Taizo Iizasa

Tissue-differential expression of two distinct genes for phosphoribosyl pyrophosphate synthetase and existence of the testis-specific transcript

Cloning of cDNA coding for rat phosphoribosyl pyrophosphate (PPRibP) synthetase (EC 2.7.6.1) revealed two distinct types of subunit, referred to as PRS I and PRS II (Taira et al. (1987) J. Biol. Chem. 262, 14867-14870). Tissue-specific expression of PRS I and PRS II genes (designated PRPS1 and PRPS2, respectively), was shown for 16 rat organs, using Northern blot analysis. The 2.3 kb PRPS1 mRNA level was high in the brain and adrenal gland, whereas the 3.7 kb PRPS2 mRNA level prevailed in the lung and spleen. Both genes were highly expressed in the thymus, adipose tissue and testis. In other mammals (mouse, calf and human), these two types of mRNA were also detected in various tissues and cell lines. Thus, the expression of each gene is regulated in a tissue-specific manner and there may be functional differences between catalytic and/or regulatory properties of subunits PRS I and II of this enzyme. In the testis, an additional PRPS1-related transcript of 1.4 kb was noted in rats, mice and humans. This transcript may belong to a group of testis-specific gene expressions or functions.

Mammalian phosphoribosyl-pyrophosphate synthetase

PRPP synthetase from rat liver exists as large molecular weight aggregates composed of at least three different components. Cloning of cDNA for the catalytic subunit revealed the presence of two highly homologous isoforms of 34 kDa, designated as PRS I and PRS II. Northern blot analysis showed tissue-differential expression of the two isoform genes. cDNA was expressed in E. coli and studies on the recombinant isoforms showed differences in sensitivity to inhibition by ADP and GDP and to heat inactivation. The rat gene for PRS I has 22 kb and is split into 7 exons. cDNAs for human enzymes were also cloned. Human genes for PRS I and PRS II are localized at different regions on the X-chromosome and their promoter regions were examined. Another component, PRPP synthetase-associated protein of 39 kDa (PAP39), was cloned from cDNA library of the rat liver. The deduced amino acid sequence of PAP39 is remarkably similar to those of PRS I and PRS II. Evidence indicated molecular interaction between PAP39 and the catalytic subunits and an inhibitory effect of PAP39 on the catalytic activity. Expression of the PAP39 gene is tissue-differential like the PRS genes, indicating that the composition of PRPP synthetase may differ with the tissue, hence properties of the enzyme would differ. Further studies on these components and their interaction are expected to reveal various mechanisms governing mammalian PRPP synthetase.

Localization of human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2) to different regions of the X chromosome and assignment of two PRPS1-related genes to autosomes

Complementary DNA clones for phosphoribosylpyrophosphate synthetase subunits I and II (PRS I and PRS II) were used to determine the chromosomal localization of the corresponding human genes. Southern blot analysis of genomic DNAs isolated from human placenta and a panel of humanmouse somatic cell hybrids revealed that the rat PRS I cDNA probe detected at least five human specific DNA segments (23, 20, 14.5, 6.7, and 4.3 kb) in BamHI digests. The 23-, 14.5-, and 6.7-kb DNA segments were detected only if the hybrids contained human chromosome X or translocation chromosome 7p+ (7qter>7p22::Xq21>Xqter), indicating the location of these segments to Xq21-qter (PRPS1). The 20- and 4.3-kb DNA segments did not cosegregate with the other three segments, and spot blot hybridization analysis using flow-sorted human chromosomes indicated that these are the PRPS1-related genes (PRPS1L1 and PRPS1L2) and could be assigned to chromosomes 7 and 9, respectively. The human-specific PRS II cDNA probe revealed a BamHI DNA segment (17 kb), which segregated condordantly with the X chromosome but not with the PRPS1 gene. We surmise that the gene for PRS II (PRPS2) is located at a different region of the X chromosome, namely Xpter-q21.

Molecular cloning and sequencing of human cDNA for phosphoribosyl pyrophosphate synthetase subunit II

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit II (PRS II) were isolated. The five overlapping clones contained 2457 base pairs (bp) covering a 954‐bp complete coding region for 318 amino acid residues. Homologies between human and rats PRS II were 99% of the amino acid and 88% of the nucleotides in the coding region. This amino acid homology seems to be the highest so far reported for enzymes involved in nucleotide metabolism and glycolysis. The highly conserved structure may be required for unique catalysis and rigid regulation of this enzyme.

