Characterization and linkage mapping often sheep microsatellite markers derived from a sheep x hamster cell hybrid

A cosmid library has been constructed from a sheep x hamster cell hybrid containing sheep chromosome t₁, rob (6;24). Clones containing sheep DNA were identified by hybridizing to a total sheep genomic DNA probe. Small fragments (<500 bp) containing (AC)_n microsatellites were subcloned and sequenced. Ten microsatellite markers were characterized and six were mapped back to chromosomes 6 and 24. The remaining microsatellites mapped to chromosome 26, which was shown to be present in a small proportion of cells of the cell line.     

Simultaneous activity of nucleolus organizer regions of human and Chinese hamster chromosomes in somatic cell hybrids

In an interspecific human-Chinese hamster hybrid that retains 13 and 85.6% of the chromosomes of each parental complement, activity of nucleolus-organizing regions (NOR) of both type chromosomes is observed in 18.9% of the cells. Interspecific chromosomal associations are also noted. Unlike the parental lines of Chinese hamster cells, the hybrids show the associations of the NOR of Chinese hamster chromosomes. In hybrid cells, there occurs partial suppression of NOR activity in human and Chinese hamster chromosomes, while the NOR of the 3d chromosome of the Chinese hamster is completely suppressed.     

THE RIBOSOMAL RNA OF HAMSTER-MOUSE HYBRID CELLS

The ribosomal RNA (rRNA) of a series of hamster-mouse somatic cell hybrids was studied. Mouse 28S rRNA was separated from its hamster counterpart by a two-step procedure involving sucrose gradient centrifugation of ribosomes and polyacrylamide gel electrophoresis of rRNA. Both hamster and mouse types of rRNA were synthesized in the 11 hybrids tested, including hybrids containing only about one-half the haploid number of either mouse or hamster chromosomes. It appears that, for both hamster and mouse rRNA, when the chromosomes of one species constituted the majority of the chromosomes of a hybrid, a disproportionately higher percentage of rRNA of that species was present in the hybrid. Some hybrid clones, having a majority of mouse chromosomes, had a mouse rRNA cell concentration approximately four to five times higher than the concentration expected from linear extrapolation of the value found for the mouse parental cell line.     

Human-hamster hybrid cells used as models to investigate species-specific factors modulating the efficiency of repair of UV-induced DNA damage

The human-Chinese hamster hybrid cell line XR-C1#8, containing human chromosome 8, was used as a model system to investigate the relative importance of cellular enzymatic environment and chromosomal structure for modulating the efficiency of repair of UV-induced DNA damage. The hybrid cells were irradiated with UVC light and the extent of cytogenetic damage, detected as frequencies of sister chromatid exchanges (SCEs), was compared between the human and the hamster chromosomes. The combination of immunofluorescent staining for SCEs and chromosome painting with fluorescence in situ hybridization allowed the simultaneous analysis of SCEs in the human and hamster chromosomes. The aim of the present study was to determine if the differences in biological response to comparable UV treatments observed between human and hamster cells were maintained in the hybrid cells in which human and hamster chromosomes are exposed in the same cellular environment. The analysis of replication time of human chromosome 8 indicated the active status of this chromosome in XR-C1#8 hybrid cells. The frequencies of SCEs for human chromosome 8 and a hamster chromosome of comparable size were 0.35 +/- 0.52, 0.80 +/- 0.73, 1.24 +/- 2.24 and 0.36 +/- 0.12, 0.71 +/- 0.2, 0.97 +/- 0.27, respectively, after irradiation with 0, 5, and 10 J/m2. The persistence of UV-induced SCEs after three cell cycles was also analyzed, both for the human and hamster chromosomes. The observed frequencies of SCEs were 0.40 +/- 0.57, 0.62 +/- 1.05, 0.58 +/- 0.83 and 0.26 +/- 0.08, 0.67 +/- 0.18, 0.69 +/- 0.24, in human and hamster chromosomes respectively, after treatment with 0, 10, and 20 J/m2 of UVC light. No significant differences could be observed between the human and hamster chromosomes. These results suggest that the enzymatic environment of human and hamster cells has the main role, in comparison to the structural organization of human and hamster chromosomes, for determining the different level of repair of UV-induced DNA damage observed in these two species.     

