Assignment of an oligomycin-resistance locus to human chromosome 10

An oligomycin-resistant variant of human fibrosarcoma HT1080 was isolated and characterized as nuclear and codominant. The mutant was stable, was not cross-resistant to respiratory inhibitors, and it contained a mitochondrial ATPase which was less sensitive to oligomycin. Hybrids formed between the human mutant and a mouse cell line expressed the resistance phenotype. By a detailed karyotypic analysis of these hybrids using trypsin-Giemsa banding it was found that resistance to oligomycin correlated with the retention of two human chromosomes 10. The hybrid lines contained only mouse mitochondrial DNA as shown by analyses of mitochondrially synthesized proteins and mitochondrial DNA. The study assigns an ATPase oligomycin-resistance locus to human chromosome 10 and suggests that mouse and human subunits can combine in a functional enzyme complex.     

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 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.     

Assignment of three human markers in chromosome 21q11 to mouse chromosome 16

Three unique sequence microclones from human chromosome region 21q11 were assigned to mouse chromosome 16 using a mouse/Chinese hamster cell hybrid 96Az2 containing a single mouse chromosome 16. This comparative mapping provides further homology between human chromosome 21 and mouse chromosome 16 to include the very proximal portion of the long arm of human chromosome 21. Since this part of human chromosome 21 is associated with mental retardation in Down syndrome individuals, its homologous mouse region should also be included in the construction of mouse models for studying Down syndrome phenotypes including mental retardation.     

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.     

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 human preprothyrotropin-releasing hormone (TRH) gene to chromosome 3

The human gene encoding preproTRH (thyrotropin-releasing hormone) was assigned to chromosome 3, using human-Chinese hamster ovary somatic cell hybrids, analyzed by Southern hybridizations. Hybridization was carried out with a³²P-labeled human preproTRH cDNA labeled by the method of random priming (3). Hybridization of the cDNA probe to a human specific 4.8-kb DNA fragment of EcoRI-digested WBC DNA was used to localize the human preproTRH gene. No hybridization, by contrast, was seen with human preproTRH cDNA probe and hamster DNA after EcoRI treatment. Results from 29 somatic cell hybrids corroborated unequivocally that the human preproTRH gene can be assigned to human chromosome 3.Supported in part by USEHS grant DK37378.     

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.     

Identification of a human member of the Ly-49 multigene family

Three classes of multigene family-encoded receptors enable NK cells to discriminate between polymorphic MHC class I molecules: Ly-49 homodimers, CD94/NKG2 heterodimers and the killer cell inhibitory receptors (KIR). Of these, CD94/NKG2 has been characterized in both rodents and humans. In contrast, Ly-49 family members have hitherto been found only in rodents, and KIR molecules only in the human. In this report, we describe a human cDNA, termed Ly-49L, that constitutes the first human member of the Ly-49 multigene family. Compared with rodent Ly-49 molecules, the Ly-49L sequence contains a premature stop codon and predicts a truncated protein that lacks the distal part of a C-terminal lectin domain. Evidence is presented that the premature stop codon results from incomplete excision of the intron between the first two lectin domain exons. Splice variants predicting a full-size Ly-49L protein were not detected. As demonstrated by Northern blot analysis, Ly-49L was transcribed by IL-2-activated NK cells, but not by freshly isolated B or T cells. PCR screening of a 22-clone yeast artificial chromosome contig localized the LY49L locus to the human NK gene complex on chromosome 12p12-p13. Southern blot analysis of genomic DNA showed a simple pattern with a full-length Ly-49L probe at low stringency hybridization conditions, suggesting that Ly-49L may be the only human member of the Ly-49 multigene family.     

Assignment ofLIPA,associated with human acid lipase deficiency,to human chromosome 10 and comparative assignment to mouse chromosome 19

The genetics of lysosomal acid lipase (LIP) has been investigated in human-Chinese hamster and mouse-Chinese hamster somatic cell hybrids. Cellulose acetate electrophoresis of human fibroblast extracts demonstrated that LIP activity consists of three isozymes. A deficiency of LIP activity has been observed in Wolman's disease (WD), cholesterol ester storage disease (CESD), and I-cell disease (ICD); this deficiency was associated with only one LIP isozyme, LIPA. We have demonstrated concordant segregation between human LIPA and human chromosome 10 and its enzyme marker glutamate oxaloacetate transaminase-1 (GOT1) in cell hybrid clones. Previous evidence suggested the different mutations associated with WD and CESD to be in the structural gene which we assign to human chromosome 10, while a different gene, involved in the processing of LIPA, is altered in ICD. These results indicate that several types of gene products are involved in the final expression of LIPA. In mouse-Chinese hamster hybrid clones, mouseLip-1 (homologous to humanLIPA) was assigned to chromosome 19. Previously, mouseGot-1 has been assigned to chromosome 19. Thus, theLIPA-GOT1 linkage group has remained intact during the 80×10⁶ years of evolution that separates humans and mice.Preliminary report of a portion of this work has been presented (30).     