Promoter regions of the human X-linked housekeeping genes PRPS1 and PRPS2 encoding phosphoribosylpyrophosphate synthetase subunit I and II isoforms

The 5 regions of the human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2, respectively) were isolated and sequenced. A comparison of the nucleotide sequences between human and rat PRPS1 genes revealed that the sequences around the transcription initiation sites were conserved over 56 nucleotides, and that a TATA-like sequence, a CCAAT box and three putative Sp1 binding sites were present at almost the same positions in the GC-rich sequences. Two major transcription initiation sites were localized in the human PRPS1, one of the two was located 27 nucleotides downstream from the TATA-like sequence, while the upstream initiation site was in the TATA-like sequence. The promoter region of the human PRPS2 gene was also GC-rich and contained a TATA-like sequence, four Sp1 binding sites and a homopyrimidine stretch. The initiation sites were localized at 90 nucleotides upstream from the ATG initiation codon. Chloramphenicol acetyltransferase (CAT)/promoter fusion assays showed that a 2.0 kb region (human PRPS1) and a 1.1 kb region (human PRPS2) possessed the promoter activities in four cell lines. The CAT activities in the three human cell lines tended to correlate with the steady-state mRNA levels of the PRPS1 and PRPS2 genes. These results suggest that the 5 flanking regions cloned contribute to the cell-differential expression of these two genes.

Deduced Amino Acid Sequence from Human Phosphoribosylpyrophosphate Synthetase Subunit II cDNA

5-Phosphoribosyl 1-pyrophosphate (PRPP) is a primary substrate in the de novo and salvage pathways of the biosyntheses of purine, pyrimidine, and pyridine nucleotides [1]. It also serves as a regulatory substrate for amidophosphoribosyltransferase and as a critical activator for carbamoylphosphate synthetase II [2], key enzymes of the purine and pyrimidine de novo syntheses, respectively. In man, abnormalities of this enzyme were reported, which causes hyperuricemia and eventually gout [3–7], This disease was transmitted as an X-linked trait, and, in fact, this PRPP synthetase gene was mapped to the human chromosome Xq22–q26, using human enzyme-specific antibody [8]. Recently we showed that rat PRPP synthetase has two distinct types of subunits, referred to as PRS I and PRS II, by cDNA cloning [9] and N-terminal amino acid sequencing [K. Kita et al., in this volume]. Studies with human and mouse somatic cell hybrids showed that human PRS I and PRS II genes were both located on different regions of the human chromosome X [10]. Superactivity of PRPP synthetase was categorized into five groups by Becker et al. [11]. However, the complex cases were reported: for instance, B-lymphoblast lines derived from individuals with the excessive maximal reaction velocity (catalytic defects) of this enzyme in erythrocytes, fibroblasts, and lymphocytes do not express the superactive phenotype [12]. This phenomenon may be caused by the presence of the two genes for this enzyme. For the precise analysis of the human PRPP synthetase genes and their abnormalities, we have attempted to clone cDNAs of the human enzyme. In this paper, we reported the cloning of cDNA for human PRPP synthetase subunit II and the deduced amino acid sequence.

60 PARTIAL CHARACTERIZATION OF PHOSPHORIBOSYL-PYROPHOSPHATE SYNTHETASE cDNA CLONES FROM HUMAN TESTIS

Phosphoribosylpyrophosphate (PRPP) synthetase is a key enzyme for de novo and salvage syntheses of nucleotides. PRPP synthetase activity is distributed in almost all tissues. cDNA cloning of rat PRPP synthetase showed the existence of two distinct types of subunits, referred to as PRS I and II (JBC 262; 14867, ′87). Northern blot analysis of RNAs from various mammalian tissues, using rat PRS I and PRS II cDNA as probes, showed that the expression of each gene is regulated in a tissue-specific manner: mRNAs of rat PRS I and/or PRS II were highly detected in brain, adrenal gland, spleen, lung, thymus, adipose tissue, and testis. The sizes of mRNAs for PRS I and II were 2.3 kb and 3.7 kb (2.7 kb for humans), respectively. In addition, in testes of rat, mouse, and humans, a unique PRS I-related 1.4 kb transcript was found, together with regular PRS I and II mRNAs. To characterize human PRS I and II mRNA as well as testis-specific one, the cDNA library was constructed in lambda phage vector λgt 10 from human testicular poly(A)+RNA and 1.35 × 106 recombinants were screened with rat PRS I and II cDNAs as probes. Two types of clones were obtained: with PRS I cDNA probe, clones with a 1.2 or 1.1 kb insert and with PRS II probe, clones with a 2.4, 2.3, 2.1, or 1.8 kb insert. The former group covered the full coding region and may correspond to the specific 1.4 kb transcript and the latter to the PRS II transcript.

Analysis of Molecular Structure of Rat Phosphoribosylpyrophosphate Synthetase Genes

Phosphoribosylpyrophosphate (PRPP) synthetase catalyzes the formation of PRPP by a pyrophosphoryl transfer from ATP to ribose 5-phosphate. The enzyme has been purified from bacteria [1, 2] and mammalian tissues [3, 4], The mammalian enzyme exists in many active forms of various molecular mass [5], reportedly composed of subunits of 31, 000–40, 500 daltons [1–4], However, our recent results showed that rat liver PRPP synthetase exists as complex aggregates of 34-kDa, 38-kDa, and 40-kDa components and the 34-kDa species is the catalytic subunit (K. Kita et al., this volume). We then carried out cDNA cloning of rat enzyme [6] in order to determine the primary structure and also to proceed to studies of gene regulation. We here describe unpublished details of part of the cloning experiments and preliminary accounts of analysis of rat genomic genes.