Gene regulation involved in C5 pathway for synthesis of delta-aminolevulinic acid.
Item
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Title
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Gene regulation involved in C5 pathway for synthesis of delta-aminolevulinic acid.
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Identifier
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AAI3037417
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identifier
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3037417
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Creator
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Lin, Fang.
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Contributor
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Advisers: Sharon Cosloy | Charlotte Russell
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Date
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2002
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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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Biology, Molecular
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Abstract
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Production of delta-aminolevulinic acid (ALA) is the rate-limiting step in the C5 pathway for tetrapyrrole biosynthesis. In the C5 pathway, glutamate is charged with tRNAGlu by glutamyl-tRNA synthetase (GTS) encoded by gltX; glutamyl-tRNA is reduced to glutamyl-1-semialdehyde by glutamyl-tRNA reductase (GTR) encoded by hemA; GSA is converted to ALA by an aminotransferase. Regulation of hemA most probably controls ALA biosynthesis. There are P1, P2 and putative P3 promoters in the hemA upstream region, and two overlapping stem-loops (SL1 and SL2) behind the putative P3. Regulation of hemA at the transcriptional level was studied by comparing the chloramphenicol acetyltransferase (CAT) expression of various parts of the hemA upstream region inserted in front of the promoterless CAT reporter gene of pKK232-8---pWH515-CAT (pKK232-8 with the 554 bp of the hemA upstream region) and pFL510-CAT (pKK232-8 inserted with the hemA upstream region with deletion of SL1 and SL2). It was found that deletion of the SL1 and SL2 did not change the hemA promoter activity. The putative P3 promoter activity was detected by measuring the CAT expression of pFL25-CAT in medium copy plasmids (pKK232-8 inserted with the hemA upstream region with deletion of P1 and P2 promoters), which accounts for 1% of the promoter activity of the whole 5 ' upstream region of hemA. The CAT expression did not change when a plasmid harboring hemA or gltX coexisted in a strain with pWH515-CAT, pFL510-CAT or pGLTX-CAT respectively. Over-expression of GTS or GTR did not change the promoter activity of hemA or gltX. HemX in Bacillus subtilis is a 32-kDa membrane protein. Expression of B. subtilis hemX down-regulates its GTR. E. coli does not have hemX. In order to understand HemX function, hemX was subcloned into modified pUC19 and transformed into SASX41B, an E. coli hemA- heme-impermeable strain. The transformants did not grow in the medium supplemented with hemin. It suggests that HemX is not a heme channel and does not make E. coli permeable to heme.
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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.
