Gene linkage analysis in the mouse by somatic cell hybridization: Assignment of adenine phosphoribosyltransferase to chromosome 8 and α-galactosidase to the X chromosome

Somatic cell hybridization techniques were applied to gene linkage analysis in the laboratory mouse. Cells of an established line of Chinese hamster lung fibroblasts were fused with mouse embryo fibroblasts and with mouse peritoneal macrophages obtained from different inbred strains: From 3 hybridization experiments, 123 primary and secondary clones were isolated in HAT selective medium and 24 were back-selected in 8-azaguanine. Hybrid clones were characterized for the expression of 16 murine isozymes by starch, acrylamide, and Cellogel electrophoresis, and on the basis of segregation data, 3 syntenic associations could be made. Malate oxidoreductase decarboxylating (MOD) and mannose phosphate isomerase (MPI) segregated concordantly, confirming an established linkage relationship;adenine phosphoribosyltransferase (APRT) segregated concordantly with glutathione reductase (GR) which is known to be on chromosome 8;α-galactosidase was observed to be syntenic with hypoxanthine phosphoribosyltransferase (HPRT), and X-linked enzyme. All other isozymes examined segregated independently of one another.     

Chromosome assignment of genes encoding theα andΒ subunits of glycoprotein hormones in man and mouse

The chromosomal locations of the genes for the common α subunit of the glycoprotein hormones and the Β subunit of chorionic gonadotropin in humans and mice have been determined by restriction enzyme analysis of DNA isolated from somatic cell hybrids. The CGα gene (CGA), detected as a 15-kb BamHI fragment in human DNA by hybridization to CGα cDNA, segregated with the chromosome 6 enzyme markers ME1 (malic enzyme, soluble) and SOD2 (superoxide dismutase, mitchondrial) and an intact chromosome 6 in human-rodent hybrids. Cell hybrids containing portions of chromosome 6 allowed the localization of CGA to the q12 → q21 region. The >30- and 6.5-kb BamHI CGB fragments hybridizing to human CGΒ cDNA segregated concordantly with the human chromosome 19 marker enzymes PEPD (peptidase D) and GPI (glucose phosphate isomerase) and a normal chromosome 19 in karyotyped hybrids. A KpnI-HindIII digest of cell hybrid DNAs indicated that the multiple copies of the CGΒ gene are all located on human chromosome 19. In the mouse, the α subunit gene, detected by a mouse thyrotropin (TSH) α subunit probe, and the CGΒ-like sequences (CGΒ-LHΒ), detected by the human CGΒ cDNA probe, are on chromosomes 4 and 7, respectively.     

Coronavirus 229E susceptibility in man-mouse hybrids is located on human chromosome 15

Human coronavirus 229E, an enveloped, RNA-containing virus, causes respiratory illness in man and is serologically related to murine coronavirus JHM, which causes acute and chronic demyelination in rodents. 229E displays a species-specific host range restriction whose genetic basis was studied in human-mouse hybrids. 229E replicated in human WI-38 cells but not in three mouse cell lines tested (RAG, LM/TK^–, and A9). Human coronavirus sensitivity (HCVS) was expressed as a dominant phenotype in hybrids, indicating that mouse cells do not actively suppress 229E replication. HCVS segregated concordantly with the human chromosome 15 enzyme markers mannose phosphate isomerase (MPI) and the muscle form of pyruvate kinase (PKM2), and analysis of hybrids containing an X/15 translocation [t(X;15)(p11;q11)] localized HCVS to the q11 qter region of chromosome 15. HCVS might code for a specific surface receptor, allowing 229E to be absorbed to and received within the host cell.     

