Conservation of the syntenic group enol - pgd - pgm in mammals: its assignment to chromosome 2 in the Chinese hamster

Hybrids between cells from mouse permanent lines and Chinese hamster thymus cells explanted from animals maintained mouse chromosomes and lost most hamster chromosomes. In twenty-seven hybrids examined for expression of enolase 1. phosphogluconate dehydrogenase, and phosphoglucomutase, the Chinese hamster forms of the three enzymes were either expressed together, or not expressed at all. Thus, the three genes eno1, pgd, and pgm appear syntenic in Chinese hamster as they are in man (chromosome 1p), and in mouse (chromosome 4). The three markers map on the Chinese hamster chromosome 2.     

Regional assignment of ADA and ITPA to mouse chromosome 2 (C1----ter). A demonstration of the conserved linkage of enzyme and proto-oncogene loci

Similarity of G-band patterns between the long arm of Chinese hamster chromosome 6 and mouse chromosome 2, combined with the assignments of AK1, ADA, and ITPA to hamster chromosome 6 and AK1 to mouse chromosome 2, suggested mouse chromosome 2 also might contain ADA and ITPA. Here, concordant segregation analysis of enzyme loci and chromosomes in mouse spleen X CHO as well as mouse microcell X CHO somatic cell hybrids established the assignments of ADA and ITPA onto mouse chromosome 2 in the region between the first G-band and the terminus (C1----ter). This assignment presents a demonstration of the conservation and evolution of enzyme and proto-oncogene loci linkage since two cellular homologs of viral oncogenes--c-src and c-abl--also map to mouse chromosome 2. In humans c-src, ADA, and ITPA remain conserved on chromosome 20, whereas AK1 and c-abl are together on chromosome 9. These observations and concepts are discussed with respect to the role of proto-oncogenes in chromosomal evolution and suggest the long arm of chromosome 6 as a fruitful place to look for c-src and c-abl in the Chinese hamster.     

Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.     

Gene mapping in Mus musculus by interspecific cell hybridization: assignment of the genes for tripeptidase-1 to chromosome 10, dipeptidase-2 to chromosome 18, acid phosphatase-1 to chromosome 12, and adenylate kinase-1 to chromosome 2.

Chinese hamster X mouse somatic cell hybrids segregating mouse chromosomes were examined for their mouse chromosome content using trypsin-Giemsa (GTG) banding and Hoechst 33258 staining techniques. Simultaneously, they were scored for the presence of 24 mouse enzymes. The results confirm the assignments of 11 genes previously mapped by sexual genetics: Dip-1 and Id-1 to chromosome 1; Pgm-2 and Pgd to 4; Pgm-1 to 5; Gpi-1 to 7; Gr-1 to 8; Mpi-1 and Mod-1 to 9; Np-1 and Es-10 to 14. They also confirm chromosomally the assignments of 3 genes that were made by other somatic cell genetic studies: Aprt to 8; Hprt and alpha-gal to the X chromosome. But most importantly, four enzyme loci are assigned to four chromosomes that until now were not known to carry a biochemical marker which is expressed in cultured cells: Trip-1 to 10; Dip-2 to 18; Acp-1 to 12; and Ak-1 to 2. Cytogenetic examination of clones showing discordant segregation of HPRT and A-GAL, suggested the assignment of alpha-gal to region XE leads to XF of the mouse X chromosome. The cytologic studies provide a comparison between data from sexual genetics and somatic cell hybrids and validate hybrid cell techniques. They provide evidence of the reliability of scoring chromosomes by GTG and Hoechst staining and stress the importance of identifying clones with multiple chromosome rearrangements. Striking examples of norandom segregation of mouse chromosomes were observed in these hybrids with preferential retention of 15 and segregation of 11 and the Y chromosome.     

