Assignment of human gene encoding thymidylate synthase to chromosome 18 using interspecific cell hybrids between thymidylate synthase-Negative mouse mutant cells and human diploid fibroblasts

We have constructed interspecific somatic cell hybrids between a thymidineauxotrophic mutant cell line of mouse FM3A cells that lacks thymidylate synthase and human diploid fibroblasts derived from a male patient with fragile X-linked mental retardation. Twenty primary hybrid clones were isolated independently, all of which exhibited the thymidine-prototrophic phenotype. Segregation of the hybrid cells in nonselective culture conditions gave rise to thymidine-auxotrophic hybrid clones. Both electrophoretic assay of thymidylate synthase activity and karyotype analysis of the segregants revealed a strong correlation between the expression of the human form of the enzyme and the presence of human chromosome 18. Thus, it is concluded that the functional gene for human thymidylate synthase, designated TS,is located on this chromosome.     

Isolation and characterization of a Chinese hamster lung cell tryptophanyl-tRNA synthetase mutant

A novel type of temperature-sensitive protein synthetic mutant was isolated from V-79 Chinese hamster lung cells using an amino acid analog suicide selection. The expression of the temperature sensitive phenotype of the mutant is greatly affected by the concentration of tryptophan in the culture medium. In addition, the activity of tryptophanyltRNA synthetase is undetectable in cellfree extracts prepared from the mutant cells. The results suggest that the mutant has an alteration in the structural gene encoding tryptophanyl-tRNA synthetase.     

Assignment of a human gene for tryptophanyl-tRNA synthetase to chromosome 14 using human-mouse somatic cell hybrids

Human tryptophanyl-tRNA synthetase (Trp-RS, EC 6.1.1.2) can be separated from its mouse counterpart by Cellogel electrophoresis. Analysis of the presence or absence of human Trp-RS and other human enzyme markers in eleven independently derived cell lines of human-mouse somatic cell hybrids revealed that the expression of Trp-RS is correlated with the expression of human nucleoside phosphorylase (NP, EC 2.4.2.1). The syntenic relationship between Trp-RS and NP permits the assignment of the structural gene for Trp-RS to human chromosome 14. Karyotype and isozyme analysis of these hybrid clones rules out other linkage assignments.     

Assignment of the humanMARS gene,encoding methioninyl-tRNA synthetase,to chromosome 12 using human × chinese hamster cell hybrids

We have isolated interspecific somatic cell hybrids between a temperature-sensitive Chinese hamster ovary (CHO) cell methioninyl-tRNA synthetase mutant and human peripheral leukocytes. The hybrids were selected at 39° C which requires the retention and expression of the human gene, MARS, which complements the defective CHO gene. In vitro heatinactivation experiments on the methioninyl-tRNA synthetase activity in cell-free extracts from heat-resistant hybrids indicate that the human form of this enzyme and, therefore, the human MARS gene is present in hybrid cells. Cytogenetic analysis of three independent temperature-resistant hybrids revealed the presence of a single human chromosome, number 12. Two other independent hybrids examined contained human chromosome 12 as well as a second human chromosome. Electrophoretic analysis of extracts from hybrid cell lines for a human chromosome 12 marker isozyme, LDH-B, showed a pattern of heterotetrameric bands consistent with the presence of the human form of this enzyme in these cells. The correlation between the presence of the human form of methioninyl-tRNA synthetase and human chromosome 12 in temperature-resistant hybrids indicates that the human MARS locus is located on this chromosome.     

Assignment of the gene for cytoplasmic glutamate-oxaloacetate transaminase to mouse chromosome 19 using Chinese hamster x mouse somatic cell hybrids

Chinese hamster and mouse cytoplasmic glutamate-oxaloacetate transaminase (GOT-1, EC 2.6.1.1) were separated by isoelectric focusing of cell extracts on thin-layer polyacrylamide plates. The expression of mouse GOT-1 was correlated with the retention of mouse chromosomes and the expression of 16 marker isozymes in 77 Chinese hamster x mouse somatic cell hybrids. Mouse GOT-1 expression was discordant to the expression of isozyme genes assigned to mouse chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, and X. Furthermore it showed no concordant expression with mouse chromosomes 3, 13, and 15. The expression of mouse GOT-1 was highly concordant to mouse chromosome 19 in 21 hybrid clones analyzed karyotypically. Our data confirm by means of somatic cell genetics the chromosomal assignment of the mouse GOT-1 gene based on Mendelian genetics by Eicher, Reynolds, and Southard (3). The appearance of a heteropolymeric band in hybrid cells expressing mouse GOT-1 suggests that this enzyme molecule is probably composed of two subunits.     

