Comparative study of pig-rodent somatic cell hybrids

The pig chromosome complement of six different types of pig-rodent hybrid cell lines was examined by means of fluorescence in situ hybridization with a porcine SINE probe. The cell lines were obtained by fusing pig lymphocytes with cells of the Chinese hamster cell lines wg3h, BK14-150 and E36, and of the mouse cell lines NSO, PU and LMTK^-. The hybrids were analysed with respect to: (1) the number of pig chromosomes, (2) the type of pig chromosomes, (3) the occurrence of pig-rodent chromosome trans-locations, and (4) the presence of pig chromsome fragments. The results show that the number of pig chromosomes varied within and among hybrid cell lines. The pig-hamster hybrids mainly retained nontelocentric pig chromosomes, whereas the pig-mouse hybrids also retained telocentric pig chromosomes. Pig-rodent chromosome translocations were found in all types of hybrids, but the incidence was in general low. Chromosome fragments were abundant in BK14-150 hybrids, and rare in most other hybrid cell lines. It is concluded that the SINE probe is a useful tool to make a preliminary characterization of the porcine chromosome complement of pig-rodent somatic cell hybrids. The results of this characterization can be used to select hybrids for further cytogenetic analysis. Furthermore, our data show that different rodent cell lines will have to be used as fusion partners for the production of hybrids when constructing a panel informative for all pig chromosomes.     

Embryonic stem cells can be used to construct hybrid cell lines containing a single, selectable murine chromosome

Microcell-mediated chromosome transfer is a useful technique for the study of gene function, gene regulation, gene mapping, and functional cloning in mammalian cells. Complete panels of donor cell lines, each containing a different human chromosome, have been developed. These donor cell lines contain a single human chromosome marked with a dominant selectable gene in a rodent cell background. However, a similar panel does not exist for murine chromosomes. To produce mouse monochromosomal donor hybrids, we have utilized embryonic stem (ES) cells with targeted gene disruptions of known chromosomal location as starting material. ES cells with mutations in aprt, fyn, and myc were utilized to generate monochromosomal hybrids with neomycin phosphotransferase-marked murine Chr 8, 10, or 15 respectively in a hamster or rat background. This same methodology can be used to generate a complete panel of marked mouse chromosomes for somatic cell genetic experimentaion. Received: 28 July 1998 / Accepted: 15 December 1998     

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.     

Segregation of the Huntington disease region of human chromosome 4 in a somatic cell hybrid

We have developed an X-irradiation:cell fusion procedure that segregates segments of human chromosomes lacking selectable markers and have used this approach to construct somatic cell hybrids retaining fragments of human chromosome 4 as the only human material. To identify hybrids retaining a small chromosomal fragment in the region of the Huntington disease (HD) gene, we used Southern blot analysis to screen 72 hybrid lines for the presence or absence of seven chromosome 4 single-copy loci. These data, combined with in situ hybridization experiments, identified three hybrids of interest. One of these cell lines, C25, stably retains a 10,000- to 20,000-kb fragment of distal 4p in the vicinity of the HD gene, translocated to a hamster chromosome. Field-inversion gel electrophoresis revealed no evidence of rearrangements in the human DNA present in C25. In combination with similar radiation hybrids, C25 is a valuable tool for isolating DNA probes near the HD gene.     

Analyses of deoxycytidylate deaminase molecular forms in human-mouse and monkey-mouse somatic cell hybrids

Disc polyacrylamide gel electrophoresis (disc PAGE) analyses have revealed that mouse, human, and monkey cytosol deoxycytidylate (dCMP) deaminases differ in electrophoretic mobility, so that mixtures of mouse and human, mouse and monkey, and human and monkey enzymes can be separated. To learn whether the genes for dCMP deaminase and thymidine (dT) kinase are genetically linked, disc PAGE analyses of cytosol fractions from human-mouse and monkey-mouse somatic cell hybrids were carried out. The interspecific somatic cell hybrids were derived from the fusion of cytosol dT kinase deficient mouse cells with cytosol dT kinase-positive human and monkey cells: they contained mostly mouse chromosomes and a few primate chromosomes, including the determinant for primate cytosol dT kinase. The disc PAGE analyses demonstrated that the human-mouse and monkey-mouse somatic cell hybrids contained a dCMP deaminase activity with an electrophoretic mobility characteristic of mouse dCMP deaminase. Enzymes with electrophoretic mobilities characteristic of human and monkey dCMP deaminases were not demonstrable. These findings suggest that primate cytosol dT kinase and dCMP deaminase are coded on different chromosomes, or that the formation in hybrid cells of an active primate dCMP deaminase is suppressed. Chick-mouse somatic cell hybrids containing chick but not mouse cytosol dT kinase were also analyzed. The chick-mouse hybrid cells contained cytosol dCMP deaminase activity, but it was not possible to establish whether the enzyme was of murine or avian origin because of the similarity in electrophoretic mobility between the chick and mouse enzymes. Human and mouse cells contained low levels of mitochondrial dCMP deaminase activity. In contrast to dT kinase isozymes, however, mitochondrial and cytosol dCMP deaminases were electrophoretically indistinguishable.This investigation was aided by Grant Q-163 from the Robert A. Welch Foundation and by USPHS Grants CA-06656-12 and 1-K6-AI 2352 from the National Cancer Institute and the National Institute of Allergy and Infectious Diseases.     

