Chinese Hamster Somatic Cell Hybrids Research Articles

Characterization of the mouse loricrin gene: linkage with profilaggrin and the flaky tail and soft coat mutant loci on chromosome 3.

Loricrin is the major component of a specialized structure, termed the cornified cell envelope, that is formed beneath the plasma membrane of stratified squamous epithelial cells and is coexpressed with profilaggrin in terminally differentiating epidermal keratinocytes. Full-length cDNAs for both mouse and human loricrin have been cloned and characterized, as has the human gene. Here we report the isolation and characterization of the mouse loricrin gene. The gene has a simple structure consisting of a single intron of 1091 bp within the 5' noncoding sequence and an uninterrupted open reading frame. Using PCR analyses of DNAs isolated from mouse x Chinese hamster somatic cell hybrids, we have mapped both the loricrin and the profilaggrin genes to chromosome 3. Genetic linkage analysis has shown that mouse loricin and profilaggrin lie within 1.5 +/- 1.1 centimorgans of each other. We have further shown that both genes map in the vicinity of the flaky tail (ft) and soft coat (soc) loci. These mouse mutants exhibit a number of changes in their integument, suggesting that abnormalities in these genes may contribute to the mutant phenotype.

Chromosomal assignments of mouse genes for connexin 50 and connexin 33 by somatic cell hybridization

The 12 known connexin genes coding for the subunit proteins of the gap junction channels and related in nucleotide sequence are widely dispersed in the mouse genome. By using a series of mouse X Chinese hamster somatic cell hybrids and molecular probes of murine connexin genes, we have assigned the connexin 50 gene to mouse chromosome 3. The connexin 33 gene has been mapped to the X chromosome, thus confirming the previous chromosomal assignment of this gene based on interspecific back-cross mapping.

Localization of the CAMKG gene encoding gamma isoforms of multifunctional calcium/calmodulin-dependent protein kinase (CaM kinase) to human chromosome 10 band q22 and mouse chromosome 14.

Multifunctional calcium/calmodulin-dependent protein kinase (CaM kinase) is an enzyme mediating calcium-dependent signal transduction pathways. CaM kinase exists in a variety of isoforms, each with a distinct tissue-specific expression pattern, that enables the kinase to regulate multiple functions in mammalian systems. Here we report the chromosomal localization of the previously cloned human gamma-CaM kinase gene (CAMKG). By using a mapping panel of human x Chinese hamster somatic cell hybrid lines and fluorescence in situ hybridization, we have assigned human CAMKG to chromosome 10q22. We have partially cloned the murine gamma-CaM kinase gene and mapped it Camkg to mouse chromosome 14 by analyzing a panel of mouse x rodent somatic cell hybrid lines. A recessive gene, asa, implicated in the control of autoimmune response, is located within the predicted region for Camkg.

A radiation hybrid map of human chromosome 18

A human x Chinese hamster (CH) somatic cell hybrid subclone deficient in HPRT and containing only human chromosome 18 was irradiated with 7000 rad and fused to a thymidine kinase deficient CH cell line. Radiation-rescued hybrid cell lines, selected in HAT medium, were analyzed for human DNA with human interspersed-repeat sequence primers. Size and number of human chromosome fragments retained in a subset of hybrids were determined by FISH. A panel of 98 radiation hybrids (RH) was selected and analyzed for 90 chromosome 18-specific STSs by PCR, and for the D18Z1 centromeric marker by Southern blotting. STSs were developed from previously mapped RFLP loci and from published sequences. In addition, 32 novel STSs were generated from an 18-specific lambda library and from 18-specific YACs previously localized to chromosome bands by FISH. Marker retention frequency varied from 8-65% with an average of 24%. In selected RH the STS typing data were correlated with the chromosome 18 regions retained using 'reverse FISH' of IRS-PCR products from the RH to normal metaphase chromosomes. The order and intermarker distances of loci were determined using two-point and multipoint maximum likelihood methods. The resulting RH map covers most of chromosome 18 with four groups of tightly linked markers and three regions of loosely linked markers, one around the centromere and two on the long arm. More than a third of the markers are polymorphic and allow integration with the linkage map. This RH map provides a framework for establishing a clone contig of the entire chromosome 18.