Assignment of the human gene for muscle-type phosphofructokinase (PFKM) to chromosome 1 (region cen→q32) using somatic cell hybrids and monoclonal anti-M antibody

Human phosphofructokinase (PFK; EC 2.7.1.11) is under the control of three structural loci which encode muscle-type (M), liver-type (L), and platelet-type (P) subunits; human diploid fibroblasts and leukocytes express all three loci. In order to assign human PFKMlocus to a specific chromosome we have analyzed human × Chinese hamster somatic cell hybrids for the expression of human M subunits, using an anti-human M subunit-specific mouse monoclonal antibody. In 18 of 19 hybrids studied, the expression of the PFKMlocus segregated concordantly with the presence of chromosome 1 (discordancy rate 0.05) as indicated by chromosome and isozyme marker analysis. The discordancy rates for all the other chromosomes were 0.32 or greater, indicating that the PFKMlocus is on chromosome 1. For the regional mapping of PFKM,eight hybrids were studied that contained one of five distinct regions of chromosome 1. These results further localize the human PFKMlocus to region cenq32 of chromosome 1.     

Intrachromosomal gene mapping in man: Assignment of nucleoside phosphorylase to region 14cen→14q21 by interspecific hybridization of cells with a t(X;14) (p22;q21) translocation

The structural gene for purine-nucleoside phosphorylase (NP) has been assigned to a subregion of chromosome 14 by somatic cell hybridization of male and female cells containing the balanced translocation t(X;14) (p22;q21). Peripheral lymphocytes were fused to a pseudodiploid HPRT-deficient established Chinese hamster cell line. 23 primary hybrid clones (10 derived from male and 13 from female cells) were isolated and maintained in HAT selective medium. Parallel subcultures from generations 16, 24, and 40 after clonal isolation were fully karyotyped and analyzed electrophoretically for expression of the human types of NP, HPRT, G6PD, and PGK. The human NP phenotype segregated discordantly with each human chromosome except chromosome 14 and the der(14),t(X;14) translocation chromosome. In all, 8 hybrids which had retained the der(X), t(X;14) translocation chromosome under HAT selective pressure and expressed human HPRT had lost the human NP phenotype. These results indicate localization of the NP gene in region 14pter14q21.     

Proximity of thyroglobulin and c-myc genes on human chromosome 8

The human thyroglobulin structural gene (TG) was mapped to the long arm of chromosome 8 by blot hydridization of a TG cDNA probe to DNA from 21 human × mouse somatic cell hybrids containing overlapping subsets of human chromosomes. In situ hybridization of the TG probe to metaphase chromosomes from a karyotypically normal human lymphoblastoid cell line, JS, localized the TG gene to within the region 8q23 q24.3. Thus, the TG and c-myc genes map to the same chromosome band in normal human cells. In a human colon carcinoma cell line (COLO 320 DM) which contains amplified c-myc, the TG gene is not amplified and hence it lies outside the amplification domain.     

Bioautographic visualization of aminoacylase-1: Assignment of the structural geneACY-1 to chromosome 3 in man

A bioautographic assay was developed for the visualization of aminoacylase-1 (N -acylamino acid aminohydrolase, ACY-1; EC 3.5.1.14) after zone electrophoresis. Bioautography and species differences in electrophoretic mobility of ACY-1 made it possible to investigate the chromosome assignment of the gene for human ACY-1 using human—mouse somatic cell hybrids. Human ACY-1 segregated concordantly with -galactosidase-A (GAL _A;EC 3.2.1.23) but showed discordant segregation with 32 other markers representing 23 linkage groups. The GAL_A gene has been previously assigned to chromosome 3. From this evidence and confirming chromosome analyses, ACY-1has been assigned to chromosome 3. A genetic polymorphism in the electrophoretic mobility of ACY was observed in mouse strains, demonstrating that this enzyme can be mapped in genetic crosses of Mus musculus.     

