The minisatellite of the GPI/AMF/NLK/MF gene: interspecies conservation and transcriptional activity.

Minisatellites are tandemly repeated DNA sequences found throughout the genomes of all eukaryotes. They are regions often prone to instability and hence hypervariability; thus repeat unit sequence is generally not conserved beyond closely related species. We have studied the minisatellite located in intron 9 of the human glucose phosphate isomerase (GPI) gene (also known as neuroleukin, autocrine motility factor, maturation and differentiation factor) and have found, by Zoo blotting coupled with PCR amplification and DNA sequencing, that similar repeat units are present in seven other species of mammal. There is also evidence for the presence of the minisatellite in chicken. The repeat unit does not appear to be present at any other locus in these genomes. Minisatellite DNA has been reported to be involved in recombination activity, control of gene expression of nearby gene(s) (both transcriptional and translational), whilst others form protein coding regions. The high level of conservation exhibited by the GPI minisatellite, coupled with the unique location, strongly suggests a functional role. Our results from transient and stable transfections using luciferase reporter constructs have shown that the GPI minisatellite region can act to increase transcription from the SV40 promoter, CMV promoter and the human GPI promoter.

Sequence characterization of ENU-induced mutants of glucose phosphate isomerase in mouse.

Four of five mutations producing GPI1 null lethal phenotypes in the homozygous state, which were previously identified from the offspring of male mice, spermatogonially treated with N-ethyl N-nitrosourea (ENU), have been characterized at the nucleotide level by reverse transcription of RNA from heterozygotes for mutant and wild-type alleles and cycle sequencing with cDNA-derived primers. In three of the mutations studied, a single nucleotide substitution, altering the predicted amino acid on translation, was observed in the mutant allele. In Gpi1-sam1H amino acid residue 277, TCA Ser (wild type), is altered to CCA Pro, and in Gpi1-sbm3H and Gpi1-sbm4H amino acid residue 510 Asp GAC (wild type) is altered to GGC Gly. These ENU-induced mutations occur at A-T base pairs in agreement with the current view of the mechanism of action for this mutagen. These changes also occur at residues implicated as being important in the catalytic functioning of the enzyme, from crystallographic studies, and may explain the loss of enzyme function. The fourth identified mutation, Gpi1-sbm2H, is a deletion of amino acid residues Arg134 to Leu162 inclusive, which may arise from incorrect splicing of mRNA; a fifth mutation has remained undetermined.

Structure and organization of the human glucose phosphate isomerase gene (GPI).

Two overlapping yeast artificial chromosome clones containing the human glucose 6-phosphate isomerase gene (GPI) have been isolated. PCR and direct sequencing were used to determine the exon/intron structure of the gene. The gene spans in excess of 40 kb and consists of 18 exons ranging in size from 44 to 153 bp. All splice sites conform to the GT/AG rule.

Sequence characterization of alleles Gpi1-Sa and Gpi1-Sb at the glucose phosphate isomerase structural locus.

The sequences of alleles Gpi1-sa and Gpi1-sb at the glucose phosphate isomerase structural locus have been determined from cDNA of the mouse inbred strains 101/H Gpi1-sa and C3H/HeH Gpi1-sb by RT PCR and direct sequencing of the amplified products. Four individual nucleotide differences were observed between the two alleles. The difference at amino acid residue 94, (Gpi1-sa GAT Asp, Gpi1-sb AAT Asn) may account for the differing electrophoretic migration, isoelectric point, and thermostability of the two alleles. Two of the other observed differences in the coding region (amino acid residue 12 Leu, Gpi1-sa CTC, Gpi1-sb CTG and amino acid residue 17 Arg, Gpi1-sa CGC, Gpi1-sb CGT) are silent and do not affect the predicted amino acid residues on translation. The fourth observed difference is located within the 3' noncoding sequences of the cDNA. The change at amino acid residue 94 is associated with the presence of a Hinf1 restriction site in Gpi1-sb, which is absent in Gpi1-sa, and may be a useful method for determining this marker.

Characterization of cDNAs coding for glucose phosphate isomerase and phosphoglycerate kinase in Chinese hamster ovary cell line CHO-K1 and identification of defects in R1.1.7, a glycolysis-deficient variant of CHO-K1.