CDNA Cloning of the Two Subunits of Human CAAX Farnesyltransferase and Chromosomal Mapping of FNTA and FNTB Loci and Related Sequences

The CAAX farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to a single cysteine in several cellular proteins. Substrates include the p21ras proteins, nuclear lamins, and several retinal proteins, all of which end with a "CAAXbox," where C is cysteine, A is an aliphatic amino acid, and X is methionine or serine. Full-length cDNAs for the alpha and beta subunits of the rat farnesyltransferase have been cloned, and both have been shown to be essential for catalytic activity. Here we have used the rat cDNAs to clone cDNAs for the human alpha and beta subunits. Comparison of the human and rat amino acid sequences revealed a remarkable degree of conservation (93% identity for the alpha subunit and 96% identity for the beta subunit). The functional genes for the alpha and beta subunits of human farnesyltransferase (gene symbols, FNTA and FNTB) were localized to human chromosome bands 8p22-q11 and 14q23-q24, respectively, by Southern blot hybridization and PCR analyses of panels of human x Chinese hamster somatic cell hybrid lines and by fluorescence chromosomal in situ hybridization. We also found several related farnesyltransferase genes. FNTAL1 was assigned to 11q13.4-q14.1, FNTAL2 to chromosome 13, and FNTBL1 to chromosome 9.

Chromosomal assignments of mouse connexin genes, coding for gap junctional proteins, by somatic cell hybridization

The connexin genes Cx31 and Cx45 coding for proteins of gap junctional subunits have been assigned to mouse chromosomes 4 and 11 by Southern blot hybridization of specific gene probes to DNA from mouse x Chinese hamster somatic cell hybrids. In addition, our results confirm the recent assignment of mouse connexin genes Cx26, Cx32, Cx37, Cx40, Cx43, and Cx46 to mouse chromosomes 14, X, 4, 3, 10, and 14, respectively, by analysis of interspecific backcrosses and by somatic cell hybridization. Our assignment of the Cx31 gene to mouse chromosome 4 locates the fourth connexin gene on this mouse chromosome to which the genes for Cx31.1, Cx37, and Cx30.3 have previously been assigned. Interestingly three of them (coding for Cx31, Cx31.1, and Cx30.3) are preferentially expressed in skin. Possibly some of the connexin genes clustered on mouse chromosome 4 may be regulated coordinately.

Expression and chromosome localization of the murine cystic fibrosis transmembrane conductance regulator

A 13.5-kb genomic fragment of the mouse cystic fibrosis transmembrane conductance regulator (CFTR) gene was isolated from a C57 BL 6 J liver DNA library, using a human CFTR exon 10 probe. This region of the human gene includes the most common cystic fibrosis mutation (deletion of the Phe 508 residue) in the first nucleotide binding domain of CFTR. Sequence analysis demonstrated 87% identity between the predicted mouse and the normal human CFTR exon 10 sequences, including conservation of the Phe 508 residue. Northern analysis revealed that the mouse gene is expressed in intestine, lung, stomach, kidney, and salivary gland. In contrast to human CFTR, murine CFTR transcripts were not detectable by Northern analysis in the liver or pancreas. More sensitive PCR analysis, however, revealed that the mouse CFTR gene is weakly expressed in other tissues, including liver and pancreas. During development, mouse CFTR transcripts were observed as early as Embryonic Day 13. Southern analysis of mouse × Chinese hamster somatic cell hybrid DNAs mapped the mouse CFTR locus ( Cftr) to Chromosome 6 (Chr 6). Subsequent typing of the progeny of an interspecies backcross revealed that Cftr is closely linked to the proto-oncogene c- met locus ( Met) in the centromeric region of mouse Chr 6, consistent with the observation that there is a conserved chromosomal segment on human chromosome 7 and mouse Chr 6.

Human cyclin B1 gene (CCNB1) assigned to chromosome 5 (q13-qter)

Cyclins play an important role in cell cycle regulation. At least five classes of cyclins have been identified--A, B, C, D, and E. B cyclins are generally of two types in most organisms--B1 and B2. We have mapped the gene for human cyclin B1 (CCNB1) to human chromosome 5 (region q13-qter) by Southern blot analysis of human x Chinese hamster somatic cell hybrid panels. Many more cyclin B-related sequences have been identified in the mouse (Cycb-1 to Cycb-10) and have been mapped to chromosomes 4, 5, 7, 8, 13, 14, 15, and 17. Based on our mapping of human CCNB1 and known evolutionary conservation of chromosomal regions, we propose that the homologous cyclin B1 locus, Cycb-4, on mouse chromosome 13 is a functional gene.

The gonadotropin-releasing hormone (Gnrh) gene maps to mouse chromosome 14 and identifies a homologous region on human chromosome 8

The murine gonadotropin-releasing hormone (Gnrh) locus has been mapped to mouse chromosome 14 using a mouse x Chinese hamster somatic cell hybrid panel. The equivalent human locus, known as luteinizing hormone-releasing hormone (LHRH), has been previously mapped to 8p21-8p11.2. Four other loci mapping to the human chromosome 8 short arm have been mapped to mouse chromosome 8; two of these (PLAT, GSR) lie proximal to LHRH, and two (LPL, DEF1) lie distal to LHRH. The localization of Gnrh, the murine homolog of LHRH, to mouse chromosome 14 therefore defines a hitherto unrecognized block of homology between man and mouse. Furthermore, it indicates that the region of homology between the human chromosome 8 short arm and mouse chromosome 8 is composed of two separate blocks.

