Molecular genetic studies of human delta-aminolevulinic acid dehydratase.
Item
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Title
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Molecular genetic studies of human delta-aminolevulinic acid dehydratase.
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Identifier
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AAI9218243
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identifier
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9218243
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Creator
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Kaya, Angela Haruko.
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Contributor
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Adviser: James G. Wetmur
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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 | Environmental Sciences
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Abstract
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The ALAD gene has been cloned and sequenced in both directions. The genomic sequence was found to be identical to the previously published cDNA sequence. The gene encodes thirteen exons: eleven coding exons, exons 2-12, and two non-coding exons, 1A and 1B. The zinc binding site was located in exon five. The active lysine was located in exon ten. Nine Alu repetitive elements were found in the gene. Additional types of repetitive elements were not found in the genomic sequence.;The most upstream of the ALAD non-coding exons, exon 1A, is presumed to be governed by a housekeeping promoter. The promoter region of exon 1A contained characteristics of a constitutive promoter. The promoter region of exon 1B contained characteristics of genes which are specifically regulated in erythrocytes.;The ALAD{dollar}\sp2{dollar} allele was cloned and sequenced. The only mutation found was a G to C transversion at nucleotide 177 of the coding sequence. This transversion created an MspI restriction endonuclease site and predicted the substitution of a positively charged lysine by a neutral asparagine in residue 59 of the ALAD 2 subunit. Because of the presence of the restriction endonuclease site, a simple PCR based method was employed for ALAD genotyping. Analysis of a 85 ALAD 1-2 individuals and eight ALAD 2-2 individuals revealed that, in all cases, the ALAD{dollar}\sp2{dollar} allele phenotype corresponded with the MspI RFLP genotype.;Analysis of a random population of 428 normal Caucasian individuals revealed the frequency of the ALAD{dollar}\sp1{dollar} and the ALAD{dollar}\sp2{dollar} allele frequencies were 0.88 and 0.12, respectively. The allele frequencies of a previously identified RsaI RFLP were 0.75(RsaI{dollar}\sp-){dollar} and 0.25(RsaI{dollar}\sp+){dollar}. Individually, the RFLPs were in Hardy-Weinberg equilibrium, however, the two RFLPs taken together were in linkage disequilibrium. The ALAD{dollar}\sp2{dollar}/{dollar}Rsa\sp+{dollar} allele was highly under represented.;Bacterial expression of an authentic, soluble, biologically active human ALAD{dollar}\sp1{dollar} was achieved using a co-cistronic vector derived from pUC9. The ALAD enzymatic activity was over 100 times that found in human erythrocytes. The recombinant protein was identical to human erythrocyte protein by polyacrylamide gel electrophoresis mobility, Western blotting, elution of the homooctamer from a gel filtration column and its sedimentation in a sucrose gradient.
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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.