Comparative mapping of seven genes in mouse, rat and Chinese hamster chromosomes by fluorescence in situ hybridization

By fluorescence in situ hybridization (FISH) using mouse probes, we assigned homologues for cathepsin E (Ctse), protocadherin 10 (Pcdh10, alias OL-protocadherin, Ol-pc), protocadherin 13 (Pcdh13, alias protocadherin 2c, Pcdh2c), neuroglycan C (Cspg5) and myosin X (Myo10) genes to rat chromosomes (RNO) 13q13, 2q24-->q25, 18p12-->p11, 8q32.1 and 2q22.1-->q22.3, respectively. Similarly, homologues for mouse Ctse, Pcdh13, Cspg5 and Myo10 genes and homologues for rat Smad2 (Madh2) and Smad4 (Madh4) genes were assigned to Chinese hamster chromosomes (CGR) 5q28, 2q17, 4q26, 2p29-->p27, 2q112-->q113 and 2q112-->q113, respectively. The chromosome assignments of homologues of Ctse and Cspg5 reinforced well-known homologous relationships among mouse chromosome (MMU) 1, RNO 13 and CGR 5q, and among MMU 9, RNO 8 and CGR 4q, respectively. The chromosome locations of homologues for Madh2, Madh4 and Pcdh13 genes suggested that inversion events were involved in chromosomal rearrangements in the differentiation of MMU 18 and RNO 18, whereas most of MMU 18 is conserved as a continuous segment in CGR 2q. Furthermore, the mapping result of Myo10 and homologues suggested an orthologous segment of MMU 15, RNO 2 and CGR 2.     

Microcell-mediated cotransfer of genes specifying methotrexate resistance, emetine sensitivity, and chromate sensitivity with Chinese hamster chromosome 2.

Many selectable mutants of somatic Chinese hamster cells have been described, but very few of the mutations have been mapped to specific chromosomes. We have utilized the microcell-mediated gene transfer technique to establish the location of three selectable genetic markers on chromosome 2 of Chinese hamster. Microcells were prepared from the methotrexate-resistant MtxRIII line of Flintoff et al. (Somatic Cell Genet. 2:245-261, 1976) and fused to wild-type CHO cells, and microcell hybrids (transferants) were selected in medium containing methotrexate. All transferants were karyotyped and found to contain a marker chromosome from the donor MtxRIII line. This marker chromosome, called 2p-, consisted of a chromosome 2 with a reduced short arm resulting from a reciprocal translocation between 2p and 5q. In experiments utilizing emetine-resistant (Emtr) or chromate-resistant (Chrr) recipient cells it was found that the emt+ and chr+ wild-type genes were cotransferred with the 2p- chromosomes. Karyotype analysis of several transferants with rearranged or broken 2p- markers allowed regional localization of the emt and chr loci to the proximal third of the long arm and localization of the gene or genes conferring methotrexate resistance to the short arm. These results confirm our earlier assignment of the emt and chr loci to chromosome 2 in Chinese hamster.     

Chinese hamster x American mink somatic cell hybrids: characterization of a clone panel and assignment of the mink genes for malate dehydrogenase,NADP-1 and malate dehydrogenase,NAD-1

Summary Chinese hamster x American mink somatic cell hybrids were obtained and examined for chromosome content and expression of mink malate dehydrogenase, NADP (MOD-1; EC 1.1.1.40), malate dehydrogenase, NAD (MOR-1; EC 1.1.1.37), glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8). All the hybrid clones examined were found to segregate mink chromosomes. A clone panel containing 25 clones was set up. The possibilities and limitations of this panel for mink gene mapping are analysed. Using this panel, it is feasible to rapidly map genes located on chromosomes 1–13 and to provisionally assign genes located on chromosome 14 and the X. Based on the data obtained, the genes for MOD-1 and MOR-1 were firmly assigned to mink chromosomes 1 and 11, respectively, and the genes for G6PD and HPRT were provisionally assigned to the X.     

Genetic effects of chromosomal rearrangements in Chinese hamster ovary cells: expression and chromosomal assignment of TK, GALK, ACP1, ADA, and ITPA loci.

