Regulation of the cardiolipin synthase gene in Escherichia coli.
Item
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Title
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Regulation of the cardiolipin synthase gene in Escherichia coli.
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Identifier
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AAI9108115
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identifier
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9108115
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Creator
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Heber, Sheldon Orly.
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Contributor
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Adviser: Burton E. Tropp
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Date
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1990
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Molecular | Biology, Microbiology
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Abstract
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To better understand the importance of cls, the structural gene for cardiolipin synthase, an extensive study was carried out. The first objective was to determine whether cls is essential and establish its chromosomal location. Using Tn10dTet several insertions into cls were obtained. These insertions caused an increased resistance to 3,4-dihydroxybutyl-1-phosphonate and a deficiency in cardiolipin content. Using the tetracycline resistance gene inside Tn10dTet as a marker, three factor crosses were carried out to determine the location of cls with respect to neighboring genes. The gene order hemA-narC-tyrT-adhC-cls was established.;The second objective was to create operon and protein fusions, between cls and lacZ. Extensive in vivo attempts to fuse cls to lacZ were unsuccessful. It therefore became necessary to clone cls and create the gene fusions in vitro. A two step approach was used to clone cls. The first step involved the insertion of a Tn10dCam next to cls. The second step was to clone cls by selecting for Tn10dCam. Plasmids containing a cloned cls increased the CL content of cls defective strains.;Using the cloned cls, in vitro operon and protein fusions between cls and lacZ were constructed. The gene fusions were transferred from multicopy plasmids into the E. coli {dollar}\lambda{dollar} attachment. Using {dollar}\beta{dollar}-galactosidase as an indication of cls expression, the regulation of cls was investigated under a number of conditions. Under most of the conditions studied, there was little or no change in cls expression. However, a two to three fold increase in expression of the operon and protein fusion was observed as the cells were grown to stationary phase, where grown in minimal media versus rich media, and when the bacteria were grown anaerobically. The anaerobic affect was further investigated using different electron acceptors. The better the electron acceptor the lower the cls expression. In the presence of two electron acceptors, cls expression reflected that of the better electron acceptor.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
