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Characterization of the A subunit mutants of Stx1 and Stx2 in Saccharomyces cerevisiae
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Title
Characterization of the A subunit mutants of Stx1 and Stx2 in Saccharomyces cerevisiae
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ETD_2168
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000051853
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eng
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theses
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Microbiology and Molecular Genetics
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(authority = ETD-LCSH)
Topic
Saccharomyces cerevisiae
Subject
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(authority = ETD-LCSH)
Topic
Mutation (Biology)
Abstract
Shiga toxins are a family of related toxins with two major groups, Stx1 and Stx2. The most common sources for Shiga toxin are the bacteria Shigella dysenteriae and the Shigatoxigenic group of Escherichia coli (STEC), which includes serotype O157:H7. Shiga-like toxin 1 and 2 (Stx1 and Stx2) are produced by some E. coli strains. Stx produced by S. dysenteriae differs from Stx1 by one amino acid. Stx1 and Stx2 have only 56% sequence homology.
E. coli O157:H7 has been the major cause of food-borne illnesses. Symptoms include abdominal cramping, bloody diarrhea, vomiting and a low-grade fever. Serious complications can lead to Hemolytic Uremic Syndrome (HUS).
Stx1 and Stx2 are categorized as and AB5 toxin. The A complex is responsible for toxicity and has enzymatic activity inside the host cell. The B subunits bind to a receptor on the surface of the host cell. Shiga toxin acts as an N-glycosidase. It cleaves a specific adenine residue in the conserved α- sarcin ricin loop in the 28S subunit of the eukaryotic rRNA. This interferes with the translocation step in protein synthesis and leads to inhibition of translation and cell death.
When Stx1A and Stx2A cDNA was cloned downstream of the GAL1 promoter and transformed into Saccharomyces cerevisiae, cells growth was greatly reduced when expression of the toxin was induced. To identify residues critical for cytotoxicity or Stx1A and Stx2A random and site-directed mutations were made. The ability of the mutant proteins to depurinate ribosomes in vivo in yeast was determined by dual primer extension analysis. The amount of translation was measured by incorporation of [35S]-methionine in the cell.
The results of these assays show mutations in N75, Y77, E167, R170, and R176 are critical for toxicity in both toxins. Also C-terminal deletions after L240 in Stx1A and I238 in Stx2A abolished toxicity. Several of these mutants, even though they are non-toxic still have the ability to depurinate ribosomes. This indicates cytotoxicity and depurination may not be directly related. This provides evidence that cytotoxicity is not necessarily a direct result of depurination, and other potential mechanisms may contribute in Stx1A and Stx2A cytotoxicity in yeast.
E. coli O157:H7 has been the major cause of food-borne illnesses. Symptoms include abdominal cramping, bloody diarrhea, vomiting and a low-grade fever. Serious complications can lead to Hemolytic Uremic Syndrome (HUS).
Stx1 and Stx2 are categorized as and AB5 toxin. The A complex is responsible for toxicity and has enzymatic activity inside the host cell. The B subunits bind to a receptor on the surface of the host cell. Shiga toxin acts as an N-glycosidase. It cleaves a specific adenine residue in the conserved α- sarcin ricin loop in the 28S subunit of the eukaryotic rRNA. This interferes with the translocation step in protein synthesis and leads to inhibition of translation and cell death.
When Stx1A and Stx2A cDNA was cloned downstream of the GAL1 promoter and transformed into Saccharomyces cerevisiae, cells growth was greatly reduced when expression of the toxin was induced. To identify residues critical for cytotoxicity or Stx1A and Stx2A random and site-directed mutations were made. The ability of the mutant proteins to depurinate ribosomes in vivo in yeast was determined by dual primer extension analysis. The amount of translation was measured by incorporation of [35S]-methionine in the cell.
The results of these assays show mutations in N75, Y77, E167, R170, and R176 are critical for toxicity in both toxins. Also C-terminal deletions after L240 in Stx1A and I238 in Stx2A abolished toxicity. Several of these mutants, even though they are non-toxic still have the ability to depurinate ribosomes. This indicates cytotoxicity and depurination may not be directly related. This provides evidence that cytotoxicity is not necessarily a direct result of depurination, and other potential mechanisms may contribute in Stx1A and Stx2A cytotoxicity in yeast.
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electronic resource
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viii, 48 p. : ill.
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M.S.
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by Eric Kyu
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Kyu
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Eric
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1982-
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Eric Kyu
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Rong
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Rong Di
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Tumer
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Nilgun
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Nilgun Tumer
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Martin
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Charles
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Charles Martin
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Rutgers University
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Graduate School - New Brunswick
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2009
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2009-10
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xx
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Rutgers University Electronic Theses and Dissertations
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ETD
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Graduate School - New Brunswick Electronic Theses and Dissertations
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doi:10.7282/T34F1QWJ
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ETD graduate
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The author owns the copyright to this work
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Eric
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Eric Kyu
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Rutgers University. Graduate School - New Brunswick
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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