Chromosomal distribution of endogenous Jaagsiekte sheep retrovirus proviral sequences in the sheep genome

A family of endogenous retroviruses (enJSRV) closely related to Jaagsiekte sheep retrovirus (JSRV) is ubiquitous in domestic and wild sheep and goats. Southern blot hybridization studies indicate that there is little active replication or movement of the enJSRV proviruses in these species. Two approaches were used to investigate the distribution of proviral loci in the sheep genome. Fluorescence in situ hybridization (FISH) to metaphase chromosome spreads using viral DNA probes was used to detect loci on chromosomes. Hybridization signals were reproducibly detected on seven sheep chromosomes and eight goat chromosomes in seven cell lines. In addition, a panel of 30 sheep-hamster hybrid cell lines, each of which carries one or more sheep chromosomes and which collectively contain the whole sheep genome, was examined for enJSRV sequences. DNA from each of the lines was used as a template for PCR with JSRV gag-specific primers. A PCR product was amplified from 27 of the hybrid lines, indicating that JSRV gag sequences are found on at least 15 of the 28 sheep chromosomes, including those identified by FISH. Thus, enJSRV proviruses are essentially randomly distributed among the chromosomes of sheep and goats. FISH and/or Southern blot hybridization on DNA from several of the sheep-hamster hybrid cell lines suggests that loci containing multiple copies of enJSRV are present on chromosomes 6 and 9. The origin and functional significance of these arrays is not known.     

Chromosomal localization of the Moloney sarcoma virus mouse cellular (c-mos) sequence.

The Moloney sarcoma virus-specific onc gene, referred to as v-mos, was used as probe to hybridize to restricted DNAs from various mouse-Chinese hamster hybrid cell lines. These hybrid cells contain, in addition to all of the Chinese hamster chromosomes, various numbers (less than a full complement) of mouse chromosomes. Comparison of the presence or absence of the mouse cellular mos gene with the known karyotype in each of the hybrid cell lines allows us to conclude that the mos gene is on mouse chromosome 4.     

Expression of differentiated functions in hepatoma cell hybrids: IX extinction and reexpression of liver-specific enzymes in rat hepatoma-Chinese hamster fibroblast hybrids.

Most of the hybrid clones derived from a cross of Chinese hamster fibroblasts (DON) with rat hepatoma cells (Faza 967) showed preferential loss of rat chromosomes. Two of the hybrid clones retained the rat chromosomes, and both showed extinction of 4 liver-specific enzymes: aldolase B, liver alcohol dehydrogenase, and the inducible enzymes tyrosine aminotransferase and alanine aminotransferase. Subcloning of 1 of these hybrids, which contained 2 sets of hepatoma chromosomes and 1 set of hamster chromosomes, permitted the isolation of some clones which reexpressed 1 or more of the liver-specific enzymes. Liver alcohol dehydrogenase was the most frequently reexpressed enzyme and aldolase B the least. Tyrosine aminotransferase inducibility was reexpressed independently of basal activity, and the enzyme produced by the reexpressing hybrid cells was precipitated by a specific antiserum. No correlation was detected between the presence or absence of the marker chromosomes (large metacentrics) of the hamster parent and the extinction and reexpression of the hepatic enzymes. The results reported confirm and extend to interspecific hybrids the observation of the stable and independent reexpression of tissue-specific enzymes.     

Relationship between the ratio of parental chromosomes and parental doubling times in Chinese hamster-mouse somatic cell hybrids

Somatic hybrids obtained by the selective method of Littlefield between a permanent line of Chinese hamster cells (Wg3) and one of mouse cells (3TP) showed a preponderance of biarmed (hamster) chromosomes. Under normal culture conditions (37°) the doubling time of the parental mouse cells was twice as long as that of the parental hamster cells. If the temperature of incubation was lowered (31°), the relative difference in doubling times was reduced; in hybrid lines obtained under these conditions, the proportion of biarmed chromosomes was also reduced. Upon extended cultivation the average number of telocentric chromosomes progressively decreased in all hybrid lines tested, regardless whether these were started and maintained at 37° or at 31°. An inverse correlation was observed in hybrid cells between doubling time and relative proportion of biarmed chromosomes, suggesting that the karyotypic changes observed after extended culture were due to the selective overgrowth of cells with a high biarmed to telocentric ratio.     

