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 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.     

Assignment of the human gene for δ aminolevulinate dehydrase to chromosome 9 by somatic cell hybridization and specific enzyme immunoassay

A non-competitive enzyme immunoassay specific for δ aminolevulinate dehydrase has been devised and applied to rodent–human hybrid cell lines. Two different conditions have been used, one specific for the human enzyme and the other indicative of both rodent and human enzymes. The ratio of the values obtained under the two conditions was used to discriminate between positive and negative clones. By this method the gene for ALA dehydrase has been assigned to chromosome 9.     

Assignment of the genes for triose phosphate isomerase to chromosome 6 and tripeptidase-1 to chromosome 10 inMus musculus by somatic cell hybridization

Evidence is presented for the assignment of the gene for triose phosphate isomerase toMus musculus chromosome 6 and tripeptidase-1 to chromosome 10 by synteny testing and chromosome assignment in Chinese hamster x mouse somatic cell hybrid clones. Neither TPI nor TRIP-1 were expressed concordantly with any known isozyme markers in 45 hybrid clones (13 primary and 32 secondary). Karyotypic analysis of 21 clones showed that the expression of TPI and chromosome 6 were concordant in all cases as was expressed of TRIP-1 and chromosome 10. Both chromosomes were previously unmarked by isozymes.     

Assignment of the ǵene for F-type phosphofructokinase to human chromosome 10 by somatic cell hybridization and specific immunoprecipitation

A method of specific immunoprecipitation of minor isozymes was developed and applied to the detection of human F-type phosphofructokinase in man-rodent somatic cell hybrids.
This method allowed us to assign the gene for this newly discovered isozyme of phosphofructokinase to chromosome 10.     

Assignment of the ZIP kinase gene to human chromosome 19p13.3 by somatic hybrid analysis and fluorescence in-situ hybridization

A cDNA for a putative member of the serine/threonine kinase family was cloned from an adult human testis cDNA library. The predicted translation product was identical to ZIP kinase, which has been suggested to play an important role in the induction of apoptosis. The messenger RNA was ubiquitously expressed in various tissues. The chromosomal location of the gene was determined by fluorescence in-situ hybridization and polymerase chain reaction-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. This gene was mapped on the q13.3 region of chromosome 19. Received: April 24, 1998/Accepted: May 29, 1998     

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.     

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 gene for human cell-surface membrane antigen Trop-4 to chromosome 11

The gene (named MFI6) for a surface membrane antigen, Trop-4, is assigned to human chromosome 11 on the basis of studies using a mouse monoclonal antibody, immunofluorescence, fluorescence-activated cell sorting (FACS), immunoprecipitation, and mouse-human lymphocyte hybrids. The Trop-4 antigen is present on all human cell lines tested, on peripheral blood monocytes and granulocytes, and on a small fraction of peripheral blood lymphocytes, but is absent from erythrocytes. The Trop-4 monoclonal antibody precipitates an 85,000-dalton glycopolypeptide from hybrid cells containing human chromosome 11. However, in a human cell line expressing this antigen, a larger-molecular-weight species, 100–105,000 daltons was coprecipitated with the 85,000-dalton glycopeptide, and under nonreducing conditions a larger compound of 110–125,000 daltons was obtained. Although the Trop-4 antigen is of similar molecular weight to the Mab-4 and F10.44.2 antigens previously assigned to chromosome 11, it is shown to be different from them.     

Assignment of the human gene for uridine 5'-monophosphate phosphohydrolase (UMPH2) to the long arm of chromosome 17

Two distinct isozymes of uridine 5'-monophosphate phosphohydrolase (UMPH) have been identified in mammalian cells. Both of these isozymes hydrolyse other pyrimidine nucleotides. One of the isozymes, UMPH2, is more active against deoxy-nucleotides than non-deoxynucleotides and has a wider substrate specificity. We examined the segregation of the human gene for UMPH2 in human-mouse somatic cell hybrids. Electrophoretic analysis of UMPH2 in hybrids suggested that the enzyme is a dimer. Human UMPH2 cosegregated with the long arm of chromosome 17 in the nineteen hybrids examined. The ease of separation of the rodent and human isozymes of UMPH2 and the relative simplicity of the enzyme assay suggest that UMPH2 may be a more useful marker gene for chromosome 17 than galactokinase.     