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.     

Somatic hybrids between Solanum etuberosum and diploid,tuber bearing Solanum clones

Electrofusion was used to obtain somatic hybrids between Solanum etuberosum (2n=2x=24) and two diploid potato lines. These hybridizations were conducted to determine if haploidxwild species hybrids are better fusion partners than conventional S. tuberosumGp. Tuberosum haploids. Restriction fragment length polymerase (RFLP) analyses of the putative somatic hybrids confirmed that each parental genome was present. The somatic hybrids between S. etuberosum and a haploid S. tuberosum clone, US-W730, were stunted and had curled, purple leaves. In contrast, somatic hybrids between S. etuberosum and a haploidxwild species hybrid (US-W 730 haploidx S. berthaultii), were vigorous and generally tuberized under field conditions. These hybrids were designated as E+BT somatic hybrids. Analyses of 23 E+BT somatic hybrids revealed a statistically significant bias towards the retention of S. etuberosum chloroplasts. Stylar incompatibilities were observed when the E+BT somatic hybrids were used as pollen donors in crosses with S. tuberosum cultivars. Reciprocal crosses did not show this incompatibility. The progeny were vigorous and had improved tuber traits when compared to the maternal E+BT parent. RFLP analyses of three sexual progeny lines confirmed the presence of all 12 S. etuberosum chromosomes. In two of these lines, RFLPs that marked each of the 24 chromosome arms of S. etuberosum were present. However, RFLP markers specific for regions on chromosomes 2, 7, and 11 were missing from the third clone. Because other markers for these chromosomes were present in the progeny line, these results indicated the likelihood of pairing and recombination between S. etuberosum and S. tuberosum chromosomes.     

Assignment of adenosine deaminase complexing protein (ADCP) gene(s) to human chromosome 2 in rodent-human somatic cell hybrids

Summary The experiments reported in this paper indicate that the expression of human adenosine deaminase complexing protein (ADCP) in the human-rodent somatic cell hybrids is influenced by the state of confluency of the cells and the background rodent genome. Thus, the complement of the L-cell derived A9 or B82 mouse parent apparently prevents the expression of human ADCP in the interspecific somatic cell hybrids. In the a3, E36, or RAG hybrids the human ADCP expression was not prevented by the rodent genome and was found to be proportional to the degree of confluency of the cell in the culture as in the case of primary human fibroblasts.An analysis of human chromosomes, chromosome specific enzyme markers, and ADCP in a panel of rodent-human somatic cell hybrids optimally maintained and harvested at full confluency has shown that the expression of human ADCP in the mouse (RAG)-human as well as in the hamster (E36 or a3)-human hybrids is determined by a gene(s) in human chromosome 2 and that neither chromosome 6 nor any other of the chromosomes of man carry any gene(s) involved in the formation of human ADCP at least in the Chinese hamster-human hybrids. A series of rodent-human hybrid clones exhibiting a mitotic separation of IDH1 and MDH1 indicated that ADCP is most probably situated between corresponding loci in human chromosome 2.A part of the results was presented at the Fifth International Conference on Human Gene Mapping, Edinburgh, July 1979 and reported as an abstract in the proceedings of this conference [Cytogenet Cell Genet 25:164 (1979)]     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

The use of cell surface antigens to characterize and select for fragments of human chromosomes retained by interspecies hybrids

We have used a mouse cell transformant generated by human chromosome-mediated gene transfer (CMGT) to explore the use of cell surface antigens in the identification of fragments of human chromosomes retained by somatic cell hybrids. The transformed line, 21-30b, contained an intact rear-ranged human chromosome, and could be shown by isozyme analysis to contain genetic material from chromosomes 9 and X. By using the transformant as an immunogen in mice, it was also possible to produce antiserum to human-specific surface antigens. Using genetically characterized human X rodent hybrid lines, the genes controlling expression of these antigens could be localized to 11per----11p13, segregating concordantly with surface antigen S3. These conclusions were possible despite the fact that the presence of chromosome 11 in the transformant was not detectable by the presence of chromosome specific isozyme LDH-A or surface antigens W6/34 and 4F2. Finally, the fluorescence-activated cell sorter (FACS) was used to fractionate the transformant cells into antigen positive and negative subpopulations. This resulted in the isolation and characterization of four additional chromosome rearrangements involving interspecies chromosome translocations. This work demonstrates the value of chromosome-specific surface antigens and the FACS in the evaluation of human chromosome fragments retained by interspecies hybrids.     

