The McCollum-Pratt Institute and Department of Biology, The Johns Hopkins University, Baltimore, MD 21218.
Proc Natl Acad Sci U S A. 1987 May;84(10):3219-23. doi: 10.1073/pnas.84.10.3219.
Bradyhizobium japonicum strain LO501 is a Tn5-induced mutant that does not express the terminal oxidase cytochrome aa(3) (cytochrome-c oxidase, EC 1.9.3.1). Two and one-half kilobase pairs of LO501 genomic DNA that flanks the transposon was isolated and used as a hybridization probe to obtain the wild-type gene from a cosmid library. Two subcloned fragments from two of the isolated cosmids were ligated into broad host range vectors, and restriction maps of these fragments were generated. The resultant plasmids, pCA1 and pBL33, each contained DNA homologous to that mutated in strain LO501. The two plasmids were each introduced into strain LO501 by conjugal transfer, and it was found that pCA1, but not pBL33, complemented the oxidase mutant. The transconjugant strain LO501[pCA1] expressed wild-type levels of cytochrome aa(3), as discerned spectrophotometrically, and had restored N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity. Furthermore, the frequency of complementation of LO501 cells that received pCA1 by conjugation was 1.0, demonstrating that pCA1 complemented the mutant in trans. The results show that pCA1 contains the entire wild-type gene that was mutated in strain LO501, and this gene is required for cytochrome aa(3) expression.
日本慢生根瘤菌 LO501 菌株是 Tn5 诱导的突变体，不表达末端氧化酶细胞色素 aa(3)（细胞色素-c 氧化酶，EC 1.9.3.1）。分离出 LO501 基因组 DNA 侧翼的 2.5 千碱基对的转座子，并将其用作杂交探针，从 cosmid 文库中获得野生型基因。从两个分离的 cosmid 中克隆了两个亚克隆片段，并将它们连接到广谱宿主载体中，生成这些片段的限制图谱。这些片段的质粒，pCA1 和 pBL33，都含有与 LO501 菌株突变相同的 DNA。将这两个质粒通过共轭转移分别引入 LO501 菌株中，发现 pCA1，但不是 pBL33，能够互补氧化酶突变体。转导子 LO501[pCA1]表达出野生型水平的细胞色素 aa(3)，可以通过分光光度法检测到，并且已经恢复了 N,N,N',N'-四甲基-p-苯二胺氧化酶活性。此外，通过共轭传递接受 pCA1 的 LO501 细胞的互补频率为 1.0，表明 pCA1 以反式互补突变体。结果表明，pCA1 包含在 LO501 菌株中突变的整个野生型基因，该基因是细胞色素 aa(3)表达所必需的。
Zotero 插件