Molecular genetic studies of mouse alpha-galactosidase A.
Item
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Title
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Molecular genetic studies of mouse alpha-galactosidase A.
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Identifier
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AAI9224817
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identifier
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9224817
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Creator
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Gotlib, Richard Warren.
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Contributor
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Adviser: David F. Bishop
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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
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Abstract
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{dollar}\alpha{dollar}-galactosidase A ({dollar}\alpha{dollar}-Gal A, E.C. 3.2.1.22) is a lysosomal hydrolase which cleaves neutral glycosphingolipids with terminal galactosyl moieties. The deficient activity of this enzyme in humans results in Fabry disease, an X-linked disorder characterized by accumulation of neutral glycosphingolipids in body fluids and most visceral tissues. Major manifestations in affected individuals include disseminated angiokeratoma corporis diffusum, acroparethesias, corneal opacities, and cardiac and renal dysfunction. Initial studies of transgenic mice expressing the human {dollar}\alpha{dollar}-Gal A gene revealed ubiquitous expression of the transgene. Subsequently, a full-length cDNA encoding mouse {dollar}\alpha{dollar}-Gal A was isolated and characterized. The full-length 1367 bp {dollar}\alpha{dollar}-Gal A sequence predicted 419 amino acids including a signal peptide sequence of 31 amino acids and four glycosylation sites. The functional integrity of the {dollar}\alpha{dollar}-Gal A cDNA was demonstrated by transient expression in COS-1 cells. Northern hybridization analysis of mouse RNA revealed two transcripts of about 1.4 and 3.3 kb. The mouse {dollar}\alpha{dollar}-Gal A cDNA had 82% nucleotide and 79% amino acid identity with the human {dollar}\alpha{dollar}-Gal A cDNA. The isolation of the cDNA facilitated the isolation and characterization of the mouse {dollar}\alpha{dollar}-Gal A structural gene. The intron/exon structure was shown to be identical to that of the human gene. All intron/exon junctions conformed to the GT/AG rule. Analysis of 193 nt of the 5{dollar}\sp\prime{dollar} flanking region revealed 67% sequence identity to the human promoter, with one Sp1 binding site, five CAAT boxes, and no TATA box. Sixteen Alu repetitive elements (twelve type 1 and four type 2) were identified. The {dollar}\alpha{dollar}-Gal A genomic sequences were used to construct vectors for homologous recombination in mouse embryonic stem (ES) cells to generate mice disrupted at the {dollar}\alpha{dollar}-Gal A locus, and thus deficient in {dollar}\alpha{dollar}-Gal A activity. Such mice may provide an animal model for Fabry disease and be useful for subsequent enzyme and gene therapy trials.
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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.
