Characterization of the human glutamine PRPP amidotransferase-AIR carboxylase locus
Abstract
Glutamine PRPP Amidotransferase catalyzes the first committed step of de novo purine biosynthesis. The regulation of this enzyme is not well understood. The amidotransferase from B. subtilis and higher eukaryotes has an 11 amino acid propeptide, which must be cleaved for activity, and an Fe-S cluster, both of which may have a regulatory function. The cDNA for this enzyme has been cloned from E. coli, B. subtilis, yeast, chicken, rat, and human. To determine if GPAT is subject to regulation on the transcriptional level the genes from both chicken and human have been cloned. As chickens do not have a urea cycle, they shuttle all of their excess nitrogen through the purine de novo pathway and excrete it as uric acid. For this reason we expected to find differences between the chicken and the human genes. Described herein is the cloning of the human gene. During the cloning process, it was noted that there is another gene involved in purine biosynthesis immediately upstream from the chicken gene. This gene was cloned from the human and found to be closely linked to GPAT. This second gene encodes the bifunctional enzyme 5$\sp \prime$-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole synthetase/aminoimidazole ribonucleotide carboxylase, abbreviated AIRC, which catalyzes steps 6 and 7 of the de novo pathway. The structures of both of these genes have been determined; GPAT is 45 kb, and is composed of 11 exons, whereas AIRC is encoded in 9 exons that span 25 kb. The structure of the human gene is similar to the chicken; the exon/intron splice sites are positioned identically, however, most of the introns are sightly larger in the human gene. These genes are separated by 621 base pairs and are transcribed on opposite strands of DNA. The intergenic region contains two separate promoters that have been localized to within 110 bp of the transcription start site for each gene. The cloning of these genes provides a solid basis for regulation studies using these promoters.
Degree
Ph.D.
Advisors
Zalkin, Purdue University.
Subject Area
Biochemistry
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