Structural and functional comparison of the Saccharomyces cerevisiae maltose-inducible transcription activator encoded by MAL63 and its nonfunctional homologue encoded by MAL64.
Item
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Title
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Structural and functional comparison of the Saccharomyces cerevisiae maltose-inducible transcription activator encoded by MAL63 and its nonfunctional homologue encoded by MAL64.
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Identifier
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AAI9405601
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identifier
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9405601
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Creator
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Wojciechowicz, Lori Ann.
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Contributor
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Adviser: Corinne A. Michels
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Date
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1993
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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, Genetics
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Abstract
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Maltose fermentation in Saccharomyces yeasts requires one of five, unlinked complex loci (MAL1-4 and MAL6) each containing the three genes required for fermentation. At the MAL6 locus, MAL63 encodes a DNA-binding protein which promotes the maltose-inducible transcription of the two structural genes, MAL61 (encoding maltose permease) and MAL62 (encoding maltase). Yeast strains carrying noninducible mal63 mutations can revert to maltose fermenters but structural gene expression in these revertant strains is constitutive. Two such constitutive mutations lie in a gene upstream of the MAL63 gene, referred to as MAL64, and the two constitutive alleles are called MAL64-C2 and MAL64-R10. Wild-type MAL64 is not essential for maltose fermentation.;MAL63 and MAL64 are highly homologous. Both genes encode proteins which are 470 amino acids long and 85% identical. Analysis of MAL64-C2 and MAL64-R10 revealed nonsense mutations at codons 307 and 282, respectively. Experiments here confirmed that the nonsense mutations in these constitutive alleles were responsible for activity.;Chimeric gene fusions between MAL63 and MAL64 fragments showed that residues 215-470 of MAL63 encoded protein (MAL63p) are responsible for responding to the inducer, maltose. Also, deletion of a short acidic stretch of 14 amino acids at the C-terminal end of MAL63p results in an uninducible activator. Therefore, unlike the constitutive MAL64p, MAL63p requires residues in its C-terminal domain for activity.;A hemagglutinin epitope tag was placed at the 5{dollar}\sp\prime{dollar}-end of MAL63, overexpressed off the GAL10 promoter and this fusion protein was detected in cells. Overexpression in galactose of either the wild-type tagged protein or a mutant tagged protein lacking the final 14 C-terminal acidic residues did not result in a constitutive phenotype. This experiment excludes the possibility of the involvement of a limiting repressor protein since overexpression of MAL63p would titrate accessory factors and thus activate structural gene expression.;Based on homology to other yeast activators, the acidic final C-terminal residues of MAL63p could be involved in transcription activation. By use of lexA-MAL63 gene fusions, these residues were required for the maltose-inducible transcription activation function of MAL63.
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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.