Assignment of structural gene for asparagine synthetase to human chromosome 7

Somatic cell hybrids obtained from the fusion of human B lymphocytes and an asparagine synthetase-deficient Chinese hamster ovary cell line were isolated after growth in asparagine-free medium. The human and hamster forms of asparagine synthetase differ significantly in their rate of inactivation at 47.5° C. The asparagine synthetase activity expressed in the hybrids was inactivated at 47.5° C at the same rate as the human form of the enzyme. Karyotypic analysis and analysis for chromosome-specific enzyme markers showed that the structural gene for asparagine synthetase is located on chromosome 7 in humans. The heat-inactivation profile for asparagine synthetase in extracts of hybrids formed between human peripheral leukocytes and a hamster cell line expressing asparagine synthetase activity was intermediate between the two parental types when human chromosome 7 was present, but was identical to the hamster parent when chromosome 7 was absent.     

Assignment of dioxin-inducible cytochrome P-450 gene family to Chinese hamster chromosome 4

The 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P-450gene family (P₁-450and P₃-450in the C57BL/6N mouse)has recently been localized to mouse chromosome 9. In the present study, HindIII-digested DNA from Chinese hamster, mouse, and 20 Chinese hamster × mouse somatic cell hybrids and subclones segregating hamster chromosomes was probed with the mouse P₁-450and P₃-450full-length cDNA clones. Hamster P-450gene fragments (6.0 and 7.4 kb) were assigned to Chinese hamster chromosome 4. These data are consistent with linkage conservation among these two P-450sequences and four other loci on mouse chromosome 9 that map to hamster chromosome 4.     

Isolation and characterization of a mutant Chinese hamster cell line resistant to the glutamine analog 6-diazo-5-oxo-l-norleucine

A mutant cell line, called don801, was isolated from a wild-type population of V79.5 Chinese hamster cells by its ability to grow in the presence of the glutamine analog 6-diazo-5-oxo-l-norleucine (DON), which is toxic for V79.5 cells. The don801 cells were found not to be cross-resistant to another glutamine analog, O-diazoacetyl-l-serine (azaserine, AS). It was shown that guanine but neither hypoxanthine nor adenine protected V79.5 cells from the toxic effects of DON, while hypoxanthine and adenine, but not guanine protected them against AS toxicity. Exposure of wild-type cells to DON was shown to result in a specific reduction of intracellular GTP pools, while in the mutant cells there was no effect on GTP levels. These results strongly suggested that DON was specifically inhibiting guanylate synthetase (GMP synthetase;xanthosine-5-phosphate:l-glutamine amidoligase, EC 6.3.5.2) in V79.5 cells and that the enzyme in don801 cells was resistant to inhibition. In vitro assays of GMP synthetase activities from V79.5 and don801 cells confirmed this hypothesis. The mutant phenotype was also found to be dominant in intraspecific cell hybrids.     

Physiological and biochemical properties of a temperature-sensitive leucyl-tRNA synthetase mutant (tsHl) and revertant from Chinese hamster cells

A relevant (rl) of the temperature-sensitive leucyl-tRNA synthetase mutant (tsHl) of Chinese hamster ovary cells has been analyzed. The enzyme in the revertant is also temperature sensitive, but its activity at every temperature tested is slightly higher than that of the mutant. Both tsHl and the revertant are sensitive to a decreased concentration of leucine in the growth medium, but the revertant is less sensitive than the mutant. The tsHl synthetase has a K_m for leucine which is 80-fold higher at 39°C than that of wildtype CHO cells, whereas the K_m of the rl enzyme is 20-fold higher than the wild type at this temperature.     

Assignment of the gene locus for human α-l-fucosidase to chromosome 1 by analysis of somatic cell hybrids

The -l-fucosidases (EC 3.2.1.51) from human and mouse cells could be separated by isoelectric focusing of neuraminidase-treated cell extracts in acrylamide slab gels. Fourteen hybrid clones derived from the fusion of mouse and human cultured fibroblasts and 37 hybrid clones derived from the fusion of human long-term lymphoid lines with mouse RAG cells were tested for expression of human -l-fucosidase. A strong correlation between the expression of the human enzyme and the presence or absence of human chromosome 1 was found. The presence of human -l-fucosidase in clones scored as positive by isoelectric focusing was confirmed by Ouchterlony double immunodiffusion against IgG from rabbits immunized with purified human -l-fucosidase. It is concluded that the structural gene locus for human -l-fucosidase is located on chromosome 1.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: Isolation and characterization of a mutant deficient in the activity of phosphoribosylaminoimidazole synthetase

