Isolation and characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyl transferase.

Abstract

We have cloned a full-length 1.6-kilobase cDNA of a human mRNA coding for hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) into a simian virus 40-based expression vector and have determined its full nucleotide sequence. The inferred amino acid sequence agrees with a partial amino acid sequence determined for authentic human HPRTprotein..Transfection of HPRTdeficient mouse IA9 cells with the purified plasmid leads to the expression of human HPRT enzyme activity in cells stably transfected and selected for enzyme activity in hypoxanthine/aminopterin/thymidine medium.Current methods of molecular biology, including the techniques of recombinant -DNA construction and cloning, rapid nucleotide sequence analysis, the design and construction of transducing vectors, and techniques oftransfection ofeukaryotic cells with foreign genes, have made it possible to clone and characterize a large number of eukaryotic genes. One gene of particular interest, not only for basic studies of eukaryotic gene regulation but also for understanding of several important human genetic diseases, is the gene encoding the enzyme hypoxanthine phosphoribosyltransferase (HPRT) (1). This enzyme catalyzes vital steps in the reutilization pathway for purine biosynthesis, and its deficiency leads to forms ofgouty arthritis and to the devastating Lesch-Nyhan disease (2,3). The HPRT locus is known to be X linked in the human and other mammalian genomes, and the availability of a cloned HPRT gene would facilitate studies ofthe organization ofa particularly interesting region of the human X chromosome and of the mechanisms of inactivation of specific and well-mapped regions of the X chromosome. Recently, we succeeded in isolating a human genomic clone containing a portion ofthe HPRT (4) by a combination of gene transfection into enzyme-deficient mouse cells and localization of human sequences by hybridization with probes of middle repetitive human (Alu) sequence, as described (4). A 15-kilobase (kb) EcoRI/BamilI fragment derived from p6B2aE2 and free ofrepetitive human sequences ,has been subcloned into the BamHI and EcoRI-sites ofpBR322 and shown to hybridize to a discrete human cytoplasmic poly(A)+RNA approximately 1.5 kb long, presumably. representing the mRNA for HPRT (4). DNA from this subcloned plasmid, called pBR1.5, was prepared by detergent lysis (5) of chloramphenicol-treated (6) transformed SF8 Escherichia coli (7) followed. by cesium chloride/ethidium bromide ultracentrifugation (8). This DNA was cleaved with EcoRI/BamrHI and the cloned insert was isolated by trough electroelution onto a dialysis membrane (9) The colonies on the replica plates were transferred to Whatman 541 paper, amplified on chloramphenicol plates overnight at 37QC by the method ofGergen et at (13), denatured and fixed to the filters, and screened for HPRT cDNA sequences by hybridization with the gel-purified insert from pBR1.5 containing the HPRT fragment free ofrepeat sequences. The filter replicas of the...

Growth factor – regulated expression of enzymes involved in nucleotide biosynthesis: a novel mechanism of growth factor action

“…This sequence is 100% identical to the sequence (nt 219 ± 511) published by Powell et al (1992). Furthermore, we cloned fragments encoding parts of PRPS subunit one (398 bp; nt 183 ± 580; Sonoda et al, 1991), APRT (368 bp; nt 220 ± 587; Iwahana et al, 1993a), HPRT (326 bp; nt 355 ± 680; Jolly et al, 1983), and of the multifunctional protein CAD (310 bp; nt 871 ± 1180; Iwahana et al, 1996).…”

Lipopolysaccharide increases cyclo-oxygenase-2 expression in a colon carcinoma cell line through nuclear factor-κB activation

“…Therefore, we selected 35 cycles for PCR detection of COX-2 and HPRT mRNA expression. Oligonucleotide primers were: COX-2, 5'-TTCAAATGAGATTGTGGGAAAAT-3' (sense) and 5'-AGATCATCTCTGCCTGAGTATCTT-3' (antisense); and HPRT, 5'-CGAGATGTGATGAAGGAGATGG-3' (sense) and 5'-GGATTATACTGCCTGACCAAGG-3' (antisense) (O'neil and Ford-Hutchinson, 1993;Jolly et al, 1983). PCR products were separated by electrophoresis on 1.5% agarose gels and stained with ethidium bromide.…”

VASA Is a Specific Marker for Both Normal and Malignant Human Germ Cells

“…Relative quantification of the VASA-transcript levels was performed by duplex PCR with VASA specific primers (primer sequences: 5' AAG AGA GGC TAT CGA GAT GGA 3' and 5' CGT TCA CTT CCA CTG CCA CTT CTG 3') compared with HPRT (primer sequences: 5' CGT GGG GTC CTT TTC ACC AGC AAG 3' and 5' AAT TAT GGA CAG GAC TGA ACG TC 3'; Jolly et al, 1983), resulting in amplification products of 238 bp and 387 bp, respectively. The expression level of HPRT is constant in most tissues, including the testis, and can therefore serve as an internal RT-PCR standard (Pannetier et al, 1993).…”