• 主题
高级搜索  >
历史搜索 清空历史
首页> 美国政府科技报告 >Cloning and Sequencing of a Plasmid-Borne Gene (opd) Encoding a Phosphotriesterase
【24h】

Cloning and Sequencing of a Plasmid-Borne Gene (opd) Encoding a Phosphotriesterase

机译：编码磷酸三酯酶的质粒携带基因（opd）的克隆和测序

获取原文
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Plasmid pCMSI was isolated from Pseudomonas diminuta MG, a strain which constitutively hydrolyzes a broad spectrum of organophosphorus compounds. The native plasmid was restricted with PstI, and individual DNA fragments were subcloned into pBR322. A recombinant plasmid transformed inot Escherichia coli possessed weak hydrolytic activity, and southern blotting with the native plasmid DNA verified that the DNA sequence originated from pCMSI. When the cloned 1.3-kilobase fragment was placed behind the lacZ' promoter of M13mp10 and retransformed into E. coli, clear-plaque isolates with correctly sized inserts exhibited isopropyl Beta-d-thiogalactopyranoside-inducible whole-cell activity. Sequence determination of the M13 constructions identified an open reading frame of 975 bases preceded by a putative ribosome-binding site appropriately positioned upstream of the first ATG codon in the open reading frame. An intragenic fusion of the opd gene with the lacZ gene produced a hybrid polypeptide which was purified by Beta-galactosidase immunoaffinity chromatography and used to confirm the open reading frame of opd. The gene product, an organophosphorus phosphotriesterase, would have a molecular weight of 35,418 if the presumed start site is correct. Eighty to ninety percent of the enzymatic activity was associated with the pseudomonad membrane fractions. When dissociated by treatment with 0.1% Triton and 1 M Sodium Chloride, the enzymatic activity was associated with a molecular weight of approximately 65,000, suggesting that the active enzyme was dimeric. Keywords; Neurotoxins, Genetic engineering, Reprints. (aw)

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号