Genetics of the cell surface: Assignment of genes coding for external membrane proteins to human chromosomes 10 and 14

Using somatic cell hybridization gene mapping methodology, genes coding for human cell-surface proteins have been assigned to specific chromosomes. Lactoperoxidase-catalyzed iodination was employed to label external membrane proteins in cell hybrids between mouse and human cultured cells. Mouse and human external membrane proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. After electrophoresis, external membrane proteins were identified by autoradiography. An external membrane protein of 130,000 molecular weight (EMP-130) segregated concordantly with glutamic oxaloacetic transaminase_s (GOT_s, EC 2.6.1.1), an enzyme marker encoded on chromosome 10. External membrane proteins of 195,000 and 175,000 molecular weight (EMP-195 and EMP-175) segregated concordantly with nucleoside phosphorylase (NP, EC 2.4.2.1), an enzyme marker encoded on chromosome 14. Limited proteolysis of the 195,000 and 175,000 molecular weight proteins suggests that these two proteins are modified forms of each other and are encoded by the same locus. These findings demonstrate the mapping of human genes coding for external proteins EMP-130 and EMP-195 to chromosomes 10 and 14, respectively. Chromosome analyses of cell hybrids supported these assignments.     

c-myc GENE ABNORMALITIES IN MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMAS

c-myc gene abnormalities associated with lymphomagenesis, including rearrangements and mutations in the regulatory region between exon I and intron I, have been studied in 54 MALT lymphomas (43 low and 11 high grade) and 36 nodal lymphomas (27 low and 9 high grade). By Southern blot analysis, none of the 54 MALT lymphomas but 2 of 36 nodal lymphomas had c-myc gene rearrangements. Defined tumour cell populations from all MALT lymphoma cases were isolated by microdissection from frozen tissue sections and analysed by polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) and direct sequencing for somatic mutations in the exon I/intron I region of the gene. Point mutations in this region were identified in nine cases of MALT lymphoma (7/43=16·2 per cent of low grade; 2/11=18·1 per cent of high grade). These mutations were located at either the exon I/intron I border of myc intron factor (MIF) binding sites, which are critical in the negative regulation of c-myc expression. Of the nodal lymphomas, only the two cases (5·6 per cent) with c-myc gene rearrangement showed scattered or clustered mutations. These results suggest that c-myc mutations in MALT lymphomas are unlikely to be associated with chromosome translocation, which is the main cause of somatic mutations observed in other types of lymphoma. The mutations involving the c-myc regulatory regions may play a pathogenetic role in at least a proportion of MALT lymphomas. © 1997 by John Wiley & Sons, Ltd.     

Assignment of the genes for thymidine kinase and galactokinase toMus musculus chromosome 11 and the preferential segregation of this chromosome in Chinese hamster/mouse somatic cell hybrids

Somatic cell genetic techniques were used to establish linkage assignments for the loci coding for galactokinase (GLK) and thymidine kinase (TK) in the laboratory mouse, Mus musculus.Four fusion experiments produced cell hybrids that segregated mouse chromosomes. Three series of hybrid clones were produced from the fusion of mouse macrophages or fibroblasts and Chinese hamster cells of the E36 line, which is deficient in hypoxanthine phosphoribosyltransferase activity. Clones were assayed for the expression of 11 mouse isozymes coded by genes on 11 chromosomes. Ten isozymes were retained at high frequencies, but the mouse GLK phenotype was absent in most primary clones and all secondary clones. Chromosome analysis of 24 of these hybrids indicated that GLK activity was lost concordantly with chromosome 11. One hybrid clone was produced from the fusion of peritoneal macrophages and hamster cells of the a3 line, which lacks TK activity. Mouse GLK activity was expressed in this primary clone and all secondary clones isolated in HAT medium, and was lost in all secondary clones backselected in medium with 5-bromodeoxyuridine. Thus, the genes coding for GLK and TK are syntenic and both can be assigned to chromosome 11. The absence of chromosome 11 in the related series of a3 hybrids, and its rapid segregation in 3 series of E36 hybrids, suggests that it carries a third locus (or loci), the retention of which is detrimental to Chinese hamster/mouse hybrids.     