Full-length cDNAs for glucose phosphate isomerase (GPI) and phosphoglycerate kinase (PGK) of the Chinese hamster ovary cell line CHO-K1 have been characterized using RT-PCR and cycle sequencing of the PCR-amplified templates. Mutations in both genes have been identified in a glycolysis-deficient Chinese hamster ovary cell line, R1.1.7, derived from CHO-K1 cells.

Identification of a novel tandemly repeated sequence present in an intron of the glucose phosphate isomerase (GPI) gene in mouse and man.

Glucose phosphate isomerase (GPI, glucose 6-phosphate ketol-isomerase, EC 5.3.1.9) is a housekeeping gene expressed in all tissues and organisms that utilize glycolysis and gluconeogenesis. Deficiency in humans leads to a rare form of nonspherocytic hemolytic anemia. We have isolated a 3.2-kb mouse cDNA containing glucose phosphate isomerase coding sequence and a 2.1-kb intronic sequence and a large proportion of the human gene (approaching 55 kb) in four phage lambda recombinants. A 4-kb intronic fragment from the human gene showing homology to the mouse intronic sequence has been isolated and sequenced. The fragment contains approximately 1.5 kb of sequence that is composed of 30 repeat units of a novel 50-bp tandemly repeated units. The mouse intronic sequence contains 18 similar units. The human consensus sequence differs from the mouse consensus sequence at only 7 positions out of 50 (positions 16, 26, 27, 42, 43, 47, and 48). A probe containing the repeat element detects polymorphisms, specific to glucose phosphate isomerase, in human DNA. The repeat element does not appear to be present at any other loci in human DNA. The conservation of this intronic repeat element extends to pig and Chinese hamster.

Measurement of androgen receptor expression in adult liver, fetal liver, and Hep-G2 cells by the polymerase chain reaction.

Hepatocellular carcinoma is the most commonly fatal malignant tumour worldwide. The role of androgen receptors, which have been found in hepatocellular carcinoma, is controversial. Sequence specific polymerase chain reaction (PCR) was used to quantify, for the first time, the expression of androgen receptor in four adult liver biopsy specimens (HL-A to HL-D), fetal liver, and Hep-G2 cells. The measurement of androgen receptor is expressed as a ratio (androgen receptor: beta-actin) of the value of androgen receptor to the value of a control gene, beta-actin. The value of the androgen receptor: beta-actin ratios for HL-A, HL-B, HL-C, HL-D, fetal liver, and Hep-G2 were 0.37, 0.86, 0.37, 0.44, 0.87, and 0.66 respectively. To verify sequence specific amplification of the androgen receptor, the PCR androgen receptor fragment was sequenced. The resultant sequence data for both strands of the double stranded PCR androgen receptor fragment had 100% similarity with the published androgen receptor mRNA sequence (complete codons).

Characterization of the 5' end of the gene for human glucose phosphate isomerase (GPI).

We have isolated the gene coding for human glucose phosphate isomerase, and here we report the characterization of its 5' end including the first two exons. The gene is greater than 50 kb in size and contains a minimum of eight exons. We have no evidence that this gene is part of a multigene family or that there are any pseudogenes. The potential transcription start site has been determined by primer extension analysis and is 52 bp upstream from the translation initiation site of the protein. Sequences 5' to the transcription initiation site and within the first intron are extremely GC rich and form part of a CpG island. Five potential Sp1 sites (GGGCGG) have been located at positions -57, -61, -95, +183, and +575. The most 5' of these (GGGGCGGGGG) is likely to be the main Sp1 binding site, as it conforms precisely to a 10-bp consensus sequence. At position -44 there is a putative TATA box (CATAAA). Thus in common with an increasing number of genes, the putative promoter region of glucose phosphate isomerase shows structural similarities to both housekeeping and facultative gene promoters.

Transport and accumulation of 2-deoxy-D-glucose in wild-type and hexokinase-deficient cultured Chinese-hamster ovary (CHO) cells.