A new purine-requiring mutant of Chinese hamster ovary cells (CHO-Kl) is described. This mutant, Ade^– G, grows on aminoimidazole carboxamide, hypoxanthine, or adenine. It complements all eight of our other previously described Ade^– mutants. Biochemical analysis of de novo purine synthesis in whole cells suggests that Ade^– G is capable of the first four reactions of de novo purine biosynthesis and that it synthesizes and accumulates phosphoribosylformylglycinamidine (FGAM). Direct enzyme assay in cell-free extracts confirms that Ade^– G is defective in phosphoribo-sylaminoimidazole synthetase activity and does not convert FGAM to phosphoribosylaminoimidazole (AIR), the next intermediate in the de novo biosynthetic pathway.Recipient of a Research Career Development Award (AM00044) from the National Institute of Arthritis, Metabolic and Digestive Diseases.     

Assignment of human nerve growth factor receptor gene to chromosome 17 and regulation of receptor expression in somatic cell hybrids

Nerve growth factor (NGF) is a polypeptide hormone which plays a central role in the development and growth of sympathetic and sensory neurons. The effects of NGF on target cells are mediated by a specific cell surface structure, nerve growth factor receptor (NGFr), which has been identified in human cells as a 75,000-mol-wt glycoprotein. We have used a monoclonal antibody to human NGFr to study cell-surface expression of the receptor on a panel of mouse-human neuroblastoma hybrids, and the serological typing results permit assignment of the gene coding for NGFr(NGFR) to chromosome 17q21-qter. In addition to mouse-human neuroblastoma hybrids, human NGFr was also detected on hybrids derived from fusions between mouse L-cell fibroblasts and human neuroblastoma and melanoma cells. Furthermore, induction of human NGFr expression was observed in hybrids derived from NGFr^– human kidney epithelial cells and mouse L cells, but not in hybrids derived from human kidney epithelial cells and mouse RAG kidney carcinoma cells. These results suggest that cell-surface expression of human NGFr is controlled by transacting regulatory signals.     

Isolation and characterization of a UV-sensitive hypermutable aphidicolin-resistant Chinese hamster cell line

Aphidicolin is a specific inhibitor of DNA polymerase and blocks DNA synthesis in vivo. The inhibition of purified -polymerase has been shown to be competitive with dCTP but not with the other three deoxynucleoside triphosphates (dNTPs). In order to study the various roles that the -polymerase might play in DNA replication and/or repair, we have attempted to isolate Chinese hamster V79 cells that are resistant to aphidicolin. Four resistant mutants were isolated from BrdU-black light- and UV-mutagenized cells. None of the mutants isolated contains an -polymerase that is resistant, in crude extract measurements, to aphidicolin. Three mutants isolated, however, were found to be resistant to araC. Two mutants tested were found to be sensitive to cytidine and have elevated levels of dCTP or all 4 dNTPs. These results indicate that they are nucleotide pool mutants instead of -polymerase mutants. One mutant, aph^r-4, is characterized by the following: (1) high level of dCTP; (2) thymidine (or CdR, UdR, auxotrophic; (3) sensitive to thymidine (and AdR, GdR); (4) slow-growing; (5) cytidine sensitive; (6) UV sensitive and hypermutable at the ouabain-resistant locus; and (7) a ninefold increase in frequency of chromatid gaps and breaks when cells are exposed to BrdU- containing medium. Revertants of aph^r-4 which are partially aphidicolin-resistant and retain the first three characteristics listed above, but not the others, have been isolated. The appearance of this type of revertant indicates that either aph^r- 4 or its revertant is a double mutant.     

Assignment of aMus musculus gene for triosephosphate isomerase to chromosome 6 and for Glyoxalase-I to chromosome 17 using somatic cell hybrids

Chinese hamster × mouse hybrid cells segregating mouse chromosomes have been used to assign a gene for triosephosphate isomerase (TPI-1, EC 5.3.1.1, McKusick #19045) to mouse chromosome 6, and a gene for Glyoxalase-I (GLO-1, EC4.4.1.5, McKusick #13875) to mouse chromosome 17. The genes for TPI-1 and lactate dehydrogenase B are syntenic in man and probably so in the dog. It is therefore likely that they are syntenic also in the mouse. It is of interest then that there is a mouse gene,Ldr-1, on chromosome 6 that regulates the level of LDH B subunits in mouse erythrocytes. The locus for GLO-1 is closely linked to the major histocompatibility complex in man. Since the major histocompatibility complex in the mouse is present on chromosome 17, this locus and theGlo-1 locus are syntenic in the mouse as well. This finding adds to the number of autosomal gene pairs which are syntenic in both mouse and man and reinforces the belief that there is considerable conservation of linkage groups during evolution.     