Polyethylene glycol-mediated fusion of Chinese hamster ovary (CHO) cells with mouse Cl1D cells produced interspecific somatic cell hybrids which slowly segregated CHO chromosomes. Cytogenetic and isozyme analysis of HAT- and bromodeoxyuridine-selected hybrid subclones and of members of a hybrid clone panel retaining different combinations of CHO chromosomes enabled provisional assignments of the following enzyme loci to CHO chromosomes: TK, GALK, and ACP1 to chromosome 7; TK and GALK to chromosome Z13; ACP1, ADA, and ITPA to chromosome Z8; and ADA and ITPA to chromosome Z9. These genetic markers reflect the origin of each of these Z group chromosomes and indicate the functional activity of alleles located on rearranged chromosomes. Identification of diploid electrophoretic shift mutations for ADA and ITPA was consistent with those observations. Assignment of the functional TK locus in TK+/- CHO-AT3-2 cells indicated that gene deletion may be responsible for TK hemizygosity in this subline.     

Chromosome assignment of four plexin A genes (Plxna1, Plxna2, Plxna3, Plxna4) in mouse, rat, Syrian hamster and Chinese hamster

We determined chromosome locations of four plexin A subfamily genes, Plxna1, Plxna2, Plxna3 and Plxna4, in four rodent species, mouse, rat, Syrian hamster and Chinese hamster, by fluorescence in situ hybridization. Plxna1, Plxna2, Plxna3 and Plxna4 were localized to Chr 6E2, 1H6, XB-C1 and 6B1 in mouse, Chr 4q34.1, 13q26-->q27, Xq37.1-->q37.2 and 4q21.3-->q22 in rat, Chr 8qb1.1-->qb1.3, 11qb8, Xpb8 and 5qb3.3 in Syrian hamster, and Chr 8q1.2, 5q3.7, Xp2.7 and 1q2.2-->q2.3 in Chinese hamster, respectively. All the mouse and rat plexin A genes were localized to chromosome regions where conserved homology has been identified among human, mouse and rat.     

Assignment of snRNA gene sequences to the large chromosomes of rat kangaroo and Chinese hamster isolated by flow cytometric sorting

Chromosomes from a rat kangaroo (Potorous tridactylus) cell line (PtK2) and from a Chinese hamster (Cricetulus griseus) cell line (CHV79) were isolated by means of fluorescence activated flow cytometric sorting. DAPI (4-6-diamino-2-phenylindole) was used as the DNA specific fluorescent dye. The karyotype of the PtK2 cells which exhibits 13 chromosomes was separated into 6, and the 22 chromosomes of the CHV79 cells were resolved into 11 fractions. DNA extracted from these chromosomal fractions was used for restriction enzyme digestion and blotting on nitrocellulose filters. The blots were challenged with gene probes corresponding to ribosomal RNA (18S and 28S) and small nuclear RNA (U1-snRNA) genes. The rRNA genes were exclusively assigned to chromosomes containing the nucleolus organizing region (in PtK2: X chromosome; in CHV79: chromosomes 4, 5, 6, and 11). — Solely the largest chromosomes in both cell lines hybridized with U1-snRNA indicating that these gene sequences are located on those chromosomes only. Further possible genetic and biochemical applications of this experimental system are discussed.     

Chinese hamster telomeres are comparable in size to mouse telomeres.

We report here the results of a telomere length analysis in four male Chinese hamsters by quantitative fluorescence in situ hybridization (Q-FISH). We were able to measure telomere length of 64 (73%) of 88 Chinese hamster telomeres. We could not measure telomere length in chromosome 10 or in the short arms of chromosomes 5, 6, 7 and 8 because of the overlaps between the interstitial and terminal telomeric signals. Our analysis in the 73% of Chinese hamster telomeres indicate that their average length is approximately 38 kb. Therefore, Chinese hamster telomeres are comparable in length to mouse telomeres, but are much longer than human telomeres. Similar to previous Q-FISH studies on human and mouse chromosomes, our results indicate that individual Chinese hamster chromosomes may have specific telomere lengths, suggesting that chromosome-specific factors may be involved in telomere length regulation.     

Synteny mapping in the bovine: genes from human chromosome 4.

