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Deletion analysis of the promoter/operator nucleotide sequence of themetF gene from Escherichia coli K-12: The use of beta-lactamase as a reporter gene
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Deletion analysis of the promoter/operator nucleotide sequence of themetF gene from Escherichia coli K-12: The use of beta-lactamase as a reporter gene
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Deletion analysis of the promoter/operator nucleotide sequence of themetF gene from Escherichia coli K-12: The use of beta-lactamase as a reporter gene
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Dissertation
Deletion analysis of the promoter/operator nucleotide sequence of themetF gene from Escherichia coli K-12: The use of beta-lactamase as a reporter gene
1991
Overview
The metF operator, which consists of five imperfect repeats of the met box (AGACGTCT), has been identified as a nucleotide sequence partially overlapping the metF promoter region. Understanding the mechanisms involved in regulation of metF gene expression by the MetJ repressor protein requires precise identification of the MetJ protein DNA binding site. Plasmids, with and without deletions in the metF promoter/operator region, were constructed and provided material which was employed to test for MetJ/MetK-mediated regulation of metF and/or bla (reporter) gene expression from the metF promoter/operator. Gene expression by plasmids with an intact metF promoter, but with less than three intact met box operator sequences, show no MetJ/MetK regulatory control on bla gene expression when only one chromosomal copy of the metJ gene was present. In the presence of two chromosomal copies of the metJ gene, gene expression by plasmids with an intact metF promoter, but with less than three intact met boxes, show a significant response to MetJ/MetK regulatory control on bla gene expression. Deletion of components of the $-$35 sequence of the metF promoter dramatically reduce the level of metF expression. The data indicates that the $-$35 sequence region of the metF promoter is essential for promoter strength but can be replaced with a poorer sequence. This alteration has a profound impact upon the level of metF expression and upon the efficiency of MetJ-mediated repression of the mutant-metF allele. The results also indicate that the metF promoter is stronger than what is predicted by Hawley-McClure homology scores. Collectively, these results demonstrate that the essential components of the metF promoter/operator sequences can be physically localized to separate, discrete regions of DNA sequence immediately upstream of the metF gene. Also as a result of these investigations a rapid, highly sensitive, and relatively inexpensive spectrophotometric assay was developed to quantify beta-lactamase enzyme activity. This assay provides the means for using beta-lactamase as a reporter gene to study bacterial promoters.
Publisher
ProQuest Dissertations & Theses
Subject
ISBN
9798207205502