Assignment of the structural genes for the α subunit of hexosaminidase A,mannosephosphate isomerase,and pyruvate kinase to the region q22-qter of human chromosome 15

Concordant segregation of the expression of the subunit of human hexosaminidase A, human mannosephosphate isomerase, and pyruvate kinase was observed in somatic cell hybrids between either thymidine kinase-deficient mouse cells or thymidine kinase-deficient Chinese hamster cells and human white blood cells carrying a translocation of the distal half (q22-qter) of the long arm of chromosome 15 to chromosome 17. A positive correlation was established between the expression of these human phenotypes and the presence of the distal half of the long arm of human chromosome 15.     

Intrachromosomal gene mapping in man: The gene for tryptophanyl-tRNA synthetase maps in region q21→qter of chromosome 14

A gene for tryptophanyl-tRNA synthetase (EC 6.1.1.2), the enzyme which attaches tryptophan to its tRNA, has previously been assigned to human chromosome 14 by analysis of man-mouse somatic cell hybrids. We report here a method for the electrophoretic separation of Chinese hamster and human tryptophanyl-tRNA synthetases and its application to a series of independently derived Chinese hamster-human hybrids in which part of the human chromosome 14 has been translocated to the human X chromosome. When this derivative der (X),t(X;14) (Xqter Xp22::14q21 14qter) chromosome carrying the human gene for hypoxanthine-guanine phosphoribosyltransferase was selected for and against in cell hybrid lines by the appropriate selective conditions, the human tryptophanyl-tRNA synthetase activity was found to segregate concordantly. These results provide additional confirmation for the assignment of the tryptophanyl-tRNA synthetase gene to human chromosome 14 and define its intrachromosomal location in the region 14q21 14qter. Our findings indicate that the genes for tryptophanyl-tRNA synthetase and for ribosomal RNA are not closely linked on chromosome 14.     

Human mitochondrial thymidine kinase is coded for by a gene on chromosome 16 of the nucleus

The expression of human mitochondrial thymidine kinase (mt TK) was investigated by polyacrylamide electrophoresis in 19 independent human—mouse somatic cell hybrids which allowed all human chromosomes to be analyzed. In 8 hybrid clones the presence of this enzymatic activity could be demonstrated. Human mt TK segregated concordantly with human adenine phosphoribosyltransferase (APRT) and human chromosome 16. Discordant segregation with all other human chromosomes was demonstrated by karyotype and isozyme analyses. These results suggest that human mt TK is coded for by a gene on chromosome 16 of the nucleus. Thus human mt TK is genetically different from human cytosol thymidine kinase which is coded for by a gene on chromosome 17. The appearance of one heteropolymer band after electrophoretic separation of human and murine mt TK supports the notion that both enzymes have dimeric structures.     

Human Luteinizing hormone-releasing hormone gene (LHRH) is located on short arm of chromosome 8 (region 8p11.2 → p21)

Luteinizing hormone-releasing hormone (LHRH) is synthesized by hypothalamic neurons and affects the release of gonadotropic hormones from the anterior pituitary gland. A cDNA clone encoding the human LHRH precursor molecule was used to assign theLHRH gene to a human chromosome by in situ hybridization and Southern blot analysis. Metaphase spreads from two normal individuals were hybridized with³H -labeled LHRH-specific sequence. Of 120 cells analyzed, 33 had silver grains over the p11.2 p21 bands of chromosome 8. No other chromosomal site was labeled above background, indicating the presence of a single site for LHRH sequences in the human genome. Independent confirmation for this location of the humanLHRH gene on chromosome 8p was provided by analysis of DNA from human × Chinese hamster somatic cell hybrids. DNA samples were digested with EcoRI, blotted, and hybridized with the³²P-labeled human LHRH precursor cDNA probe. The single 11.5-kb human-specific band was detected only in hybrids containing human chromosome 8. Also, hybridization was observed in DNA from hybrids in which a portion of human chromosome 8 (region 8pter 8q21) had been spontaneously translocated onto a Chinese hamster chromosome.Presented in part at the 8th Human Gene Mapping Conference, Helsinki, August 1985.     

