Subchromosomal localization and order of GLA, PGK1, HPRT, and G6PD loci on the X chromosome of the American mink (Mustela vison)

Segregation of the X-linked mink markers alpha-galactosidase (GLA), phosphoglycerate kinase-1 (PGK1), hypoxanthine phosphoribosyltransferase (HPRT), and glucose-6-phosphate dehydrogenase (G6PD) was analyzed in hybrids of gamma-irradiated mink fibroblasts and Chinese hamster cells and in hybrids of nonirradiated mink fibroblasts and mouse hepatoma cells. Based on this analysis, the order of the four genes is GLA-PGK1-HPRT-G6PD on the mink X chromosome. Cytogenetic analysis of five mink x Chinese hamster hybrid clones containing mink GLA, PGK1, and HPRT, but lacking G6PD, tentatively localized mink G6PD to Xq15.22----qter and also confirmed the gene order as GLA-PGK1-HPRT-G6PD-qter. Comparison of this order with its counterpart in man and the mouse, as well as an analysis of the G-band patterns of their X chromosomes, demonstrated putative similarities between mink and man and differences in the mouse. These differences may be due to a different rate of X-chromosomal rearrangement in mammalian evolution.     

Frequency of reactivation and variability in expression of X-linked enzyme loci.

A mouse-human cell hybrid clone retaining an inactive human X chromosome was treated with 5-azacytidine. Following treatment, expression of the X-linked enzyme markers, hypoxanthine-guanine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and alpha-galactosidase A (GLA) was examined. Results presented here show that 45 of the 62 clones positive for human HPRT expressed human GLA, while only four of 68 clones negative for human HPRT expressed human GLA. These results strongly suggest that there is coordinate reactivation of GLA and HPRT. Reactivated expression of G6PD was studied in detail. The studies show that 5-azacytidine can induce heritable changes in the inactive human X chromosome resulting in the expression of G6PD activity at a level lower than that from an active human X chromosome.     

Molecular studies of marsupial X chromosomes reveal limited sequence homology of mammalian X-linked genes

To explore the extent to which the X chromosome has been conserved during mammalian evolution, we compared six loci that are X-linked in the human genome with the corresponding genes of the North American marsupial, the Virginia opossum (Didelphis virginiana). Our analysis shows that in the opossum genome there are sequences highly homologous to those of human cDNAs for housekeeping genes, glucose-6-phosphoribosyltransferase (HPRT), phosphoglycerate kinase A (PGK1), and alpha-galactosidase A (GLA). However, ornithine transcarbamylase and blood clotting Factor IX--tissue-specific genes that are X-linked in eutherians mammals--have no highly conserved homologs in the marsupial genome. By cloning opossum G6PD and HPRT, we found that these genes are X-linked in the opossum and that homologous sequences are limited to coding regions. As all genomic fragments hybridizing with the human GLA probe show dosage effects, it is likely that the opossum counterpart is X-linked. Finally, the pattern of hybridization suggests that the autosomal pseudogenes of HPRT and PGK1 in the opossum have remained highly homologous to the human X-linked genes.     

Regional localization of the genes for human HEXB. PGK, GALA. HPRT, G6PD by somatic cell hybridization

Twenty independent man-mouse (Cl1D,LA/TK-, HPRT-) and man-hamster (CH,HPRT-) hybrids using female human cells with balanced reciprocal translocation XX,t(X;5)(q21;q11) were analyzed for human genes localized on chromosome 5 (HEXB), on chromosome X (PGK, GALA, HPRT, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.5, chr.5q-, chr.Xq+, chr.X). The different results obtained indicate that the genes for human markers HEXB, PGK are on Xq+, and that the genes for human markers GALA, G6PD are on 5q-. These data implicate finally the following localizations: HEXB on 5q11 leads to 5qter; PGK on Xq21 leads to Xpter; GALA, HPRT, G6PD on Xq21 leads to Xqter.     

Marsupial--mouse cell hybrids containing fragments of the marsupial X chromosome.

Hybrids were obtained from fusions of HPRT-deficient mouse fibroblasts and marsupial lymphocytes. These hybrids retained no identifiable marsupial chromosomes, but all expressed the marsupial form of HPRT. Half the clones also expressed marsupial PGK-A, and half of these also marsupial G6PD; no other marsupial allozyme markers were detected. Since G6PD is known to be sex linked in these species, we conclude that Hpt and Pgk-A are also located on the X chromosome and the markers lie in the order Hpt-Pgk-A-Gpd.     