Esterase gene expression in Chinese hamster and mouse lymphoma hybrids isolated under nonselective pressure

Hybrids between a fibroblastic Chinese hamster cell line (CH23) and a mouse lymphoma cell line (P388F36) were produced and isolated by a simple new method without using selective media and avoiding contact with the parental cells. The chromosomal situation in the two hybrid types (PCM and PCS) isolated suggested that growth on glass surface (PCM) or in suspension (PCS) depended on the number of hamster and mouse chromosomes which existed in the hybrids. Chromosomal stability in hybrids grown as monolayers (PCM) was reached at a stage in which two to four mouse chromosomes coexisted with no fewer than 19 hamster chromosomes. In a study of gene linkage utilizing clones of this hybrid population, five out of nine genes regulating the synthesis of different esterases in the mouse cells used were found to be unlinked.     

Chromosomally depleted interspecific hybrid cell clones selected with cytotoxic antisera: Utilization in the study of control of murine leukemia virus host-range

A chromosomally stable mouse-Chinese hamster hybrid cell line was subjected to five rounds of selection with cytotoxic antisera raised in rabbits against either the parental mouse 3T3 cells or the parental Chinese hamster Wg-1 cells. Routine karyological analysis of clones isolated at each stage of serum selection revealed that treatment with either serum resulted in a limited loss of chromosomes (compared to the untreated hybrid cell cultured in parallel) and that the pattern of chromosome loss could not be correlated with the particular antiserum used for selection. However, more detailed analysis with the SSC-formamide C-banding technique, which identifies chromosomes containing a mouse centromere region, demonstrated that while large-scale chromosome loss was not achieved as a result of antiserum selection, the limited loss of chromosomes did, in fact, reflect a specific depletion of chromosomes in response to treatment with cytotoxic antiserum. Specific chromosomal elimination was shown to occur as early as the first round of antiserum treatment. Antigenic analysis of the serum-selected clones revealed a quantitative decrease in the expression of the species-specific surface antigens selected against, but no qualitative loss of antigens was detected. The results suggest that treatment with cytotoxic antiserum may select for clones that have lost specific chromosomes bearing genes regulating the expression of species-specific surface antigens, rather than for those demonstrating large-scale depletion of chromosomes bearing the corresponding structural genes. Some of these chromosomally depleted hybrid cell clones have been used (along with pseudotype viruses containing the genome of vesicular stomatitis virus within the envelope of murine leukemia virus, VSV [MuLV]), to study the mechanisms regulating MuLV replication in Chinese hamster cells. The results indicate that the restriction of MuLV replication in Chinese hamster cells operates at two levels: (a) an inability to adsorb to or penetrate Chinese hamster cells; and (b) an additional intracellular block which is dominant in the mouse-Chinese hamster hybrid cell clones examined. This latter block is presently under study.     

Insertion of muntjac gene segment into hamster cells by cell fusion

Indian muntjac diploid cells that have only three pairs of easily discernible large chromosomes were fused with hamster cells deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) using polyethylene glycol. Cells that survived in hypoxanthine-aminopterine-thymidine (HAT)-oubaine medium were analyzed. Hybrid cells containing both muntjac and hamster chromosomes in a given cell were not found. Instead, the cells had the same chromosomal sets as those of either parental muntjac or hamster cells. A clonal isolate that had the same chromosomal sets as those of parental hamster cells was analyzed in detail and showed the following characteristics: (1) portions of the survival curve in various concentrations of HAT medium were intermediate between those of parental cells; (2) expressions of both muntjac and hamster antigen(s) were detected by immunofluorescence staining; (3) the mobility of the enzyme HGPRT in gel electrophoresis differed from that of parental hamster or muntjac cells. These results indicate that the clonal isolate (AD202h) is a somatic cell hybrid of hamster and muntjac that contains chromosomal sets of hamster with an inserted segment of the muntjac genome, including HGPRT. The formation of such an unusal hybrid and a possible explanation of transfer of some gene segments in the hybrid cell in this system are discussed.     

Sorting of chromosomes by magnetic separation

Summary Chromosomes were isolated from Chinese hamster x human hybrid cell lines containing four and nine human chromosomes. Human genomic DNA was biotinylated by nick translation and used to label the human chromosomes by in situ hybridization in suspension. Streptavidin was covalently coupled to the surface of magnetic beads and these were incubated with the hybridized chromosomes. The human chromosomes were bound to the magnetic beads through the strong biotin-streptavidin complex and then rapidly separated from nonlabeled Chinese hamster chromosomes by a simple permanent magnet. The hybridization was visualized by additional binding of avidin-FITC (fluorescein) to the unoccupied biotinylated human DNA bound to the human chromosomes. After magnetic separation, up to 98% of the individual chromosomes attached to magnetic beads were classified as human chromosomes by fluorescence microscopy.     

