CLONING OF HUMAN ALPHA-GALACTOSIDASE A AND MOLECULAR GENETIC STUDIES OF FABRY DISEASE (LYSOSOME, CDNA, MUTATION).
Item
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Title
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CLONING OF HUMAN ALPHA-GALACTOSIDASE A AND MOLECULAR GENETIC STUDIES OF FABRY DISEASE (LYSOSOME, CDNA, MUTATION).
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Identifier
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AAI8629673
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identifier
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8629673
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Creator
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BERNSTEIN, HAROLD SETH.
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Contributor
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Robert J. Desnick
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Date
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1986
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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
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Abstract
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In order to characterize the nature of the molecular defect in Fabry disease, a cDNA encoding the entire mature lysosomal form of human (alpha)-galactosidase A (GALA; EC 3.2.1.22) was isolated from a (lamda)gt11 human liver expression library using monospecific antibody and synthetic oligonucleotide probes, which were synthesized corresponding to amino-terminal and internal tryptic peptide sequences. Four positive clones initially were identified by antibody screening of 1.4 x 10('7) plaques. Of these, only one clone demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to the synthetic oligonucleotide mixtures.;The complete nucleotide sequence was determined for the GALA cDNA by enzymatic sequencing of M13mp18 deletion subclones. The cDNA contained a 1211 base pair open reading frame encoding 398 amino acids of the mature polypeptide and the last five amino acids of the propeptide sequence. The predicted amino acid sequence was colinear with 86 nonoverlapping residues (22% of the mature subunit) determined by microsequencing of amino-terminal, trypic and cyanogen bromide peptides of the purified mature enzyme. Transfer hybridization analysis of HeLa poly(A)('+) RNA demonstrated a single 1.45 kilobase transcript. Nucleic acid and protein data base searches did not reveal significant homology even with the limited sequences available for mammalian lysosomal enzymes.;Analysis of the GALA gene in unrelated Fabry families demonstrated genetic heterogeneity in the molecular lesions which cause the disease. Dot blot analysis of total RNA with a cRNA probe revealed significantly decreased levels of GALA transcription in several individuals. Southern hybridization studies of genomic DNA with the cDNA identified two different partial gene deletions. Using M13mp18 deletion subclones, the extent of one was determined to be (TURN)3.5 kb at the 5' end of the gene, while the other was intragenic spanning (TURN)6 kb. A third abnormality was identified as an alteration of the Msp I cleavage site in the coding sequence. This lesion, which was localized by hybridization with a site-specific oligonucleotide, was expressed as a mutant protein with 30% residual activity in plasma, only 1-3% of normal activity in leukocytes and fibroblasts, and markedly decreased stability at lysosomal pH. The identification of these mutations allowed for the definitive diagnosis of heterozygotes and hemizygotes in these families.
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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