Tumor necrosis factor receptor genes, TNFR1 and TNFR2, on human chromosomes 12 and 1.

Tumor necrosis factor, TNF, is a 17-kDa protein secreted by macrophages and classified as a cytokine. TNF binds to high-affinity receptors on the cell surface and is involved in a wide variety of biological responses. There are at least two types of receptors, tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2). The genes for TNFR1 a 55-kDa protein, and TNFR2, a 70-kDa protein, have been mapped to human chromosomes 1 12 (12pter-cen) and (1pter-p32), respectively, by Southern blot analysis of human x Chinese hamster somatic cell hybrid panels. Recently, the corresponding genes in the mouse have been mapped to chromosomes 4 and 6 in regions that are conserved on human chromosomes 1 and 12.

Structure of the human cytochrome c oxidase subunit Vb gene and chromosomal mapping of the coding gene and of seven pseudogenes

Subunit Vb of mammalian cytochrome c oxidase (COX; EC 1.9.3.1) is encoded by a nuclear gene and assembled with the other 12 COX subunits encoded in both mitochondrial and nuclear DNA. We have cloned the gene for human COX subunit Vb ( COX5B) and determined the exon-intron structure by both hybridization analysis and DNA sequencing. The gene contains five exons and four introns; the four coding exons span a region of approximately 2.4 kb. The 5′ end of the COX5B gene is GC-rich and contains many HpaII sites. Genomic Southern blot analysis of human DNA probed with the human COX Vb cDNA identified eight restriction fragments containing COX Vb-related sequences that were mapped to different chromosomes with panels of human × Chinese hamster somatic cell hybrids. Because only one of these fragments hybridized with a 210-bp probe from intron 4, we conclude that there is a single expressed gene for COX subunit Vb in the human genome. We have mapped this gene to chromosome 2, region cen-q13.

Isolation of 37 single-copy DNA probes from human chromosome 6 and physical mapping of 11 probes by in situ hybridization

Fifty-five single-copy DNA probes were isolated from the library LL06NS01, which was constructed from a complete HindIII digest of a flow-sorted human chromosome 6. Because chromosomes from a human × Chinese hamster somatic cell hybrid were used as the starting material for the flow-sorting, the library could be expected to contain some contaminating Chinese hamster DNA as well as DNA from human chromosomes other than 6. Thirty-seven of the 55 probes, however, were shown to map to human chromosome 6 by Southern blot hybridization with DNA from a panel of somatic cell hybrids. Eleven of the probes were mapped further by in situ hybridization. Four probes were localized to the short arm of chromosome 6, six to the long arm, and one to the centromeric region.

Mapping of silver fox genes: chromosomal localization of the genes for GOT2, AK1, ALDOC, ACP1, ITPA, PGP, and BLVR.

Evidence is presented for the chromosome localization of seven silver fox genes by the use of a panel of fox x Chinese hamster somatic cell hybrids. AK1, GOT2, and ALDOC are assigned to chromosome VFU2, PGP to chromosome VFU38, BLVR to chromosome VFU5, ACP1 to chromosome VFU8, and ITPA to chromosome VFU14. The genetic map of 29 fox genes is compared with those reported for man and other mammals. The results we obtained support and extend our previous suggestion that the formation of the Canidae branch of the Carnivora phylogenic tree was associated with a great increase in the rate of reorganization of the ancestral karyotype.

Mapping of the silver fox genes: assignments of the genes for ME1, ADK, PP, PEPA, GSR, MPI, and GOT1

Evidence is presented for the assignment of seven fox genes on the basis of the segregation data for chromosomes and enzymes of fox x Chinese hamster somatic cell hybrids. The chromosomal loci of the following enzyme genes were determined: ME1, VFU1; ADK and PP, VFU4; PEPA, VFU5; GSR, VFU7; and MPI and GOT1, VFU15. The localization of these genes now extends the fox genetic map to 22 mapped genes. Based on comparative analysis of mammalian genetic maps, karyotype evolution in Carnivora is discussed.

Trans-acting factors in chromosomal instability

The hypothesis that trans-acting factors affect chromosome stability was explored using human X Chinese hamster somatic cell hybrids. Two types of hybrids were examined. In either case, the human parent consisted of human diploid fibroblasts, the chromosomes of which tended to be lost from the hybrid cell. Comparisons were made between hybrid clones in which the hamster parent had a very stable karyotype (line CHO) and clones from a hamster parent with an unusual ongoing unstable karyotype (line CHX). Chinese hamster-human hybrid cell clones were expanded, and metaphase spreads were analyzed with an in situ hybridization procedure that uses biotin-labeled human genomic DNA as probe. Analyses of chromosome numbers and interspecies translocations were made after 20, 60, and 100 population doublings. Throughout the experiments, the generation of human-hamster—translocated chromosomes was more frequent in the hybrid cells with the CHX background. In addition, these cells also generated human acentric fragments, which were rare in cells with the CHO background. These results favor explanations for the instability of the CHX line that involve ongoing production of a diffusible clastogenic factor.