Association of human chromosome 14 with a ts defect in G1 of Chinese hamster K12 cells.

Somatic cell hybrids between human lymphoblastoid cells (Raji) and temperature-sensitive Chinese hamster cells (K12) were selected from monolayer cultures in MEM at 40 degrees C. A total of 21 hybrid clones were isolated and karyotyped. All clones contained a near complete set of Chinese hamster chromosomes and 1 to 5 human chromosomes. Human chromosome 14 present in the hybrid cells of all clones; and was the only human chromosome retained in 10 clones. The presence of human chromosome 14 in hybrids was further confirmed by the demonstration of human nucleoside phosphorylase activity in the hybrid cells. Only one hybrid clone was positive for EBNA, the Epstein-Barr virus antigen present in Raji cells. These findings indicate that human chromosome 14 contains the necessary information for the K12 cells to overcome their G1 defect in the cell cycle and grow at non-permissive temperature. The present study lends strong support to the possibility that different steps in the G1 phase of the cell cycle are controlled by genes located on different chromosomes.     

A method for nucleic acid hybridization to isolated chromosomes in suspension

Summary A procedure was developed to provide differential fluorescent staining of metaphase chromosomes in suspension following nucleic acid hybridization. For this purpose metaphase chromosomes were isolated from a Chinese hamster x human hybrid cell line. After hybridization with biotinylated human genomic DNA, the human chromosomes were visualized by indirect immunofluorescence using antibodies against biotin and fluoresceine-isothiocyanate-(FITC)-labeled second antibodies. This resulted in green fluorescent human chromosomes. In contrast, Chinese hamster chromosomes revealed red fluorescent staining only when counterstained with propidium iodide. Notably, interspecies chromosomal rearrangements could be easily detected. After hybridization and fluorescent staining, chromosomes still showed a well-preserved morphology under the light microscope. We suggest that this procedure may have a useful application in flow cytometry and sorting.     

Preparation of monosomal lines containing individual human 15, 21, and X chromosomes]

Sorting of human--mouse or human--hamster hybrid cells with particular human chromosomes was performed by in situ hybridization. Total human genomic DNA was heavily labelled with. H and hybridized to metaphase spreads from hybrid clone cells. The method allowed us to not only identify human chromosomes in hybrid cells but also to detect terminal translocations and insertions from 1-2 bands in length to large ones. Biochemical markers of some human chromosomes were analysed using electrophoretic technique in the clones selected. Cytogenetic analysis (G staining) of these clones was made to visualize human chromosomes. Total 99 initial hybrid human--hamster and 26 human--mouse clones were obtained. 53 clones were analysed by in situ hybridization, only one of them being monochromosomal; the latter contained human X chromosome on the background of Chinese hamster chromosomes. Two other monochromosomal clones containing particular 15 and 21 chromosomes, respectively, were obtained by more complicated way from human--mouse hybrid clones using back selection, repeated hybridization and passing through a number of subsequent subclonings.     

Characteristics of somatic cell hybrids (mouse X Chinese hamster) with different ratios of parental species chromosome sets. IV. Electrophoretic analysis of several enzymes of the dehydrogenase class]

Electrophoretic mobilities in polyacrylamide gel of five dehydrogenases: NADP-dependent malate dehydrogenase (NADP-MDH), 6-phosphogluconate dehydrogenase (6PGD), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) were investigated in a series of mouse X Chinese hamster somatic cell hybrids. Seven hybrid lines with different ratio of chromosome sets of hamster and mouse: 1:1, 2:1, 3:1 and 1:2 respectively were studied. NADP-MDH and 6PGD of both parental species and intermediate hybrid bands were present in all hybrids except two lines. These lines had only hamster MDH due to the elimination of mouse chromosomes. A correlation was found between the gene dose and the intensity of the expression of the MDH bands. The mouse type ADH was detected in all hybrids. The hamster ADH was found in one of the hybrid lines that lost all mouse chromosomes during cultivation. It is suggested that hamster ADH activity was suppressed in hybrids by the mouse genome. The species origin of GDH and G6PD could not be established due to similarity of electrophoretic mobilities of respective enzymes in parental cells.     