Genes homologous to those located on human chromosome 4 (HSA4) were mapped in the bovine to determine regions of syntenic conservation among humans, mice, and cattle. Previous studies have shown that two homologs of genes on HSA4, PGM2 and PEPS, are located in bovine syntenic group U15 (chromosome 6). The homologous mouse genes, Pgm-1 and Pep-7, are on MMU5. Using a panel of bovine x hamster hybrid somatic cells, we have assigned homologs of 11 additional HSA4 loci to their respective bovine syntenic groups. D4S43, D4S10, QDPR, IGJ, ADH2, KIT, and IF were assigned to syntenic group U15. This syntenic arrangement is not conserved in the mouse, where D4s43, D4s10, Qdpr, and Igj are on MMU5 while Adh-2 is on MMU3. IL-2, FGB, FGG, and F11, which also reside on MMU3, were assigned to bovine syntenic group U23. These data suggest that breaks and/or fusions of ancestral chromosomes carrying these genes occurred at different places during the evolution of humans, cattle, and mice.     

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-aminopterin-thymidine (HAT) 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 alpha 1 (MCOLA1) and pro alpha 2 (MCOLA2) chains to mouse chromosome 16.     

Development of arm-specific and subtelomeric region-specific painting probes for Chinese hamster chromosomes and their utility in chromosome identification of Chinese hamster cell lines

Arm-specific and subtelomeric region-specific painting probes for Chinese hamster chromosomes have been generated by microdissection and use of the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). Fluorescence in situ hybridization (FISH) analyses using these probes demonstrated their specificity. These probes painted every chromosome arm and a total of 15 subtelomeric regions, namely, both ends of chromosomes 1, 2, 3, 4, and 8 and one end of chromosome arms 5q, 6q, 7q, 9p, and Xp. Many cryptic chromosomal rearrangements in the CHO-9 and V79 cell lines that were not detectable with whole chromosome paints could be recognized when these newly developed probes were used.     

Identification of two murine loci homologous to the v-cbl oncogene.

The virally transduced oncogene v-cbl transforms fibroblasts in vitro and induces early B-cell-lineage lymphomas in vivo. A series of probes derived from a molecular clone of v-cbl were used to map related sequences in the mouse genome. Analyses of Chinese hamster x mouse somatic-cell hybrids showed that two related genes, cbl-1 and cbl-2, were located on chromosomes 6 and 9, respectively. Restriction enzyme studies of DNA from hybrid cells containing either chromosome 6 or 9 suggested that cbl-1 resembles v-cbl and may be a processed gene, whereas cbl-2 has a complex genomic structure. Analyses of Mus domesticus/M. spretus interspecific backcross mice showed that Cbl-1 maps between the immunoglobulin kappa light chain and T-cell receptor beta chain loci and that Cbl-2 is tightly linked to Thy-1.     

Provisional assignment of TPI, GPI, and PEPD to Chinese hamster autosomes 8 and 9: a cytogenetic basis for functional haploidy of an autosomal linkage group in CHO cells

Concordant segregation analysis of Chinese hamster (Cricetulus griseus) isozymes and chromosomes segregating from interspecific somatic cell hybrids made with mouse C11D cells revealed the locations of GPI and PEPD on chromosome 9 and TPI on chromosome 8 in both euploid Chinese hamster and CHO cells. The patterns of electrophoretically detectable shift mutants of these loci in CHO cells were consistent with the observed presence of two normally banded chromosome 8's and monosomy for chromosome 9. These findings and the isolation of three independent, null PEPD mutants in only 527 ethyl methansulfonate-exposed clones indicate that the high frequency of recovery of recessive drug resistant mutants in CHO cells may be due not only to haploidy caused by deletions and monosomy but also by great sensitivity of certain loci to particular mutagens.     

Characteristics of somatic cell hybrids (mouse x Chinese hamster) with a different ratio of chromosome sets from the parent species. II. Thymidine kinase activity]

The activity of thymidine kinase (TK) was studied in series of somatic cell hybrids between the mouse cell line 3T3-4E (TK-) and Chinese hamster cells M-15-1 (HGPRT-). Four groups of hybrid lines with different ratio of parental chromosome sets have been investigated: 1) three lines containing one hamster and one mouse chromosome set (1 hs+1 ms); 2) one line with 2 hs+1 ms; 3) one line containing 3 hs+1 ms and 4) one line containing 1 hs+2 ms. Mixtures of extracts from the parental cells were shown to possess the expected TK activity. The calculation of the activity per cell revealed that the 1 hs+1 ms and 2 hs+1 ms hybrid lines possessed about 50% of the initial hamster cell TK activity. The decreased TK activity in these hybrids might be due either to a loss of hamster chromosomes or to some inhibitory effect of mouse genome in cells with the studied ratio of parental sets. The enzyme activity in the 3 hs+1 ms hybrid was as expected, about three times greater than that of hamster cells.     

