Molecular genetics of acute intermittent porphyria and studies of the human cytosolic heme biosynthetic enzymes.
Item
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Title
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Molecular genetics of acute intermittent porphyria and studies of the human cytosolic heme biosynthetic enzymes.
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Identifier
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AAI9432332
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identifier
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9432332
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Creator
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Chen, Chia-Hsiang.
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Contributor
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Adviser: Robert J. Desnick
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Date
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1994
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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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Health Sciences, Medicine and Surgery
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Abstract
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Acute intermittent porphyria (AIP), an autosomal dominant disease, results from the defective heme biosynthetic enzyme, hydroxymethylbilane synthase (EC 4.3.1.8; HMBS). Asymptomatic heterozygotes are induced to manifest symptoms by various factors. We cloned and determined the complete sequences of human HMBS gene. Four new polymorphic sites were identified by the single strand conformation polymorphism analysis, a common one in intron 2 (3119 G/T), one common BsmAI site in intron 3 (3581 A/G), another common HinfI in intron 10 (7064 C/A), and a rare Mn/I site in intron 14 (7998 G/A). Six exonic mutations causing AIP were identified: an initiating methionine to isoleucine substitution (M1I), which causing variant AIP. Four missense mutations, V93F, R116W, R201W, C247F; and a nonsense mutation W283X, which cause classical AIP. Expression studies revealed that R201W, C247F, and W283X had residual activity.;Large quantities of recombinant human HMBS isozymes were purified by using immobilized metal affinity chromatography. Purified HMBS isozymes have similar physical and kinetic properties.;To determine if HMBS and UROS form a complex, polyclonal anti-HMBS antibodies were used to immunoprecipitate HMBS from human HepG2 and K562 cells labeled with ({dollar}\sp{lcub}35{rcub}{dollar}S) -methionine. UROS did not coprecipitate with HMBS. Furthermore, a two-hybrid protein interaction assay shows negative result for the presence of enzyme complex between HMBS and UROS.;In summary, these studies have provided further delineation and understanding of the molecular genetics and cellular biochemistry of human HMBS, a critical cytosolic enzyme in the heme biosynthetic pathway.
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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.
