Molecular genetic analysis of constitutive and noninducible mutants of the Saccharomyces MAL-activator.
Item
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Title
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Molecular genetic analysis of constitutive and noninducible mutants of the Saccharomyces MAL-activator.
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Identifier
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AAI9820524
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identifier
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9820524
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Creator
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Danzi, Sara Ellen.
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Contributor
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Adviser: Corinne A. Michels
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Date
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1998
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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, Microbiology | Chemistry, Biochemistry
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Abstract
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The C-terminal 220 amino acid residues of the MAL-activator protein function as a regulatory domain. Previous studies which characterized constitutive and noninducible MAL-activator mutations found several of both phenotypes which contained multiple alterations and deletions within this region suggesting that both negative and positive regulatory subdomains were present but the number, location, and function of these subdomains within this regulatory region were unknown. Using site directed in vitro mutagenesis of the inducible MAL63 and MAL63/23 genes, negative subdomains were identified at 2 sites (called NSD-1 and NSD-2). Multiple mutations within each of these regions relieve the negative regulation of MAL-activator activity in the absence of inducer, producing a constitutive phenotype. Intragenic suppressors of the constitutive mutations in NSD-2 and NSD-3, a third negative subdomain previously identified by Wang and Needleman (1996), also were identified at closely linked sites. Constitutive mutations of NSD-2 and NSD-3 are partially dominant and dominant, respectively. Additional suppressors of NSD-3 mutations were revealed at more distant sites within NSD-1. These results suggest that complex intramolecular interactions within this regulatory domain are required to maintain the inactive state of the MAL-activator in the absence of inducer. Clustered-charge to alanine mutagenesis of the C-terminal regulatory domain of the inducible Mal63 MAL-activator was undertaken to identify positive subdomains. Noninducible mutations were obtained when residues in the region from 331-469 were altered. In order to distinguish a role for these residues in induction from other required functions of the activator, the noninducible alterations were introduced into the constitutive hybrid gene MAL63/43. Those mutations found to affect only the inducible activator localized to residues 331-423 and are proposed to be involved in induction. Mutations affecting both the inducible and the constitutive activators localized to residues 438-470 and are proposed to be involved in transactivation and/or maintenance of a transcriptionally active state. Three essential residues at 364, 367, and 391 also were identified. A model for maltose induction of the MAL-activator 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.