Segregation of rat chromosomes in somatic cell hybrids between rat cells and HT 1080 human fibrosarcoma cells

Summary We produced somatic cell hybrids between HT 1080-6TG human fibrosarcoma cells and either rat white blood cells (WBC) or cells directly derived from rat spleen. Karyologic and isozyme analyses of hybrid cells indicated that they preferentially lose rat chromosomes. Hypoxanthine-aminopterine thymidine-selected hybrid clones expressing rat hypoxanthine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), and phosphoglycerate kinase (PGK) and containing the rat X chromosome were counterselected in a medium containing 30 g/ml of 6-thioguanine. Concordant loss of the rat X chromosome and of the expression of rat HPRT and G6PD was observed in the hybrid clones.     

The localization of genes for HPRT, G6PD, and alpha-GAL onto the X-chromosome of domestic pig (Sus scrofa domesticus)

Pig--mouse somatic cell hybrids were obtained from fusion of HPRT--mouse cells (RAG) and pig lymphocytes. The pig-mouse hybrids examined apparently retained on the average only 9 to 15 pig chromosomes. Seven of the hybrid clones were karyotyped to determine the pig chromosome constitution, and the same hybrid clones were tested electrophoretically for the expression of pig hypoxanthine-guanine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), and alpha-galactosidase (alpha-GAL) phenotypes. All five of the hybrid clones which had retained the pig X-chromosome exhibited concordant expression of pig HPRT, G6PD, and alpha-GAL enzymes. These data indicate that the genes HPRT, G6PD, and alpha-GAL are located on the X-chromosome of the domestic pig.     

Human gene mapping using an X/autosome translocation.

Human fibroblasts containing a translocation between the X chromosome and chromosome 15 were fused with the 6-thioguanine-resistant mouse cell line, IR. Resulting hybrids, selected in HAT medium, retained the X/15 chromosome. Hybrids which were counterselected in 6-thioguanine lost this chromosome. The X-linked markers glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and hypoxanthine phosphoribosyl transferase (HPRT), and the non-X-linked markers pyruvate kinase (PKM2) mannose phosphate isomerase (MPI), N-acetyl hexosaminidase A (HEXA) and beta2-microglubulin (beta2-m) all segregated in concordance with the X/15 translocation chromosome. The latter markers have been assigned to chromosome 15. Selection against the X/15 chromosome was done using antihuman beta2-m serum. Electrophoretic and immunochemical analyses of the N-acetyl hexosaminidases A and B in these hybrids were performed.     

Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and deletion mapping: evolutionary significance

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.     

Evidence for linkage of 3-phosphoglycerate kinase,hypoxanthine-guanine-phosphoribosyl transferase,and glucose 6-phosphate dehydrogenase loci in Chinese hamster cells studied by using a relationship between gene multiplicity and enzyme activity

The PGK activity was assayed in diploid, hyperdiploid, tetraploid, and hybrid cells all originating from the same Chinese hamster cell line (DON line). A relationship between gene multiplicity and enzyme activity was observed. Selective pressure on the HGPRT locus by growth of hybrid cells in the presence of 8-azaguanine resulted in decreased levels of PGK activity. Growth of these hybrids in the presence of 5-BUdR did not influence the enzyme activity. It was concluded that the genes coding for HGPRT, PGK, and G6PD are linked in the Chinese hamster. The TK locus seems to be linked neither to the HGPRT, PGK, and G6PD loci nor to the 6PGD locus.     

Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and delection mapping: Evolutionary significance

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.     

Gene mapping in marsupials and monotremes

Somatic cell genetic mapping of marsupial and monotreme species will greatly extend the power of comparative gene mapping to detect ancient mammalian gene arrangements. The use of eutherian-marsupial cell hybrids for such mapping is complicated by the frequent retention of deleted and rearranged marsupial chromosomes. We used staining techniques, involving the fluorochromes Hoechst 33258 and chromomycin A₃, to facilitate rapid and unequivocal identification of marsupial chromosomes and chromosome segments and to make chromosome assignment and regional localization of marsupial genes possible. Chromosome segregation in rodent-macropod hybrids was consistent with preferential loss of the marsupial complement. The extent of loss was very variable. Some hybrids retained 30% of the marsupial complement; some retained small centric fragments; and some, no cytologically identifiable marsupial material. We examined the chromosomes and gene products of a number of rodent-grey kangaroo Macropus giganteus hybrids, and have assigned the genes Pgk-A (phosphoglycerate kinase-A), Hpt (Hypoxanthine phosphoribosyl transferase), and Gpd (Glucose-6-phosphate dehydrogenase) to the long arm of the kangaroo X chromosome, and provisionally established the gene order Pgk-A -Hpt -Gpd.     

Bovine chromosome mapping with the cell hybridization technic. Localization on the x chromosome of glucose-6-phosphate dehydrogenase, phosphoglycerate kinase, alpha-galactosidase A and hypoxanthine phosphoribosyltransferase]

Twelve hamster x cattle (Bos taurus) cell hybrids showed loss of cattle chromosomes, which allows their use for chromosome mapping of cattle. Genes coding for G6PD, alpha-GAL A, PGK, and (indirectly) HGPRT are synteneic and localized on the X chromosome.     