Somatic cell mapping of the human cyclophilin B gene (PPIB) to chromosome 15.

The polymerase chain reaction (PCR) technique was used to generate a unique probe complementary to the hydrophobic 5' end of the human cyclophilin B gene. This unique probe was hybridized to DNAs from human x hamster hybrid somatic cell lines retaining different combinations of human chromosomes. The gene was assigned to chromosome 15.     

Intertribal hybrid cell lines of Atropa belladonna (X) Nicotiana chinensis obtained by cloning individual protoplast fusion products

Summary After fusion of isolated mesophyll protoplasts of belladonna (Atropa belladonna) with callus protoplasts of Chinese tobacco (Nicotiana chinensis) followed by mechanical isolation and cloning of individual heteroplasmic fusion products, 13 cell clones were obtained. The hybrid nature of most of the clones has been confirmed by biochemical (studies of amylase isozymes), cytogenetic (size and morphology of chromosomes) and physiological (peculiarities of cell-growth in vitro) analyses. Study of chromosomes and isozyme patterns in the hybrid cell lines revealed the presence of both parental genomes, without an indication of chromosome elimination, six months after hybridization. In 4 cell lines shootlike structures and plantlets have been produced by means of transfer to organogenesis-inducing media. The data obtained are interpreted as new evidence for the possibility of using non-sexual hybridization for the production of intergeneric, intertribal plant hybrids which cannot be obtained by sexual crossing. From these results the potential of Atropa (X) Nicotiana hybrids as a model system for genetic studies of distantly related plant species is discussed.This work is part of a joint project between Institute of Botany of the Ukrainian Academy of Sciences, Kiev, USSR, and Institute of Pharmaceutical Biology, University of Munich, FRG     

Regional assignment of the human acid sphingomyelinase gene (SMPD1) by PCR analysis of somatic cell hybrids and in situ hybridization to 11p15.1----p15.4

Human acid sphingomyelinase (SMPD1) is the lysosomal phosphodiesterase that cleaves sphingomyelin to ceramide and phosphocholine. The deficient activity of SMPD1 is the enzymatic defect in Types A and B Niemann-Pick disease. Previously, the gene encoding human SMPD1 was assigned to chromosome 17 by the differential thermostability of human and hamster SMPD1 in somatic cell hybrids. The recent isolation of the human SMPD1 cDNA (L. E. Quintern, E. H. Schuchman, O. Levran, M. Suchi, K. Ferlinz, H. Reinke, K. Sandhoff, and R. J. Desnick, 1989, EMBO J. 8: 2469-2473) permitted the mapping of this gene by molecular techniques. Oligonucleotide primers were synthesized to PCR amplify the human, but not murine, SMPD1 sequences in man-mouse somatic cell hybrids. In a panel of 15 hybrid cell lines, amplification of the human SMPD1 sequence was 100% concordant with the presence of human chromosome 11. For each of the other human chromosomes there were at least 6 discordant hybrid lines. Further analysis of somatic cell hybrids containing only chromosome 11 or chromosome 11 rearrangements localized the human SMPD1 gene to the region 11p15.1----p15.4. To provide an independent regional gene assignment, in situ hybridization was performed using the radiolabeled human SMPD1 cDNA. In the 58 metaphase cells examined, 34% of the 122 hybridization sites scored were located in the distal end of chromosome 11 with the major peak of hybridization at band 11p15. The absence of any other in situ hybridization site indicated the absence of pseudogenes or homologous sequences elsewhere in the genome. In contrast to the previous provisional localization to chromosome 17, these results assign a single locus for human SMPD1 to 11p15.1----p15.4.     

Isolation of a human chromosome 14-only somatic cell hybrid: analysis using Alu and LINE-based PCR.

Interspecific somatic cell hybrids containing single human chromosomes are valuable reagents for localization of cloned genes and DNA fragments to specific chromosomes, for the development of chromosome-specific libraries, and for generation of hybrid cell lines containing subchromosomal regions. A CHO somatic cell hybrid containing a single, intact human chromosome 14 (MHR14) was developed and confirmed by LINE PCR amplification gel pattern, by Alu-517 PCR product dot blot hybridization, and by cytogenetic analysis. MHR14 will serve as the chromosome source for the development of a radiation map of human chromosome 14.     

