Structure of the rat PRPS1 gene encoding phosphoribosylpyrophosphate synthetase subunit I.

H Shimada,M Taira,K Yamada,T Iizasa,M Tatibana

doi:10.1016/s0021-9258(19)39687-5

Abstract

Phosphoribosylpyrophosphate (PP-Rib-P) synthetase (EC 2.7.6.1) subunit I gene (PRPS1) is constitutively expressed in various tissues (Taira, M., Iizasa, T., Yamada, K., Shimada, H., and Tatibana, M. (1989) Biochim. Biophys. Acta 1007, 203-208). We report here the exon-intron organization and the transcription promoter sequence of rat PRPS1 gene. This gene has 22 kilobases and is split into 7 exons ranging in size from 99 to 251 base pairs (bp), except for exon 7 (1008 bp). A putative PP-Rib-P binding site is encoded in exon 5. The exon-intron boundaries are similar to the consensus sequences for mammalian introns. S1 nuclease and primer extension assays with the use of RNA from rat Yoshida ascites sarcoma cells led to the identification of four possible transcription start points closely spaced between 126 and 129 bp from the ATG initiation codon. In the upstream region from the transcriptional start sites, we observed a TATA-like sequence (TAATTTAAT) at nucleotides -28, a CCAAT element (AGCCAATC) at nucleotides -80, and three GC boxes (putative Sp1-binding sites) at nucleotides -103, -43, and -10. A comparison of the promoter region for PRPS1 with those of other housekeeping genes revealed a homology resembling that of the beta-actin gene.

Highlights

Sl nuclease and primer extension assays with the use of RNA from rat Yoshida ascites sarcoma cells led to the identification of four possible transcription start points closely spaced between 126 and 129 bp from the ATG
The enzyme is an oligomeric complex composed of about 34-kDa subunits, and we reported that the rat liver enzyme exists as complex aggregates of 34, 38, and 40-kDa components, the 34-kDa species being the catalytic subunit (Kita et al, 1989)
Thirteen positive recombinant phages were obtained; the PRPSl cDNA probe was strongly hybridized to 12 clones, whereas the PRPSS probe only hybridized to one

Summary

PROCEDURES

EMBL3 genomic library was constructed from female Sprague-Dawley rat liver DNA partially digested with MboI. This library was screened by hybridization with nick-translated fragments of rat PRPSl and PRPSZ cDNAs DNAs were isolated from recombinant phages amplified by growing on a 0.8% agarose plate They were mapped by double or partial digestion and by Southern blot analysis. Primer 1 was 5’ end-labeled with [+y-32P]ATP (ICN Biomedicals Inc.) and T4 polynucleotide kinase (Takara) at 37 “C for 30 min (a specific activity of 3 x lo cpm/pg). SI Nuclease Mapping-A genomic DNA fragment (RsaI/SacI 382 bp; positions -226 to +156) covering the putative transcription initiation site DNA prior to labeling a recessed 5’ end at the Sac site, the DNA fragment was strand-separated by electrophoresis on a. The horizontal arrows indicate the direction and extent of nucleotide sequencing from the universal primer (closed circles) or from the specific primers (open circles; Primer 2 used for exon 2, Primer 3 for exon 4, Primer 4 for exon 5)

E IGESVRGEDVYI

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RESULTS AND DISCUSSION