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Title: Molecular analysis of the promoter region of the Clostridium difficile toxin B gene that is functional in Escherichia coli
Authors: Song, K.-P. 
Faust, C.
Issue Date: 1998
Source: Song, K.-P.,Faust, C. (1998). Molecular analysis of the promoter region of the Clostridium difficile toxin B gene that is functional in Escherichia coli. Journal of Medical Microbiology 47 (4) : 309-316. ScholarBank@NUS Repository.
Abstract: Clostridium difficile is a human pathogen that produces two types of toxins, A and B, that cause a potentially lethal gastrointestinal syndrome termed pseudomembranous colitis. Virtually nothing is known about the mechanism of regulation of toxin production in this organism, and cis-regulatory regions of neither toxin have yet been identified, thus prompting this investigation. A motif homologous with the Shine-Dalgarno sequence of Escherichia coli occurs upstream from the putative initiation codon of toxin B, making this region also a candidate to contain a promoter. Therefore, a subgenomic DNA library of C. difficile in a plasmid vector was first constructed encompassing the 5'-end of the toxin B gene. A 450-bp DNA fragment was excised from the subgenomic DNA library clone and subcloned into a promoter-probe plasmid vector that contains two divergently oriented, promoterless genes to assay for promoter function. This subcloned DNA fragment directed the expression of alkaline phosphatase, a reporter gene product of the promoterless vector, thus indicating the presence of a functional promoter. To locate the promoter more precisely, a series of nested deletions of the toxin B promoter subclone was constructed with exonuclease III. The promoter that facilitates expression of the toxin B gene in E. coli was localised, based on alkaline phosphatase activity. The transcriptional initiation site of toxin B mRNA in E. coli was mapped by primer extension analysis, suggesting two closely associated tandem start sites directed by two similarly spaced promoters within this localised region.
Source Title: Journal of Medical Microbiology
ISSN: 00222615
Appears in Collections:Staff Publications

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