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.     

Assignment of the gene coding for phosphoribosylglycineamide formyltransferase to human chromosome 14

Purine-requiring Chinese hamster ovary cell auxotrophs of the complementation class ade^– E were hybridized with various human cells, and hybrids were isolated under selective conditions in which the retention of the complementing gene on the human chromosome is necessary for survival. Synteny analysis in 72 primary and secondary hybrid clones using isozyme, karyotypic, and biochemical methods provides evidence for an assignment of the gene for phosphoribosylglycineamide formyltransferase (GART, EC 2.1.2.2), deficient in ade^– E mutants, to human chromosome 14. The importance of this gene assignment to the development of hypotheses regarding the organization, structure, and regulation of genes involved in the same biosynthetic pathway in mammalian cells is discussed.     

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 the lactotransferrin gene to human chromosome 3 and to mouse chromosome 9

Lactotransferrin (LTF), a member of the transferrin family of genes, is the major iron-binding protein in milk and body secretions. The amino acid sequence of LTF consists of two homologous domains homologous to proteins in the transferrin family. Recent isolation of cDNA encoding mouse LTF has expedited the mapping of both mouse and human LTF genes. Southern blot analysis of DNA from mouse-Chinese hamster and human-mouse somatic cell hybrids maps the LTF gene to mouse chromosome 9 and to human chromosome 3, respectively. Furthermore, analysis of cell hybrids containing defined segments of human chromosome 3 demonstrates that the gene is located in the 3q21-qter region. These results suggest that LTF and associated genes of the transferrin family have existed together on the same chromosomal region for 300–500 million years.     

Assignment of the gene for cytosolic alanine aminotransferase (AAT1) to human chromosome 8

The segregation of human cytosolic alanine aminotransferase (AAT1) and the individual human chromosomes has been studied in 27 secondary and tertiary rat hepatoma-human (liver) fibroblast hybrids. The staining solution used to visualize AAT activity on starch gels was specific for AAT since it was visualized only when all components of the stain were present. The locus for human AAT1 has been assigned to chromosome 8.     

Assignment of the gene for NADH diaphoraseDia-1 to mouse chromosome 15

We have examined 14 Chinese hamster x mouse somatic cell hybrids segregating mouse chromosomes for the expression of NADH-dependent diaphorase (Dia-1, cytochrome b ₅ reductase, EC 1.6.2.2). Isoelectric focusing allowed the discrimination of mouse Dia-1 isozymes from those of the Chinese hamster in the hybrids. Correlation of the loss or retention of individual mouse chromosomes with expression of mouse diaphorase isozymes resulted in the assignment of the gene for Dia-1 to chromosome 15. Dia-1is the first biochemical gene that can be detected in cultured cells assigned to this chromosome and will therefore provide a convenient marker for future somatic cell genetic studies.     

Assignment of the human acid α-glucosidase gene (αGLU) to chromosome 17 using somatic cell hybrids

Hybrid clones (MOGs) were made between the mouse line RAG and a primary fibroblast line from an individual of the rare alphaGLU 2 phenotype. Fifteen independent primary clones and 32 subclones were tested for the presence of human alphaGLU after separation of the human and rodent enzymes by starch gel electrophoresis. Twenty-three other human-mouse hybrids from six different crosses were analysed for the presence of human alphaGLU by exploiting a difference in the thermostability of the human and mouse enzymes. The hybrids were also analysed for up to 25 other enzymes which were used as markers for different human chromosomes. Two of the MOG hybrids were karyotyped and karyotype data were already available for a number of the other hybrids. The combined results demonstrate that alphaGLU is located on chromosome 17, and probably on 17q.