核酸酶分析确定了位于启动子区域的phbA结构基因上游85bp处的一个转录起始位点。
