Woehlke G, Wifling K, Dimroth P
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
J Biol Chem. 1992 Nov 15;267(32):22798-803.
A genomic library of Salmonella typhimurium DNA was constructed in the lambda-phage EMBL3 and screened by immunoblotting for expression of the oxaloacetate decarboxylase alpha-subunit. After subcloning on plasmids the entire sequence of the oxaloacetate decarboxylase was determined. The genes encoding subunits gamma (oadG), alpha (oadA), and beta (oadB) of the decarboxylase are clustered on the chromosome in that order. A typical consensus sequence of a promoter is not found upstream of the oadG gene, but putative ribosome binding regions can be identified before each subunit gene. The amino acid sequences are highly homologous to those of oxaloacetate decarboxylase from Klebsiella pneumoniae with 71% identity between the gamma-subunits, 92% identity between the alpha-subunits, and 93% identity between the beta-subunits. The homology between the corresponding beta-subunits appeared to exist only between the 312 N-terminal amino acid residues. It was shown that a cloning artifact has occurred during DNA sequence determination of the beta-subunit from K. pneumoniae and has led to erroneous results. The sequence of this polypeptide is corrected in the Appendix to this paper. A plasmid encoding the three oad genes and that for the anaerobic citrate carrier (citS) was cloned from the chromosomal DNA and used for sequence determination.
构建了鼠伤寒沙门氏菌DNA的基因组文库，该文库采用λ噬菌体EMBL3构建，并通过免疫印迹法筛选草酰乙酸脱羧酶α亚基的表达。在质粒上进行亚克隆后，测定了草酰乙酸脱羧酶的完整序列。编码脱羧酶γ亚基（oadG）、α亚基（oadA）和β亚基（oadB）的基因按此顺序聚集在染色体上。在oadG基因上游未发现典型的启动子共有序列，但在每个亚基基因之前可鉴定出假定的核糖体结合区域。其氨基酸序列与肺炎克雷伯菌的草酰乙酸脱羧酶高度同源，γ亚基之间的同一性为71%，α亚基之间的同一性为92%，β亚基之间的同一性为93%。相应β亚基之间的同源性似乎仅存在于N端312个氨基酸残基之间。结果表明，在肺炎克雷伯菌β亚基的DNA序列测定过程中出现了克隆假象，并导致了错误的结果。该多肽的序列在本文附录中进行了校正。从染色体DNA中克隆了一个编码三个oad基因和厌氧柠檬酸载体（citS）的质粒，并用于序列测定。
