Heterochromatin as a factor influencing X-chromosome inactivation in hybrids of the common vole (Microtinae, Rodentia)]

Expression of X-linked genes for G6PD and alpha-GAL was studied in female interspecific hybrids of Microtus. The G6PD and alpha-GAL isozymes of Microtus arvalis were found to predominate in all cases when a species carrying a heterochromatin block on the X-chromosome served as one partner of hybridization and M. arvalis containing no heterochromatin block served as another. The proportions of G6PD and alpha-GAL parental forms were approx. equal in hybrid females when both species participating in hybridization contained heterochromatin blocks on X-chromosomes. Cytological analysis for revealing active and nonactive X-chromosomes on metaphase spreads of hybrid females supports the biochemical data. Non-random inactivation of X-chromosomes carrying the heterochromatin blocks in the interspecific hybrids with M. arvalis and a random one, when both parents contain heterochromatin blocks on the X-chromosomes are supposed to be the cause for the phenomenon observed. The study provided data supporting our previous hypothesis that heterochromatin affects the X-chromosome inactivation process in interspecific hybrid voles.     

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.     

Chinese hamster x American mink somatic cell hybrids: characterization of a clone panel and assignment of the mink genes for malate dehydrogenase,NADP-1 and malate dehydrogenase,NAD-1

Summary Chinese hamster x American mink somatic cell hybrids were obtained and examined for chromosome content and expression of mink malate dehydrogenase, NADP (MOD-1; EC 1.1.1.40), malate dehydrogenase, NAD (MOR-1; EC 1.1.1.37), glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8). All the hybrid clones examined were found to segregate mink chromosomes. A clone panel containing 25 clones was set up. The possibilities and limitations of this panel for mink gene mapping are analysed. Using this panel, it is feasible to rapidly map genes located on chromosomes 1–13 and to provisionally assign genes located on chromosome 14 and the X. Based on the data obtained, the genes for MOD-1 and MOR-1 were firmly assigned to mink chromosomes 1 and 11, respectively, and the genes for G6PD and HPRT were provisionally assigned to the X.     

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.     

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.     

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.     

The gene coding the human S11 surface antigens maps between the loci for HPRT and G6PD on the X-chromosome

The human S11 surface antigens are expressed on fibroblasts and are coded by a gene on the X-chromosome. We have regionally mapped this gene by examining S11 expression on a panel of hybrid lines which had fragmented the X-chromosome either during chromosome-mediated gene transfer, or by interspecies translocation during hybrid cell expansion. Using indirect immunofluorescence and the fluorescence-activated cell sorter (FACS), it was possible to isolate antigen-positive and -negative hybrid subpopulations for subsequent genetic analysis. The gene coding S11 could be localized to Xq27–28, between the loci for HPRT and G6PD where genes for the S10 and S12 antigens have been previously mapped. This work demonstrates the value of cell surface antigens and the FACS in somatic cell genetic analysis, and provides evidence for regional clustering of surface antigen loci on the human X-chromosome.     

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.     

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.     

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.     

Gene mapping in marsupials and monotremes. II. Assignments to the X chromosome of dasyurid marsupials

Somatic cell hybrids have been obtained between HPRT Chinese hamster cells and cells from several dasyurid marsupial species. These hybrids show the extensive loss of marsupial chromosomes characteristic of the majority of marsupial-eutherian somatic cell hybrids. Although all of the hybrids expressed the selected marsupial marker, HPRT, the only other markers observed were PGK, GLA, and G6PD, consistent with the conservation of X-linked genes extending to this major group of marsupials. Counterselection confirmed the synteny of PGK and GLA with HPRT, whereas G6PD showed decreased concordance.     

Presence of two active X chromosomes in hybrids between normal human and SV40-transformed fibroblasts from patients with the Lesch-Nyhan syndrome

Skin fibroblasts (LNSV) derived from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient patient with the Lesch-Nyhan syndrome, who has glucose-6-phosphate dehydrogenase (G6PD) type A, were transformed with SV40 and hybridized with WI38 human diploid fibroblasts derived from a female embryo which have normal HGPRT and G6PD type B activities. The hybrid clones selected in hypoxanthine, aminopterin and thymidine (HAT) medium, were essentially tetraploid and contained three X and one Y chromosomes. These hybrids contained HGPRT, types A and B and the AB heteropolymeric form of G6PD enzymes which were indicative that in these cells X linked genes of both parental cells were fully active. Hybrids back-selected in medium containing 8-azaguanine (8-AG) contained only two X chromosomes. They had no HGPRT activity and they contained only G6PD type A enzyme. It is concluded that the hybrid cells which grew in the presence of 8-AG retained the X chromosome of the LNSV parental cell and apparently the inactive X of the WI 38 cell.     

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.     

Statistical methods of radiation hybrid mapping: analysis of a small number of genes]

A statistical method for radiation hybrid (RH) mapping of a small number of genes detected in clones by means of isozyme analysis is suggested. This method allows analytical solution of the problem of gene ordering and qualitative comparison of relative distances between the genes that are being mapped. The possibilities offered by the suggested method are illustrated using earlier data on the distribution of four isozyme markers in human and vole (Microtus rossiaemeridionalis) clones. These markers are hypoxanthine phosphoribosyl transferase (HPRT), PGK, G6PD, and GALA.     

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.     

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.     

Stability of inactive X-chromosome in mouse embryoid body-mule cell and transformed mouse cell-mule cell heterokaryons

Cloned fibroblast cells of female mule derivation, expressing only the horse G6PD phenotype, were fused with established mouse cells, with spontaneously and virally transformed mouse cells, and with embryoid bodies from a transplantable mouse teratoma. Heterokaryons were harvested at various intervals after fusion, and tested for their G6PD activity patterns by electrophoresis on Cellogel sheets. No expression of the donkey G6PD phenotype in these heterokaryons could be detected, although hybrid G6PD bands formed between mouse and horse subunits were clearly visible. These observations imply that neither the cytoplasm of the embryoid bodies, nor of the DNA and RNA tumour virus-transformed mouse cells can induce depression of the G6PD locus on an inactive X-chromosome and provide further evidence for the genetic stability of the inactive X-chromosome in mammalian somatic cells.     

Regional mapping of the human No. 1 and X chromosome in interspecific cell hybrids using an X/1 translocation.

Fibroblasts from a carrier of an X/1 translocation, 46,XY,t(X;1)(q28;q31), were fused with Chinese hamster cells. The resulting hybrids were analyzed for human No. 1 and X-chromosome markers. The data indicate that the loci for PGM1, PGD, PPH, and GuK1 are situated either in the long arm proximal to a break point in band 1q31 or in the short arm. The loci for Pep-C, FH, and GuK1 are located distal to the break point. HPRT and G6PD are probably situated distal to a break point in band q28 of the X chromosome; alpha-Gal A is situated proximal to the break point, either on the long or short arm of the chromosome.     

Regulation of glucose 6-phosphate dehydrogenase expression in CHO-human fibroblast somatic cell hybrids

Human--hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human--hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.