Molecular genetic analysis of the functional domains of the MAL-activator of Saccharomyces cerevisiae.
Item
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Title
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Molecular genetic analysis of the functional domains of the MAL-activator of Saccharomyces cerevisiae.
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Identifier
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AAI9521272
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identifier
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9521272
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Creator
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Gibson, Andrew Winston.
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Contributor
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Adviser: Corinne A. Michels
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Date
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1995
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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, Genetics | Biology, Molecular | Biology, Cell
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Abstract
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Maltose fermentation in Saccharomyces cerevisiae requires any one of five dominant, unlinked MAL loci (MAL1, MAL2, MAL3, MAL4, MAL6). Three genes are encoded at each locus: Gene 1 encodes maltose permease, a membrane protein which transports maltose across the plasma membrane; Gene 2 encodes maltase, an {dollar}\alpha{dollar}-glucosidase which hydrolyzes maltose to give two glucose monomers; and Gene 3 which encodes the MAL-activator, a positive regulator of the expression of the structural genes.;MAL63, which encodes the activator at the MAL6 locus, encodes a predicted 470 amino acid residue DNA-binding protein with a cysteine-rich zinc cluster (residues 8-38). A peptide containing this zinc cluster was shown to bind to the UAS{dollar}\rm\sb{lcub}MAL{rcub}{dollar} in the promoter region of the structural genes to activate their transcription.;To determine the other functional domains of the Mal63p activator, we carried out a deletion analysis using LexA-MAL63 gene fusions. The analysis indicates that the DNA-binding domain of Mal63p extends to residue 100, that a transcription activation domain lies within residues 100-250, and that a maltose-responsive regulatory domain exists in residues 283-470. Our analysis also indicates that the fourteen C-terminal residues are required for function.;A molecular genetic analysis of the Mal43-Cp constitutive activator, encoded at the MAL4 locus, showed that C-terminal alterations between residues 215-470 were responsible for its unregulated mutant phenotype. However, the phenotype of Mal43-Cp could not be limited to one or even a few of the 27 amino acid residue alterations in this region.;Overexpression of MAL63 on a multi-copy plasmid failed to uncover the existence of any titratable Gal80p-like inhibitor of Mal63p activity.;A Two-Hybrid analysis, performed in order to test the hypothesis that different regions of Mal63p interacted revealed no such interaction.;A model of MAL-activator induction by maltose is discussed.
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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.