Transfer of the human genes coding for thymidine kinase and galactokinase to Chinese hamster cells and human-Chinese hamster cell hybrids.

Cotransfer of two linked human genes, coding for the enzymes thymidine kinase (TK) and galactokinase (Gak) was demonstrated following incubation of Chinese hamster TK-deficient cells with isolated human chromosomes. The 5 colonies which were isolated all expressed a stable TK-positive phenotype. Cotransfer of the human genes coding for TK and Gak has also been observed in experiments in which isolated human chromosomes were incubated with TK-deficient human-Chinese hamster cell hybrids. These receipient hybrids had lost all human chromosomes at the time of incubation. From these experiments, four colonies were isolated, all expressing an unstable TK-positive phenotype. Using chromosome staining techniques, the presence of human chromosomes could not be demonstrated in either of the transformed clonal lines obtained with the Chinese hamster and the hybrid recipient cells. This indicates that incorporation of only the fragment of the human chromosome 17, bearing the genes for TK and Gak, has occurred in the recipient cells.     

A scaffold for the Chinese hamster genome

Chinese hamster ovary (CHO) cells are a prevalent tool in biological research and are among the most widely used host cell lines for production of recombinant therapeutic proteins. While research in other organisms has been revolutionized through the development of DNA sequence-based tools, the lack of comparable genomic resources for the Chinese hamster has impeded similar work in CHO cell lines. A comparative genomics approach, based upon the completely sequenced mouse genome, can facilitate genomic work in this important organism. Using chromosome synteny to define regions of conserved linkage between Chinese hamster and mouse chromosomes, a working scaffold for the Chinese hamster genome has been developed. Mapping CHO and Chinese hamster sequences to the mouse genome creates direct access to relevant information in public databases. Additionally, mapping gene expression data onto a chromosome scaffold affords the ability to interpret information in a genomic context, potentially revealing important structural and regulatory features in the Chinese hamster genome. Further development of this genomic scaffold will provide opportunities to use biomolecular tools for research in CHO cell lines today and will be an asset to future efforts to sequence the Chinese hamster genome.     

Mapping of aminoacylase-1 and beta-galactosidase-A to homologous regions of human chromosome 3 and mouse chromosome 9 suggests location of additional genes.

Conserved linkage groups have been found on the X and autosomal chromosomes in several mammalian species. The identification of conserved chromosomal regions has potential for predicting gene location in mammals, particularly in humans. The genes for human aminoacylase-1 (ACY1, N-acylamino acid aminohydrolase, E.C.3.5.1.14), an enzyme in amino acid metabolism, and beta-galactosidase-A (GLB1, E.C.3.2.1.23), deficient in GM1-gangliosidosis, have been assigned to human chromosome 3. Using human-mouse somatic cell hybrids segregating translocations of human chromosome 3, expression of both ACY1 and GLB1 correlated with the presence of the p21 leads to q21 region of chromosome 3. In a previous study, assignment of these genes to mouse chromosome 9 used mouse-Chinese hamster somatic cell hybrids, eliminating mouse chromosomes. To approximate the size of the conserved region in the mouse, experiments were performed with recombinant inbred mouse strains. An electrophoretic variant of ACY-1 in mouse strains was used to map the Acy-1 gene 10.7 map U from the beta-galactosidase locus. These data suggest that there is a region of homology within the p21 leads to q21 region of human chromosome 3 and a segment of mouse chromosome 9. Since the mouse transferrin gene (Trf) is closely linked to the aminoacylase and beta-galactosidase loci, we predict that the human transferrin (TF) gene is on chromosome 3.