https://www.abbs.info
E-mail: [email protected]
ISSN
1672-9145
Acta Biochim Biophys Sin
2004, 36(12): 832–839
CN 31-1940/Q
Identification and Analysis of Genes Present in Leptospira
interrogans serovar lai but Absent in L. biflexa
serovar monvalerio
Ping HE, Xiang-Yan ZHANG, Xiao-Kui
GUO*, Bao-Yu HU, Xiao-Tian HUANG, Yang YANG, and Guo-Ping ZHAO1,2*
Department of
Microbiology and Parasitology, Shanghai Second Medical University, Shanghai
200025, China;
1Research Center of
Biotechnology, Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, China;
2Chinese National Human Genome Center at
Shanghai, Zhangjiang High Tech Park, Shanghai 201203, China
Abstract Genes present in virulent
bacterial strains but absent in avirulent close relatives can be of great
biologic and clinical interest. This project aimed to identify strain specific
DNA sequences of Leptospira interrogans serovar lai, which is absent in
the saprophytic L. biflexa serovar monvalerio, via suppression
subtractive hybridization with the former as the tester while the latter as the
driver. The mixture of PCR amplified DNA fragments from two subtractive
hybridization experiments were cloned into pMD18-T vector and the positive
clones were identified by dot blotting against the chromosome DNA of the two
strains individually. After DNA sequencing and analysis, the distribution of
these genomic fragment sequences in a panel of pathogenic and nonpathogenic
leptospires was investigated employing dot blot analysis. Among the 188
positive clones randomly chosen, 24 contained the tester strain specific
genomic regions, of which, 5 were non-coding fragments while the others
contained 23 distinct protein coding sequences. Besides 9 genes encoding
functional proteins, 12 genes encode unknown proteins and the rest two genes
encode proteins with recognizable domain structures, one for a putative
leucine-rich repeats (LRR) family protein while the other as an outer-membrane
protein. Our experiment results indicated that suppression subtractive
hybridization is effective for screening specific DNA sequences between two
leptospiral strains, and some of these sequences might be responsible for
virulence determination. Further analysis of these DNA sequences will provide
important information on the pathogenesis of Leptospira.
Key words Leptospira interrogans serovar
lai; suppression subtractive hybridization (SSH); specific genes
—————–
Received: October 9, 2004 Accepted: November 16,
2004
This work was supported in part by the grants from the National
Natural Science Foundation of China (No. 30370071) and the National High
Technology Research and Development Program of China (No. 2003AA223031)
*Corresponding authors:
Xiao-Kui GUO: Tel, +86-21-64453285; Fax, +86-21-64453285; E-mail, [email protected]
Guo-Ping ZHAO: Tel, +86-21-50801919; Fax, +86-21-50801922; E-mail, [email protected]