Assignment of the genes for mouse type I procollagen to chromosome 16 using mouse fibroblast-Chinese hamster somatic cell hybrids

Somatic cell hybrids between mouse and Chinese hamster fibroblasts have been used to identify the chromosome responsible for the synthesis of both mouse type I procollagen subunit chains (MCOLA1 and MCOLA2). Thirty-one separate hybrid clones and subclones from ten separate hybridization events were isolated in hypoxanthine-aminopterinthymidine (HA T) selection medium and were used for detailed gene-mapping studies. ELISA and Western blotting immunochemical analysis were used to detect the production of mouse type I procollagen in each hybrid clone. Mouse and Chinese hamster chromosomes were identified in each hybrid clone by trypsin-Giemsa banding of metaphase chromosome spreads and by isozyme analysis. We have found that mouse type I procollagen production segregates concordantly with mouse superoxide dismutase-1, previously mapped to mouse chromosome 16, and with the presence of mouse chromosome 16 karyotypically. Western blotting immunochemical analysis of the separated mouse procollagen chains produced by each hybrid line demonstrated that apparently the genes for both subunit chains are located on the same chromosome. These studies, therefore, assign the structural genes for mouse type I procollagen pro 1 (MCOLA1) and pro 2 (MCOLA2) chains to mouse chromosome 16.     

Characterization and use of somatic cell hybrids with interspecific translocations involving the human X chromosome

Two hybrid cell lines, whose only human material was a portion of the X translocated on to a mouse chromosome, have been characterized by cytogenetics, in situ hybridization and Southern blotting. In one hybrid (HORL911R8B) the region Xpter to Xq2(2–4) was identified. In the other (PIP) the single human fragment was found to contain sequences from two separate X chromosomal regions (corresponding approximately to Xp11.4–Xp22.1 and Xq26–Xqter). These two hybrids in combination with a third (WAG 8) retaining Xqter to Xp21 as a human X-autosome translocation chromosome, form a mapping panel for rapid subregional assignments to the human X chromosome. This mapping panel has been used to provide information about the order of DNA sequences derived from the X chromosome and to provide an assignment for an anonymous DNA segment, M201γ, to Xp11.4–Xp21.1.     

Assignment of low-molecular-weight human (2′, 5′)a synthetase to chromosome 11

Human low-molecular-weight (2, 5)A synthetase is induced in certain human x mouse somatic hybrid cell lines when these cells are treated with mouse interferon. We have assigned the gene coding for this interferon-inducible antiviral enzyme to human chromosome 11 by somatic cell genetic techniques (1). Fluorescence-activated cell sorting for cells expressing or lacking 4F2 antigen in two independently derived, chromosome 11-containing hybrid cell lines separated the cells into subpopulations of cells that had retained or segregated chromosome 11, respectively (2). We used these subpopulations to confirm our gene assignment by demonstrating that retention of chromosome 11 was required for expression of human (2, 5) A synthetase.     

Isolation and characterization of Chinese hamster cell line resistant to monofunctional alkylating agents

A clonal derivative of a Chinese hamster Don D-6 cell line resistant to methyl methane sulfonate (MMS) has been isolated following mutagenesis by ethyl methane sulfonate (EMS). The clone, designated as MMS^r-1, exhibited high resistance to killing by the monofunctional alkylating agents MMS and EMS. This characteristic had not been acquired by a transient adaptation to the alkylating agents but was found to be a stable heritable trait. MMS^r-1 was more sensitive to high-molecular-weight chemicals, such as colchicine and puromycin, than Don D-6. Both MMS^r-1 and its parental cells showed the same ability to take up radioactive MMS. The resistance of MMS^r-1 appears not to be due to altered uptake of MMS. The resistance was accompanied by low chromosomal aberration and sister chromatid exchange (SCE) induction but not by mutability. Protein synthesis inhibitors such as cycloheximide and puromycin reduced the resistance to the same level as that in Don D-6. SCE induction by MMS in this clone was not antagonized by the protein synthesis inhibitors, whereas mutagenesis was reversed to the normal parental cell level by these inhibitors. Aphidicolin, a DNA-synthesis inhibitor, exhibited no such effects. These results suggest that MMS^r-1 might have modified repair capacity, which can be normalized by treatment with the protein-synthesis inhibitors, for lethal DNA damage by monofunctional alkylating agents, and that SCE formation by the alkylating agents is closely correlated with chromosomal aberration and cell lethality.