Blm10p of Saccharomyces cerevisiae is a nuclear protein that functions in relieving cellular stresses caused by bleomycin.
Item
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Title
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Blm10p of Saccharomyces cerevisiae is a nuclear protein that functions in relieving cellular stresses caused by bleomycin.
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Identifier
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AAI3115241
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identifier
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3115241
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Creator
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Doherty, Kevin.
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Contributor
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Adviser: Carol Wood Moore
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Date
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2004
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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, Cell
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Abstract
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Blm10p has been one of many uncharacterized proteins of Saccharomyces cerevisiae. The work presented here provides an initial characterization of the protein. BLM10 acts as a multi-copy suppressor of the hypersensitivities conferred by the blm3-1 mutation to lethal effects of the bleomycin-phleomycin family of glycopeptides. Mutant blm3-1/blm3-1 cells suffered extremely high DNA damage, assayed in pulsed field gel electrophoretic analyses, and killing. BLM10 was found to be nonessential for viability, but essential for protection against lethal effects of the drug family. The 6432 bp gene (YFL007w) was sequenced in three strains, and was actually larger than originally predicted. The size was confirmed in northern analyses. Deletion of BLM10 conferred complete growth inhibition and high killing in the presence of 5 mug/ml phleomycin. Overexpression of Blm10p restored growth and survival.;A Blm10p-YFP fusion protein was created. It localized to nuclei throughout the cell cycle, and co-localized with Spc42p-CFP. After drug treatments, Blm10p also localized to nuclei, but did not form foci. Nuclei were fragmented after drug treatment. A truncated Blm10p-GST fusion, missing 340 amino acids at the Blm10p carboxy terminus, also localized to nuclei in stationary phase cells, but localized to bud necks in budded cells. This indicated the importance of the carboxyl terminal region of the protein for its retention in the nucleus. Blm10p was found highly conserved among seven lower and higher eukaryotes, and evaluation of each region of the proteins revealed five conserved regions in similar spacial arrangements. The conserved carboxyl-terminal region contained the strongest homology, further indicating the importance of this region. Only one homolog, human PA200, a 20S proteasomal activator, is characterized. Based on this knowledge, proteasomal function was investigated in BLM10 and b1m10Delta cells, but found to be equivalent in both genotypes for the degradation of whole proteins. Finally, extensive two hybrid screens were carried out to determine specific, direct interactions of Blm10p with other proteins, and should be informative when analyses are completed.
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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.
