FABRY DISEASE: ISOLATION, CLONING, AND SEQUENCE ANALYSES OF COMPLEMENTARY-DNA AND GENOMIC CLONES ENCODING ALPHA-GALACTOSIDASE A.
Item
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Title
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FABRY DISEASE: ISOLATION, CLONING, AND SEQUENCE ANALYSES OF COMPLEMENTARY-DNA AND GENOMIC CLONES ENCODING ALPHA-GALACTOSIDASE A.
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Identifier
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AAI8713787
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identifier
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8713787
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Creator
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QUINN, MERRIGENE.
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Contributor
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David B. Calhoun
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Date
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1987
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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, Molecular
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Abstract
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Fabry disease, an X-linked error of glycosphingolipid metabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A. To investigate the nature of the molecular mutations involved with Fabry disease and to characterize the structure, organization, and expression of the alpha-galactosidase A locus, this study reports the isolation of cDNA and genomic clones specific for human alpha-galactosidase A. In addition, since this is the first genomic clone described to date for a lysosomal enzyme, it establishes a reference for future analyses of the molecular events that mediate the expression of lysosomal hydrolases. The human alpha-galactosidase A cDNA clone (designated (lamda)AG18) was isolated from a (lamda)gt11 expression library using the antibody detection method. Phage (lamda)AG18 contains an EcoRI cDNA insert of 1226 nucleotides with an open reading frame encoding 398 amino acids and a protein with a predicted molecular weight of 45,346. This clone also includes sequences encoding the last five amino acids of the signal peptide. Two polyadenylation signals, AATACA and ATTAAA, are located 28 and 11 nucleotides, respectively, before the TAA stop codon. Phage (lamda)AG18 lacks a 3'-untranslated region since the poly(A) sequence immediately follows the TAA codon. Four possible N-glycosylation sites were identified within the propeptide sequence. Hybridization of HeLa cell mRNA with nick translated cDNA insert revealed a single band of approximately 1.45 kilobases. A genomic clone specific for human alpha-galactosidase A, was isolated from a lymphoblast 4X Charon 30 library. Nucleotide sequence analysis of a 1243 nucleotide genomic fragment includes sequences for the promoter, complete signal peptide, first exon, and 104 nucleotides of the first intron. Direct and inverted repeat elements of 10, 11, 16, 19, and 22 nucleotides flank the promoter site. A GA-rich repeat element of approximately 60 nucleotides, found to be homologous to similar elements in several species, is located upstream of the promoter. A GGGCGG site specific for the DNA binding protein Sp1 is located next to a CAAT box, and the inverted Sp1 binding site, CCGCCC, is located next to the TATA box. The sequence immediately flanking the ATG initiation codon of the human alpha-galactosidase A gene was shown to be highly homologous to sequences flanking the ATG initiation codons of four of the other five human lysosomal hydrolases for which sequence information is available, but not for any of the other 133 human signal peptides, examined. Information is presented which indicates conversion to the mature enzyme occurs by cleavage of a carboxy-terminal propeptide fragment.
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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.
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Program
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Biomedical Sciences
