Peroxisome biogenesis mutants and gene analysis in Saccharomyces cerevisiae.
Item
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Title
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Peroxisome biogenesis mutants and gene analysis in Saccharomyces cerevisiae.
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Identifier
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AAI9431378
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identifier
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9431378
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Creator
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Zhang, Jing Wei.
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Contributor
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Adviser: Paul B Lazarow
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Date
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1994
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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, Cell | Biology, Molecular | Biology, Microbiology
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Abstract
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The peroxisome is a nearly ubiquitous organelle in eukaryotic cells. It has vital functions in the cell, including {dollar}\rm H\sb2O\sb2{dollar}-based respiration, fatty acid {dollar}\beta{dollar}-oxidation, plasmalogen synthesis and cholesterol metabolism. A class of human genetic diseases is caused by the deficiency of peroxisome biogenesis. The goal of this project is to use the yeast, Saccharomyces cerevisiae, as a model system to study peroxisome biogenesis.;I first devised a positive selection procedure that identifies mutants lacking peroxisomes or peroxisomal functions. Immunofluorescence methods for yeast were simplified so that these mutants could be efficiently analyzed for impairments in peroxisome biogenesis. With these tools, I have identified eleven peroxisome biogenesis (peb) mutants which were sorted into five complementation groups.;I then developed a new gentle cell fractionation procedure which employs digitonin titration for the selective permeabilization of yeast plasma and intracellular membranes. This permits the intracellular distribution of proteins to be determined accurately, without mechanical breakage of membranes.;These five groups of peb mutants were analyzed with immunofluorescence, electron microscopy, immunoelectron microscopy and this new cell fractionation method. The biochemical and morphological data were consistent. Mutants from two groups lack recognizable peroxisomes. All the peroxisomal proteins examined are mislocated in the cytosol in them. Two groups contain peroxisomes, but are selectively defective in packaging newly synthesized peroxisomal proteins: peb1 fails to package thiolase, but the import of catalase and acyl-CoA oxidase into peroxisomes is normal; peb5 does not package catalase, but packages thiolase and acyl-CoA oxidase. These two mutants also show striking intracellular clustering of peroxisomes. The three peroxisomal proteins analyzed are thought to each use different topogenic information. These data suggest there are at least three pathways (or branches in a pathway), each represented by one of these three peroxisomal proteins, involved in the import of peroxisomal proteins.;Finally I cloned the PEB1 gene by complementing the peb1 mutant with a yeast genomic library. PEB1 encodes a 42 kD hydrophilic protein. The PEB1 gene product was epitope-tagged and localized to peroxisomes by fluorescence microscopy and cell fractionation. A model for its role in peroxisome biogenesis is proposed.
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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.