Assignment of the vascular smooth muscle actin gene ACTSA to human chromosome 10

Human vascular smooth muscle actin gene (ACTSA) was cloned and its unique sequence was used as the hybridization probe for Southern blot analysis of DNAs from 18 rodent-human somatic cell hybrids; the gene was assigned to human chromosome 10. Regional mapping by in situ hybridization showed that the gene is located on the long arm (q22-q24) of the chromosome. Thus, the gene is on a different chromosome from the other four actin genes so far examined.     

Assignment of a structural gene forβ-glucuronidase to human chromosome C7

An electrophoretic technique was developed which allows the separation of human -glucuronidase (GUS EC 3.2.1.31) from the enzyme present in cultured murine, Chinese and Syrian hamster cells in one buffer system on Cellogel. Using this technique a number of independent human-mouse somatic cell hybrids have been analyzed for the segregation of GUS, other enzyme markers, and all human chromosomes. The results indicate that a structural gene for human -glucuronidase is located on chromosome C7.     

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 human thyrotropin-releasing hormone (TRH) receptor gene to chromosome 8

The human gene encoding thyrotropin-releasing hormone receptor was assigned to chromosome 8, using human-Chinese hamster ovary somatic cell hybrids, analyzed by Southern hybridizations. Hybridization was carried out with a³²P-labeled fragment of the human TRH-R genomic DNA. Hybridization of this probe to a human specific 10.5-kb DNA fragment of EcoRI-digested WBC DNA was used to localize the human TRH-R gene. No hybridization, by contrast, was seen with this probe and hamster DNA after EcoRI treatment. Results from 18 somatic cell hybrids corroborated unequivocally that the human TRH-R gene can be assigned to human chromosome 8.     

Assignment of the gene determining human carbonic anhydrase, CAI, to chromosome 8

A cDNA clone complementary to the mRNA encoding the rabbit erythrocyte specific carbonic anhydrase, CAI, has been used as probe for human CAI sequences in the analysis of DNA from panels of rodent/human somatic cell hybrids. The presence of the human CAI gene in all hybrids correlates with the presence of chromosome 8. Together with published mapping data, this assignment indicates that three CA loci are situated on chromosome 8.     

Assignment of mouse beta-spectrin gene to chromosome 12

The structural gene for the Β-subunit of the mouse erythrocyte spectrin, hereinafter designated as Sp-b, was assigned to the mouse chromosome 12. This assignment was made by Southern analysis of genomic DNA from mouse X Chinese hamster hybrid cells using cloned mouse erythrocyte Β-spectrin cDNA as a probe. In the PstI-digested genomic hamster cell DNA a single band of 2.0 kb was detected, whereas PstI-digested mouse DNA gave a band of 4.2 kb, when probed with the mouse erythroid Β-spectrin cDNA clone. This allowed us to analyze a panel of mouse X Chinese hamster somatic cell hybrids to map this gene to chromosome 12. Interestingly, this assignment is different from that observed for the α-subunit of spectrin, which has been mapped to chromosome 1 in mouse. These results serve as a basis for further genetic characterization of the mouse hemolytic anemias.     

Assignment of the human gene for β-glycerol phosphatase to chromosome 8

The segregation of the human gene for β-glycerol phosphatase (GPB) was examined in human-Syrian hamster and human-Chinese hamster somatic cell hybrids. Electrophoretic analysis of the GPB in hybrids suggested that the enzyme is a dimer. Human GPB cosegregated with chromosome 8 in the twenty-five hybrids examined.     

Assignment of the human locus determining phosphoglycolate phosphatase (PGP) to chromosome 16

The segregation of human phosphoglycolate phosphatase has been studied in 52 independent human-rodent hybrids and 69 subclones. The results suggest that human PGP is on chromosome 16. Family data suggest that PGP is not close to 16qh or alpha Hp. The most likely regional assignment for PGP would appear to be 16p13 or 16p12, but a site on 16q cannot be entirely excluded. New data on 16qh and alpha Hp suggest that the male recombination fraction between these loci is about 0.2.