Hexokinase-deficient mutants and wild-type Chinese-hamster ovary cells have been used to investigate the role of hexokinase in uptake and accumulation of 2-D-deoxyglucose (2-dGlc). The evidence for a specific sugar transport system in both types of cells is that there is similar saturable phloretin-sensitive uptake of 2-dGlc and 3-O-methyl-D-glucose (3-OMG) in both types of cell. In wild-type cells, 2-dGlc is accumulated to a tissue:medium ratio of 10- and in the mutant only 3-fold; 3-OMG is not accumulated by either mutant or wild-type cells. The evidence that hexokinase affects the membrane transport process is that the rate of exit of free 2-dGlc from wild-type cells is 5-fold less than from mutant cells, whereas there is no difference in the rate of loss of 3-OMG between mutant and wild-type cells.

Complementation of metabolic defect in phosphoglycerate kinase (PGK)-deficient line of Chinese hamster ovary cells by introduction of human PGK cDNA.

A full-length copy of the coding sequence of the human phosphoglycerate kinase (PGK) gene was introduced into a glycolysis-negative, PGK-deficient line of Chinese hamster ovary (CHO-K1) cells by gene transfer. Transformants were isolated either by cotransfer of the bacterial aminoglycoside phosphotransferase (AGPT) gene, or by direct selection for expression of the PGK gene. Integration of the human PGK gene has been demonstrated by Southern blot analysis and its expression by starch gel electrophoresis. PGK transformants behaved phenotypically as predicted by the properties of their wild type parent: mannose was no longer toxic, but instead was metabolized via glycolysis to lactic acid, and cell growth was no longer dependent on glutamine oxidation. Thus a complex phenotypic change has been mediated by gene transfer. The combination of R1.1.7 cells and the PGK plasmid provides another system to facilitate the study of mammalian gene expression.

Mouse glucose-6-phosphate isomerase and neuroleukin have identical 3' sequences.

Neuroleukin is a neurotrophic factor of relative molecular mass (Mr) 56,000 (56K) found in skeletal muscle, brain, heart and kidneys which supports the survival of embryonic spinal neurones, skeletal motor neurones and sensory neurones. Neuroleukin is also a lymphokine product of lectin-stimulated T cells and induces immunoglobulin secretion by cultured human peripheral blood mononuclear cells. Mouse neuroleukin has been cloned, the complete nucleotide sequence has been determined and its complementary DNA has been transiently expressed in monkey COS-1 cells. The serum-free supernatant of the transfected, but not of control mock-transfected, cells was shown to mimic the properties of neuroleukin isolated from mouse salivary glands. In our work on the molecular genetics of carbohydrate metabolism we have recently isolated a mouse glucose-6-phosphate isomerase (or phosphoglucose isomerase, PGI) cDNA clone using the yeast PGI gene (PGI 1) as a probe. We report here that there is complete sequence identity between the 759 nucleotides at the 3' end of this clone (coding and non-coding) and the sequence of mouse neuroleukin.

Regulation of carbohydrate metabolism in cultured mammalian cells: energy provision in a glycolytic mutant.

The metabolism of radioactively labelled D-glucose, L-glutamine, and L-glutamate has been determined in a glycolytic mutant of Chinese-hamster ovary cells, R1.1.7, and in its parent, CHO-K1. The complete oxidation of glucose via the TCA-cycle is negligible in both cell types, but there is significant oxidation of carbon-1. CHO-K1 cells derive most of their energy from glycolysis and are independent of respiration in the short term. R1.1.7 cells are respiration-dependent and are rapidly killed by respiratory inhibitors. Both cell types oxidize L-glutamine and L-glutamate, but oxidation of these substrates does not appear sufficient to satisfy completely the energy requirements of R1.1.7 cells.

The regulation of carbohydrate metabolism in animal cells: isolation of a glycolytic variant of Chinese hamster ovary cells.

A variant of Chinese hamster ovary cells is described which is deficient in two glycolytic enzymes: glucosephosphate isomerase and phosphoglycerate kinase. The variant grows readily on glucose, but in contrast to the parental cell it does not excrete lactic acid. The impaired ability of the variant to perform aerobic glycolysis both in vivo and in vitro is correlated with the deficiency in both glycolytic enzymes.