Masanori Taira

Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor

The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the probands lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.

Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene

The insulin-receptor genes from a 16-year-old girl with type A insulin resistance, who presented with fasting hyperinsulinaemia, acanthosis nigricans, and reduced insulin binding, and from her family were examined. One allele of her insulin-receptor gene inherited from her mother contained a 1.2 kb deletion arising from a recombination between two Alu elements. The deletion removed the 14th exon in the beta subunit and altered the reading frame, to produce a stop codon after aminoacid 867. Pedigree analysis indicated that this mutation alone will not cause diabetes, and the proband is possibly a compound heterozygote. 4 other members of her family were heterozygous for the same mutation; all 4 had a decrease in insulin binding and slight impairment of glucose tolerance. Perhaps the same mutation is an underlying feature of some cases of non-insulin-dependent diabetes mellitus.

Expression of c‐myc oncogene in colorectal polyps as a biological marker for monitoring malignant potential

The expression of oncogenes (c‐myc, c‐fos, c‐Ki‐ras, c‐Ha‐ras, and p53) was examined by Northern blot analysis using freshly isolated human colorectal and gastric cancers and noncancerous portions as the controls. Remarkably high levels of c‐myc expression were found in colorectal cancers (eight of 11), but not in gastric cancers. High levels of c‐myc expression were also detected in colorectal polyps and in metastatic liver tumors. In colorectal polyps, the transcript levels significantly correlated with the histologic malignancy and the size. In contrast, neither c‐fos nor c‐Ki‐ras was overexpressed in colorectal and gastric cancers, and transcripts of c‐Ha‐ras and p53 were not evident in any tissue examined. In light of these observations the c‐myc expression may be specifically associated with the evolution of colorectal cancer as well as progression and maintenance stages, hence may prove to be a useful marker to evaluate the malignant potential of colorectal polyps.

Type 2 (non-insulin-dependent) diabetes mellitus associated with a mutation of the glucokinase gene in a Japanese family.

SummaryMutations were screened for in the glucokinase gene of 25 Japanese patients with Type 2 (non-insulin-dependent) diabetes mellitus. Each exon was scanned by electrophoresis of enzymatically amplified DNA segments under non-denaturing conditions and variants were sequenced. A variant pattern was detected in exon 5 of one patient. Direct sequencing of this exon revealed a single nucleotide substitution in codon 188 (GCT→ACT) of one of two alleles resulting in the mutation of Ala188→Thr, an invariant residue in the sequence of all mammalian glucokinases and hexokinases. This mutation was not found in 40 normal control subjects. The proband had been diagnosed with Type 2 diabetes at the age of 62 years. Four other members of her family have the same mutation and all have Type 2 diabetes or impaired glucose tolerance. The youngest age at diagnosis of Type 2 diabetes in these other members was 13 years, suggesting that her pedigree was maturity-onset diabetes of the young (MODY). All subjects with the Thr188 mutation show a decreased insulin secretory response during oral glucose tolerance testing. Mutations in the glucokinase gene associated with Type 2 diabetes have been previously identified in Caucasian (French and British) subjects. This study indicates that mutations in this gene are also implicated in the development of Type 2 diabetes in Asians. Further studies are required to determine the frequency of mutations in glucokinase among Japanese patients with Type 2 diabetes.

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.

Depression of liver-specific gene expression in regenerating rat liver: A putative cause for liver dysfunction after hepatectomy

We carried out studies on the expression of liver-specific genes during regeneration of the liver and searched for changes in the expression of oncogenes and housekeeping genes. Albumin and ornithine transcarbamylase genes were the liver-specific genes examined by Northern blot analysis, using total RNAs isolated from residual livers of Sprague-Dawley rats subjected to a 68% partial hepatectomy. The mRNA levels of both genes began to decrease 8 hr after hepatectomy, both reaching the lowest levels at 24 hr, and then recovered to some extent at 48 hr. In contrast, these levels in the housekeeping and growth-related genes were augmented during this period. This would suggest that there is a selective expression of growth-related and housekeeping genes, in preference to liver-specific genes during liver regeneration. The expression of these genes in the regenerating liver was simulated in primary cultured hepatocytes during the dedifferentiation processes. It would appear that the first step in regeneration of the residual liver is dedifferentiation, in which the depression of liver-specific genes may be linked to liver dysfunction following hepatectomy.

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.

Selective activation of testis-specific genes in cultured rat spermatogenic cells

During mammalian spermatogenesis the isozyme pattern of a glycolytic enzyme, phosphoglycerate kinase (PGK; ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), changes from the somatic-type PGK-1 to the testis-specific PGK-2, and this change has been suggested to involve transcription switch. We have isolated genomic DNA fragments which code for the mouse PGK isozymes and determined the transcription start site of each gene. The results demonstrate that transcriptions of the two PGK genes are initiated at multiple sites under the control of TATA box-lacking promoters. The putative promoter regions of the two genes contain several distinct sequences known as the CCAAT box and the GC box which possibly bind CCAAT-binding proteins and Sp1, respectively. We next developed a culture system in which spermatogenic gene expression is partly reproduced. When spermatogenic cells of 20-day-old rats were cultured, transcripts from PGK-2 and another spermatogenic gene PRPS3 became detectable, while expression of other non-spermatogenic genes did not significantly change during culture. These results suggest that two spermatogenic genes PGK-2 and PRPS3 were activated in culture according to a developmental program of spermatogenesis. Thus, this culture system may be useful for studying the molecular mechanism underlying mammalian spermatogenic gene expression.