Regional characterization of a hamster–sheep somatic cell hybrid panel

The regional characterization of a previously obtained hamster–sheep hybrid panel is reported. Using data available from ruminant maps (sheep, cattle, and goat), we have selected a set of 300 markers and have analyzed them by PCR in this hybrid panel. Results obtained for 204 markers show the presence of all sheep chromosomes (including gonosomes) in entire or fragmented form. Analysis of syntenies has given 130 types of answer defining segments of variable sizes. This study has led to the regional characterization of this panel and provides comparative data on a set of bovine and caprine markers. With the level of characterization now achieved for this hybrid panel, the regional assignment of new genes or markers to sheep chromosomes can be rapidly obtained. Finally, this panel will help to collect new data for comparative mapping of domestic animals and to highlight the conservation of syntenic groups between closely related species, that is, sheep, cattle, and goat. Received: 14 May 1999 / Accepted: 23 August 1999     

Construction of microcell hybrid clones containing specific mouse chromosomes: application to autosomes 8 and 17.

Fibroblast cultures prepared from mice homozygous for a Robertsonian translocation (centric fusion) between autosomes 8 and 17 [Rb(8.17)] were used as donors in microcell-mediated chromosome transfer experiments. By using hamster recipient cells deficient in adenine phosphoribosyltransferase (APRT-) and selecting for expression of murine APRT (a chromosome 8 marker), microcell hybrids were isolated which retained only the mouse Rb(8.17) translocation in addition to the hamster chromosome complement. The translocation was stable in cells maintained under APRT+ selective pressure, and mouse marker traits encoded by genes on both chromosomes 8 and 17 segregated concordantly. A second family of hybrid clones was constructed by fusing microcells derived from wild-type mouse fibroblasts with APRT- hamster cells. Four of six clones analyzed retained only mouse chromosome 8. These studies demonstrated that microcell hybrids containing specific Robertsonian translocations as the only donor-derived genetic material can be obtained. Furthermore, a number of Robertsonian translocations between chromosomes which carry selectable markers (chromosomes 3, 8, and 11) and other autosomes have been described. By using fibroblast cultures prepared from mice containing these translocations as donors in microcell fusions, 18 of the 20 mouse chromosomes could be selectively fixed in different hybrid clones. Thus, a collection of 20 hybrid clones, each containing a single, specific mouse chromosome, can be constructed by using the strategy described in this report. The potential utility of such a monochromosomal hybrid panel is discussed.     

Cytogenetic characteristics of 26 polyethylene glycol-induced human-hamster hybrid cell lines

A cytological analysis of 26 polyethylene glycol (PEG) induced human/hamster hybrid lines has shown that such lines are similar to inactivated Sendai virus (ISV) induced hybrids in respect to stability, retention of specific chromosomes, and cell selection. The evolution of stable hybrid cell lines carrying variable human chromosome complements depends upon a balance being established between the retained human and hamster genomes. This balance is a result of random loss of human and hamster chromosomes followed by selection of the fittest stem lines. A major mechanism ofchromosome loss may be fragmentation and elimination of acentric fragments. Twelve of the 26 lines had stabilized by the 30th passage, an incidence similar to that found with ISV-induced hybrids studied in this laboratory. Thus, PEG may be considered to be an ideal chemical for inducing somatic cell hybrids for genetic analysis.     

Cytogenetical anchoring of sheep linkage map and syntenic groups using a sheep BAC library

In order to simultaneously integrate linkage and syntenic groups to the ovine chromosomal map, a sheep bacterial artificial chromosome (BAC) library was screened with previously assigned microsatellites using a sheep-hamster hybrid panel and genetic linkage. Thirty-three BACs were obtained, fluorescently labelled and hybridised on sheep-goat hybrid metaphases (2n = 57). This study allowed us, (i), to anchor all linkage groups on sheep chromosomes, (ii), to give information on the probable position of the centromere on the linkage map for the centromeric chromosomes, (iii), to contradict the previous orientation of the ovine × linkage group by the mapping of BMS1008 on OARXq38. Concerning our somatic cell hybrid panel, this study resulted in the assignment of all the previously unassigned groups to ovine chromosomes and a complete characterisation of the hybrid panel. In addition, since hybridisations were performed on a sheep-goat hybrid, new marker/anchoring points were added to the caprine cytogenetic map.