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Volume 153, Issue 9

Isolation and characterization of putative promoters Free

Abstract

Novel plasmids were constructed for the analysis of DNA fragments from the rumen bacterium . Five previously unidentified promoters were characterized using a novel primer extension method to identify transcription start sites. The genes downstream of these promoters were not identified, and their activity in expression of genomic traits in wild-type remains putative. Comparison with promoters from this and closely related species revealed a consensus sequence resembling the binding motif for the RNA polymerase -like factor complex. Consensus −35 and −10 sequences within these elements were and AA respectively, interspaced by 15–16 bp. The consensus for the −10 element was extended by one nucleotide upstream and downstream of the standard hexamer (indicated in bold). Promoter strengths were measured by reverse transcription quantitative PCR and -glucuronidase assays. No correlation was found between the composition and context of elements within promoters, and promoter strength. However, a mutation within the −35 element of one promoter revealed that transcriptional strength and choice of transcription start site were sensitive to this single nucleotide change.

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Keyword(s): GUS, β-glucuronidase , HEX, hexachlorofluorescein , MCS, multiple cloning site and RT-qPCR, reverse transcription quantitative PCR
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2007-09-01
2024-04-19
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Isolation and characterization of putative Pseudobutyrivibrio ruminis promoters
Microbiology 153, 3071 (2007); https://doi.org/10.1099/mic.0.2007/006502-0
/content/journal/micro/10.1099/mic.0.2007/006502-0
/content/journal/micro/10.1099/mic.0.2007/006502-0
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