Isolation and DNA sequence of a full-length cDNA clone for human X chromosome-encoded phosphoglycerate kinase.

Abstract

Phosphoglycerate kinase (PGK), a major enzyme in glycolysis, is encoded by the X chromosome in mammals. We have initiated molecular analysis of the PGK structural gene by isolating a full-length cDNA clone from a human fetal liver cDNA library. Synthetic oligonucleotide. mixtures encoding two different portions of PGK were used as direct in situ hybridization probes for the cDNA library. Several classes of clones were obtained based on their hybridization at different stringencies to one or both of the PGK oligonucleotide mixtures. One clone, designated pHPGK-7e, which hybridized at-high stringency to each of the synthetic probes, encoded the complete PGK protein sequence as well as 82 base pairs of 5' and,437 base pairs of 3' untranslated regions. Southern blot analysis ofhuman genomic DNAs revealed a complex pattern of hybridizing fragments, two of which were non-X in origin. These results suggest that the human genome contains a small family of dispersed PGK or PGK-like genes.Phosphoglycerate kinase (PGK; ATP:3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3), a major enzyme in glycolysis, catalyzes the reversible conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate, generating one molecule of ATP (1). In mammals, this enzyme is encoded by a single active locus on the long arm of the X chromosome (2-6). This locus is subject to dosage compensation by X chromosome inactivation (7).The structures of PGK proteins from several species have been analyzed in detail. Complete amino acid sequences of horse muscle and human erythrocyte enzymes reveal extensive interspecies homology (8,9). X-ray crystallography of horse PGK demonstrated two separate protein domains that may be of functional significance to its mechanism of catalysis (8). Because the yeast enzyme has a. similar primary and tertiary structure (8, 10), PGK appears to be highly conserved during the course of evolution. Specific human PGK variants, some of which lead to enzyme deficiency and hemolytic anemia, have also been described (11)(12)(13)(14). In addition to the X-linked protein, a testis-specific isozyme, whose structural relationship to the X chromosome-encoded enzyme is largely unknown, is found in a number of mammalian species (15-18). In the mouse, this isozyme has been mapped to the major histocompatibility complex on chromosome 17 (19), whereas in the human its chromosomal location has not been determined.Given this wealth of biochemical and genetic information, we have initiated molecular analysis of the PGK locus. A probe for PGK sequences would, facilitate studies of X-chromosome inactivation, gene evolution, and the tissue-specific expression of homologous nonallelic loci. As a first step we have isolated a full-length cDNA for the X-linked PGK structural gene. MATERIALS AND METHODSConstruction of a Human Fetal Liver cDNA Library. Liver from a human fetus of 20-22 weeks gestation was homogenized in the presence of guanidine hydrochloride, and poly(A)+RNA was isolated by two passages through oligo(dT)-cellulose. cDNA (2.2 ...

Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH

“…In general, the temporal pattern and magnitude of enhanced AEG expression was similar in cells infected with HIV-1 or treated with gp120. However, expression of AEG-2 (G-binding protein), AEG-5 (hGNT-IV-H) (Furukawa et al, 1999), AEG-7 (human CTL2) (O'Regan et al, 2000), AEG-8 (acidic ribosomal phosphoprotein) (Rich and Stietz, 1987) and AEG-15 (PGK-1) (Michelson et al, 1983;Tsukada et al, 1991) was elevated in fetal astrocyte cultures more rapidly following HIV-1 infection than gp120 treatment (Figures 4 ± 6). AEG-2 (G-binding protein), AEG-5 (hGNT-IV-H) and AEG-7 (human CTL2) expression was elevated by 3 day post-infection with HIV-1, whereas enhanced expression of these genes was not apparent until 7 days after gp120 treatment.…”

Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in Cos-1 cells.

“…RNA was prepared from human alveolar macrophages (gift of Dr. R. Rose, Harvard Medical School), human umbilical vein endothelial cells, Hela cells, and HepG2 cells as described . The RNA was size fractionated on an agarose gel and transferred to Nytran hybond membrane (Amersham Corp., Arlington Heights, IL) and hybridized with a radiolabeled MR cDNA and cl)NA for phosphoglycerate kinase (PGK) (30) .…”

Antisense RNA of proto-oncogene c-fos blocks renewed growth of quiescent 3T3 cells.

“…The level of the sense c-fos mRNAs and antisense c-fos RNAs were analyzed by Si nuclease mapping (3) by using specific probes. The amount of RNA used in each sample (10 ,ug of total RNA) was checked by monitoring the mRNA level of a housekeeping gene, mouse phosphoglycerate kinase (PGK), by using a mouse PGK cDNA plasmid (pMPGK-5b) (36) (10 ,ug) from each time point with and without dexamethasone treatment was analyzed for mouse sense c-fos mRNAs by S1 nuclease mapping (3 (Fig. 5A, lane b) or presence (data not shown) of dexamethasone was very low, as reported previously (21,29,38).…”