Rearrangements in the mitochondrial genome of somatic hybrid cell lines of Pennisetum americanum (L.) K. Schum. + Panicum maximum Jacq.

Summary Mitochondrial DNA from three somatic hybrid cell lines of Pennisetum americanum + Panicum maximum was compared with mitochondrial DNA of the parents. Gel electrophoresis of BamHI-restricted mitochondrial DNA indicated that extensive rearrangements had occurred in each of the three hybrid lines. The hybrid restriction patterns showed a combination of some bands from each parent plus novel fragments not present in either parent. Additional evidence for rearrangements was obtained by hybridization of eight DNA probes, carrying sequences of known coding regions, to Southern blots. Each of the somatic hybrids exhibited a partial combination of the parental mitochondrial genomes. These data suggest recombination or amplification of the mitochondrial genomes in the somatic hybrids.     

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.     

Linkage relationship between the genes for thymidine kinase and galactokinase in different primates.

In this study we investigated the expression of primate galactokinase in somatic cell hybrids between a thymidine kinase-deficient mouse cell line and two different primate cell lines, one of which was derived from African green monkey kidney cells and the other from chimpanzee fibroblasts. All the African green monkey-mouse hybrid clones, selected in HAT medium, expressed monkey galactokinase activity and contained a monkey chromosome similar to a human E-group chromosome. When these clones were backselected in medium containing 5-bromodeoxyuridine, both this chromosome and the monkey galactokinase activity were lost. All the hybrid clones between mouse and chimpanzee cells, which were selected in HAT medium, contained the chimpanzee chromosome 17 and expressed chimpanzee galactokinase activity. These results indicate that the linkage relationship between galactokinase and thymidine kinase has been maintained in 3 divergent primate species--man, chimpanzee, and Old World monkey.     

Characterization of pig-mink cell hybrids: assignment of the TK1 and UMPH2 genes to pig chromosome 12

By fusion of thymidine kinase-deficient mink cells with pig leukocytes, a new type of cell hybrid was produced. It was demonstrated that pig chromosomes segregate in pig-mink hybrids and that hybrid cells contain no cytologically visible rearrangements between the chromosomes of parental species, or chromosome fragmentation. With a set of subclones of two primary hybrid clones, the genes for thymidine kinase-1 (TK1) and uridine 5-monophosphate hydrolase-2 (UMPH2) were assigned to pig Chromosome (Chr) 12. A cell line with a single pig Chr 8 on the background of mink chromosomes was established. This clone could serve as a source of DNA for building a chromosome-specific library of pig Chr 8. The data obtained suggest that pig-mink cell hybrids can be used for mapping of pig chromosomes.     

The chromosome variability of the Indian muntjac in somatic cell hybrids]

Hybrids were produced between the Indian muntjak fibroblasts and rat Jensen sarcoma cell line (JF1) auxotrophic for asparagine. They were selected without cloning under conditions providing survival of parental Indian muntjak and hybrid cells. This allowed to compare the Indian muntjak chromosome variability in the parental cells and hybrids under identical culture conditions. The frequency of muntjak chromosome aberrations proved to de higher in the hybrids (up to 47%) than in the parental cells (6.5%). Predominant are chromosomal breaks and dicentrics. The latter are mainly formed by fusion of chromosomes 1 and 2. The most fragile are 1 and X-chromosomes. Chromosomal breaks are evenly distributed along chromosome 1, and "hot" points are observed in X-chromosome. Possible mechanisms of the Indian muntjak chromosome rearrangements induced by somatic cell hybridization are discussed.     

Normal X chromosome induced reversion in the direction of chromosome segregation in mouse-Chinese hamster somatic cell hybrids

The effect of a normal mouse X chromosome on the chromosome segregation of mouse-Chinese hamster somatic cell hybrids was determined by (i) producing hybrids between the mouse sarcoma line CMS4 and a microcell hybrid (mfe4) of the hamster line E36, containing a mouse X chromosome from a normal cell; (ii) isolating hybrids between CMS4 and a 6-thioguanine selected (X minus) mfe4 subpopulation; (iii) comparing the direction of segregation in the two sets of hybrids. It was found that the normal X chromosome, like the X chromosomes from two MCA-transformed sarcoma lines reported previously [9], has the ability to switch the chromosome segregation of mouse-Chinese hamster somatic cell hybrids. We conclude that the reversal in chromosome segregation is mediated by factors located on the X chromosome. We designate these genetic elements as segregation reversal genes or sr genes.