Synthesis and incorporation of human ribosomal protein S14 into functional ribosomes in human-Chinese hamster cell hybrids containing human chromosome 5: HumanRPS14 gene is the structural gene for ribosomal protein S14

In Chinese hamster ovary cells, mutations in the RPS14 gene (which was previously designated emtB) render cells resistant to normally cytotoxic concentrations of the protein synthesis inhibitor, emetine. Several lines of evidence indicate the RPS14 gene in Chinese hamster is the structural gene for ribosomal protein S14, including the finding that mutants with alterations in this gene produce an electrophoretically altered form of this protein. A human gene which complements the defect in CHO RPS14 mutants and renders them sensitive to emetine has previously been assigned to the long arm of chromosome 5. The analysis of ribosomal proteins extracted from CHO Emt^r x human cell hybrids, which contain human chromosome 5 and are emetine sensitive, demonstrated the presence of both the normal human and altered hamster forms of ribosomal protein S14. Human chromosome 5, the emetine-sensitive phenotype, and the human form of ribosomal protein S14 segregate concordantly from hybrids, confirming that the human gene in question is the structural gene for this protein. In addition, the results indicate that in interspecific cell hybrids, the human form of S14 is either incorporated into functional ribosomes more efficiently than the altered hamster protein or the human gene is overexpressed relative to the corresponding hamster gene.     

Isolation and characterization of interspecific heat-resistant hybrids between a temperature-sensitive Chinese hamster cell asparaginyl-tRNA synthetase mutant and normal human leukocytes: Assignment of humanasnS gene to chromosome 18

We isolated interspecific somatic cell hybrids between human peripheral leukocytes and a temperature-sensitive CHO cell line with a thermolabile asparaginyl-tRNA synthetase. The hybrids were selected at 39° C so as to require the expression of the human gene complementing the deficient CHO enzyme. In vitro heat-inactivation profiles of cell-free extracts from temperature-resistant hybrid cells indicate the presence of two forms of asparaginyl-tRNA synthetase. One form is very resistant to thermal inactivation, like the normal human enzyme, while the other form is very thermolabile, like the altered enzyme from the CHO parent. Hybrids and temperature-sensitive segregants derived from them were analyzed for the expression of known human chromosomal marker enzymes. The strong correlation between the expression of the human form of asparaginyl-tRNA synthetase and the presence of human chromosome 18 in hybrids suggests that the human gene, asnS,which corrects the heat-sensitive phenotype of the CHO asparaginyl-tRNA synthetase mutant, is located on chromosome 18.     

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicolresistant (CAP^R)human × mouse hybrids and one CAP^R cybrid derived from CAP^R HeLa cells and CAP^S mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with ³² P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [³²P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited reverse chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human mouse hybrid is to retain human mitochondrial DNA.     

Transformation of temperature-sensitive growth mutant of BHK21 cell line to wild-type phenotype with hamster and mouse DNA

A temperature-sensitive (ts) mutant, tsBN2, which was derived from BHK21 and is defective in the regulatory mechanism for chromosome condensation, was transformed to the temperature-resistant (ts⁺) phenotype by means of DNA-mediated gene transfer with hamster and mouse DNA. Treatment of mouse DNA with the restriction enzymesEcoRI, HindIII, PstI andSall, but not withXhoI, almost completely abolished the transforming activity. A fluctuation test, originally devised by Luria and Delbrück, was used to estimate the reversion and transformation frequencies of tsBN2 cultures.     

Human parathyroid hormone gene (PTH) is on short arm of chromosome 11

The human gene for parathyroid hormone (PTH) was chromosomally mapped using human-rodent hybrids and Southern filter hybridization of cell hybrid DNA. A recombinant DNA probe containing human PTHcDNA insert (pPTHm122) hybridized to a 3.7-kb fragment in human DNA cleaved with the restriction enzyme EcoRI. By correlating the presence of this fragment in somatic cell hybrid DNA with the human chromosomal content of the hybrid cells, the PTHgene was mapped to the short arm of the chromosome 11.