Sequence analysis and identification of the DNA-binding domain of the Saccharomyces MAL-activator encoded by MAL63.
Item
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Title
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Sequence analysis and identification of the DNA-binding domain of the Saccharomyces MAL-activator encoded by MAL63.
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Identifier
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AAI9218244
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identifier
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9218244
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Creator
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Kim, Jeong Hwan.
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Contributor
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Adviser: Corinne A. Michels
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Date
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1992
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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 | Chemistry, Biochemistry
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Abstract
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Inducible maltose fermentation by Saccharomyces species requires three products of any one of the five identified MAL loci. Two of these gene products are maltose permease (GENE 1) and maltase (GENE 2), the proteins needed for transport of the sugar into the cell and its cleavage to glucose. The third gene product GENE 3 has been suggested to be an activator protein controlling the expression of the structural genes encoding the maltose fermentative enzymes perhaps by binding to DNA sequences upstream of these genes. We report the sequence of GENE 3 of the MAL6 locus, MAL63. A single open reading frame is seen capable of encoding a protein of 470 amino acid residues. A cysteine-basic amino acid-rich sequence at the N-terminal end of the protein is characteristic of one class of DNA-binding proteins supporting the hypothesis that the MAL63 gene product is a DNA-binding transcriptional activator.;To test if the cysteine-basic amino acid-rich region is a part of DNA-binding domain, we mutagenized cysteine residues 18, 27 and 34 to leucine, serine and glycine, respectively, using site-directed mutagenesis. The resulting mal63 mutant alleles were incapable of activating maltose fermentation in yeast. Also, using a gel mobility shift assay, protein extracts from E. coli strains expressing the mal63 mutant proteins all failed to bind to an oligonucleotide from the UAS{dollar}\sb{lcub}\rm MAL{rcub}{dollar} under conditions where the wild type MAL63 protein was able to bind. In addition, we altered the invariant proline found in this cysteine-rich region to leucine and found that the encoded product was partially functional in vivo but unable to bind DNA in vitro suggesting that the DNA binding domain was structurally altered. These results provide genetic evidence that the cysteine-basic amino acid-rich region is required for DNA-binding by the MAL63 protein and support the proposal that the DNA-binding domain by the MAL63 protein forms a zinc cluster similar to the structure proposed for the DNA-binding domain of the GAL4 protein (Pan and Coleman, 1990; Gardner et al., 1991).
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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.