Creation of a clone panel of fox x Chinese hamster somatic cell hybrids and chromosome mapping of genes for LDHA, LDHB, GPI, ESD, G6PD, HPRT, alpha-GALA in the silver fox

A clone panel of fox-hamster somatic cell hybrids which can be used for fox gene mapping was set up. Analysis of patterns of chromosome-enzyme segregation made it possible to assign gene GPI to chromosome 1, LDHA to chromosome 11, LDHB to chromosome 8, ESD to chromosome 6 and G6PD, HPRT, alpha-GALA to chromosome X.     

Assignment of Three Gene Loci (Pgk, Hgprt, G6pd) to the Long Arm of the Human X Chromosome by Somatic Cell Genetics

The intrachromosomal localization of three X-linked gene loci (PGK, HGPRT and G6PD) has been determined using a somatic cell genetic approach. A human cell line possessing an X/14 translocation was used as one parent in the formation of human/mouse hybrids. The translocation separates the human X into two parts: Xp and t(Xq14q). The data indicate that all three X-linked loci segregate with the t(Xq14q) rearrangement product thus permitting their assignment to the X chromosome's long arm. Secondary rearrangements and data from other laboratories suggest that the order of the the three markers from the centromere to the distal end of the X long arm is PGK, HGPRT, G6PD. It was also observed that NP, an autosomal locus, segregated with the t(Xq14q) chromosome. This provides strong support for the assignment of NP to 14.     

Transfer of the human X chromosome to human--Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes.

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack H P R T activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in H A T medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of H A T-resistant clones was 32 x 10(-6) when 10(7) cells were incubated with 10(8) HeLa chromosomes. Potential reversion of the hybrid cells in H A T medium was less than 5 x 10(-7). The 16 isolated cell lines all contained activity of the human X-linked marker enzymes H P R T, P G K,alpha-Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Induction of the glucose-6-phosphate dehydrogenase gene expression by chronic hypoxia in PC12 cells

We studied the regulation of glucose-6-phosphate dehydrogenase (G6PD) gene expression by chronic hypoxia. G6PD mRNA level and activity were increased in PC12 cells by hypoxia in a dose- and time-dependent manner. Cobalt chloride and dimethyloxalylglycine, which can mimic hypoxia, also activated G6PD gene expression. Interestingly, hypoxia-induced G6PD expression followed a time course much slower than that of phosphoglycerate kinase 1 (PGK1), a hypoxia-inducible factor (HIF)-dependent glycolytic enzyme. Hypoxic-G6PD induction was almost negligible in non-excitable Buffalo rat liver cells, although in these cells PGK1 was strongly upregulated by low PO(2). Furthermore, G6PD but not PGK1 induction was blocked by the antioxidants glutathione and N-acetylcysteine. These results suggest the dependence of G6PD gene expression on HIF and intracellular redox status and the differential hypoxic regulation of glucose-metabolizing enzymes.     

Genome Instability in interspecific cell hybrids II. Aminopterin resistance and gene amplification in lines arising from fusions of cells from divergent mammalian species

In order to investigate instances of genetic instability in divergent cell hybrids, we studied several RAT-resistant colonies recovered from fusions between HPRT or TK-deficient rodent cells and marsupial or monotreme cells. Most of these colonies proved to lack HPRT or TK activity and to have survived by acquiring resistance to aminopterin; such aminopterin-resistant lines were never recovered from parent cells subjected to HAT selection. Two of the aminopterin-resistant hybrids over-produced DHFR, and possessed either double minutes or an abnormally banded region, the cytological manifestations of gene amplification. Selection in higher aminopterin concentrations yielded a highly resistant line with 100X wild-type DHFR activity and a large homogeneously staining region. We suggest that interspecific cell hybrids are predisposed to gene amplification and may also show many other types of genetic and chromosomal instability, possibly thein vitro equivalent of the “genomic shock” phenomena described for interstrain or interspecies hybrids of plants or animals. This paper, no. II in a series by these authors, reached the Editorial Office on the date given, although it had been mailed earlier than paper no. III; the latter paper also appears in this number — Eds.     

Regional assignment of arylsulfatase A,mitochondrial aconitase and NADH-cytochrome b5 reductase by somatic cell hybridization

Summary By using somatic cell hybrids between HPRT deficient hamster cells and fibroblasts derived from a patient with a X/22 translocation t(X;22)(q13;q112), we have assigned the genes for human ARSA, DIA 1, and ACO 2 to region q112qter of human chromosome 22 and the gene for human PGK close to the breakpoint in band Xq13.