结核分枝杆菌dnaA基因的扩增与克隆

Amplification and cloning of the Mycobacterium tuberculosis dnaA gene.

作者信息

Rajagopalan M, Qin M H, Steingrube V A, Nash D R, Wallace R J, Madiraju M V

机构信息

Department of Microbiology, University of Texas Health Center at Tyler 75710, USA.

出版信息

Gene. 1995 Sep 22;163(1):75-9. doi: 10.1016/0378-1119(95)00403-s.

DOI:10.1016/0378-1119(95)00403-s

PMID:7557482

Abstract

To identify and subsequently clone the gene encoding the DnaA protein, degenerate oligodeoxyribonucleotide (oligo) primers targeted against two highly conserved domains of the eubacterial DnaA were used to amplify a 780-bp DNA region spanning the two primers from genomic DNA preparations of Mycobacterium tuberculosis (Mt), M. bovis (Mb) and M. avium (Ma). Nucleotide (nt) sequences and deduced amino acid (aa) sequences of these fragments revealed homologies with each other and with the corresponding regions from other bacteria. Using an oligo specific to Mt dnaA as a probe, the Mt genomic DNA cosmid libraries propagated in Escherichia coli were screened and a cosmid DNA clone hybridizing with the oligo was identified. Furthermore, a 5-kb DNA fragment containing the Mt dnaA was subcloned into a pUC18 vector.

摘要

为了鉴定并随后克隆编码DnaA蛋白的基因，针对真细菌DnaA的两个高度保守结构域设计的简并寡脱氧核糖核苷酸（oligo）引物，被用于从结核分枝杆菌（Mt）、牛分枝杆菌（Mb）和鸟分枝杆菌（Ma）的基因组DNA制剂中扩增跨越这两个引物的780bp DNA区域。这些片段的核苷酸（nt）序列和推导的氨基酸（aa）序列显示它们彼此之间以及与其他细菌的相应区域具有同源性。使用针对Mt dnaA的特异性oligo作为探针，筛选了在大肠杆菌中繁殖的Mt基因组DNA黏粒文库，并鉴定出一个与该oligo杂交的黏粒DNA克隆。此外，将包含Mt dnaA的5kb DNA片段亚克隆到pUC18载体中。