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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

Properties of carbohydrate utilising variants of Chinese hamster cells.

Variants of Chinese hamster ovary cells (CHO-K1) have been isolated which can grow on one of the following carbohydrates: lactose, sucrose, ribose or lactate. The ribose+ clones grow at the same rate on glucose as the parental cells whereas the others grow more slowly. With the exception of one ribose+ clone all excrete lactic acid while growing on glucose; none excrete significant amounts of lactic acid while growing on the alternative energy source.

A method for the isolation of Chinese hamster cell variants with an altered ability to utilise carbohydrates.

Chinese hamster ovary cells (CHO-K1) are able to utilise only a few carbohydrates for growth such as glucose, mannose, fructose and galactose. They do not grow on ribose, lactose, sucrose, glycerol, lactate, pyruvate, citrate, succinate, fumarate or malate nor on glycogenic or ketogenic amino acids. After mutagenesis and selection in glucose free medium supplemented with various, individual, growth substrates, we have isolated single-cell derived clones which are now able to grow on one of the following energy source: ribose, lactose, sucrose or lactate.

Utilization of gluconate by Escherichia coli. Uptake of D-gluconate by a mutant impaired in gluconate kinase activity and by membrane vesicles derived therefrom.

1. From Escherichia coli strain K2.1.