Regional assignment of the mouse αA2-crystallin gene (<i>Crya-</i><i>1</i>) to chromosome 17A3→B by in situ hybridization

Previously, we assigned the alpha A2-crystallin (Crya-1) structural gene to mouse chromosome 17 via Southern blot hybridization analysis of mouse x Chinese hamster somatic cell hybrids. Using in situ hybridization, we have now localized this gene to 17A3----B, a subchromosomal region containing several genes whose linkage relationships have been shown to be conserved on human chromosome 6. In man, however, the homologous gene (CRYA1) is located on human chromosome 21, indicating that internal rearrangements can occur within highly conserved chromosomal regions during the divergence of man and mouse.

Localization of the Murine Leukemia Inhibitory Factor Gene Near the Centromere on Chromosome 11

Leukemia inhibitory factor (LIF) is a glycoprotein with divergent activities: It induces the differentiation of certain myeloid leukemic cells, inhibits the differentiation of embryonic stem cells, and promotes bone remodelling in vivo and in vitro. The murine LIF gene has been assigned to the proximal region of chromosome 11 at sub-bands A1-A2, by analysis of a panel of mouse x Chinese hamster somatic cell hybrids and by in situ hybridization. Interestingly, the proximal portion of chromosome 11 has been shown, by virtue of its parental origin effects, to contain gene(s) involved in fetal growth. It is also interesting that there is a preponderance of chromosome 11 abnormalities in embryonal carcinoma cells. The localization of the murine LIF gene confirms the homology of a portion of murine chromosome 11 with human chromosome 22q, the site of the human LIF gene.

Chromosomal localization of murine interleukin-1 α and β genes

DNA analyses of mouse × Chinese hamster somatic cell hybrids and of recombinant inbred mouse strains have previously shown that the interleukin-1 α and β genes are tightly linked on murine chromosome 2, approximately 4.7 cM distal to β-2-microglobulin. In this study, using in situ chromosome hybridization, we show that the two interleukin-1 genes are located in the F region of murine chromsome 2 and discuss this physical map position in relation to conserved genetic linkage groups.

Chromosomal localization to 3q21----qter and two TaqI RFLPs of the human prostate-specific acid phosphatase gene (ACPP).

The chromosomal location of the gene encoding human prostate-specific acid phosphatase (ACPP) was determined by Southern blotting analysis of panels of human x rodent (mouse or Chinese hamster) somatic cell hybrids, using the PAP-1007 and PAP-1004EP ACPP cDNA probes. The ACPP gene was assigned to chromosome 3, which was confirmed by screening a chromosome 3-specific genomic library. Sublocalization of this gene was carried out using hybrids that had retained only various portions of human chromosome 3. The ACPP gene was found to segregate specifically with the chromosomal segment 3q21----qter. Analysis of Southern blots of TaqI-digested DNAs from unrelated individuals and members of large families from northern Finland revealed two simultaneous diallelic restriction fragment length polymorphisms (RFLPs), A and B, when using either PAP-1004EP or PAP-1006A ACPP cDNA probes, but not the 5' flanking PAP-1007 probe. Allele frequencies for polymorphism A were .09 (A1) and .91 (A2), and for polymorphism B, .38 (B1) and .62 (B2). There appears to be only a very minor linkage disequilibrium (chi 2 = 1.12, 0.35 greater than P greater than 0.25) between the two TaqI RFLPs at the ACPP locus. For reasons presently unknown, homozygotes for polymorphism B appear to be overrepresented. These polymorphisms could be of importance in characterizing human prostate cancer.

Localization of UDP glucuronosyltransferase gene(s) on mouse chromosome 5.

The drug-metabolizing UDP glucuronosyltransferases are encoded by genes which constitute a multigene family. One rat gene subfamily codes for at least four constitutive enzyme forms, including those which glucuronidate the androgenic steroids testosterone (UDPGTr-3) and androsterone (UDPGTr-4). In the present study, UDPGTr-3 and UDPGTr-4 cDNAs were used to demonstrate that an homologous subfamily is present in the mouse genome. Using mouse X Chinese hamster somatic cell hybrids, we mapped at least one gene of this UDP glucuronosyltransferase subfamily (UDPGTr-3) to mouse chromosome 5 and suggest the name as the Udpgt-3 locus.