Chromosome replication in somatic hybrids of mouse and temperature sensitive Chinese hamster cells

Hybrid clones were obtained between a mouse cell line (3TP) and a temperature-sensitive Chinese hamster cell line (K12) unable to grow at 40° because of a ts defect apparently located at the G1/S transition. The great majority of hybrid clones grew at 40°, showing the ts defect to be “recessive.” Chromosome DNA replication was analyzed in some detail in three hybrid clones with balanced complements. Although the S period of these hybrids was longer than that of K12, DNA replication in mouse and hamster chromosomes started and ended synchronously. Upon prolonged culture, mouse chromosomes were lost as they are in hybrids involving a non ts Chinese hamster partner, in which case asynchronous chromosome replication appears to be the rule. It seems therefore that asynchronous replication is not the determining factor in chromosome loss from cell hybrids.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

Requirement of human chromosomes 19, 6 and possibly 3 for infection of hamster x human hybrid cells with baboon M7 type C virus.

The replication pattern of the endogenous baboon type C virus M7 was studied in 29 primary Chinese hamster × human hybrid clones generated with leukemic cells from two different patients with acute lymphoblastic or myeloblastic leukemia. There was no evidence of viral particulate RDDP or M7 antigen before viral infection. M7 virus replicated in human and some hybrid cells but not in Chinese hamster cells, indicating that M7 requires dominantly expressed human gene(s) for replication. Enzyme and cytogenetic analyses show that a gene(s) coded for by human chromosome 19 is necessary for M7 infection of these hybrids. Detailed cytogenetic correlations revealed, however, that the chromosome 19+/M7 + hybrid clones with intact chromosomes also had copies of chromosomes 3 and 6. Previously, Bevi, the putative integration site for M7 virus, has been assigned to human chromosome 6. Many clones with various combinations of chromosomes 3 and 6 lacked chromosome 19, however, and failed to replicate exogenously applied M7 virus, while tests of 27 secondary clones showed that M7 markers co-segregated with chromosome 19 markers. These findings all confirm the need for a chromosome 19-coded function in Chinese hamster × human hybrids. In addition, the yield of viral particulate RDDP produced into the culture fluid was 50–100 fold less per viral antigen-positive cell in the hybrids compared with human cells. Thus some form of regulation of viral components exists in the hybrid cells. When the virus replicating in hybrid cells was transferred back to human cells, this regulation was relaxed and the yield of RDDP per FA(+) cell greatly increased. We conclude that human chromosomes 6 and 19 code for functions involved in M7 virus metabolism, and we cannot exclude a function coded for by chromosome 3.     

Transfer of the human X chromosome to human--Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes.

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack H P R T activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in H A T medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of H A T-resistant clones was 32 x 10(-6) when 10(7) cells were incubated with 10(8) HeLa chromosomes. Potential reversion of the hybrid cells in H A T medium was less than 5 x 10(-7). The 16 isolated cell lines all contained activity of the human X-linked marker enzymes H P R T, P G K,alpha-Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Assignment of the structural gene encoding human aspartylglucosaminidase to the long arm of chromosome 4 (4q21----4qter)

The structural gene for the human lysosomal enzyme aspartylglucosaminidase (AGA) has been assigned to chromosome 4 using somatic cell hybridization techniques. The human monomeric enzyme was detected in Chinese hamster-human cell hybrids by a thermal denaturation assay that selectively inactivated the Chinese hamster isozyme, while the thermostable human enzyme retained activity. Twenty informative hybrid clones, derived from seven independent fusions, were analyzed for the presence of human AGA activity and their human chromosomal constitutions. Without exception, the presence of human AGA in these hybrids was correlated with the presence of human chromosome 4. All other human chromosomes were excluded by discordant segregation of the human enzyme and other chromosomes. Two hybrid clones, with interspecific Chinese hamster-human chromosome translocations involving the long arm of human chromosome 4, permitted the assignment of human AGA to the region 4q21----4qter.     

Hybridization of Chinese hamster cells sensitive to ultraviolet irradiation

Resistance to UV-light was studied in two UV-sensitive aneuploid Chinese hamster cell clones to different origin and degree of sensitivity, their respective polyploids and somatic cell hybrids. The karyotype of the parental clones, cell hybrids and polyploids was analyzed in parallel. A great variability of karyotypes was detected in hybrid cells. Serial cultivation of hybrids was accompanied by chromosome loss. Soon after fusion the hybrid clones proved to be more resistant to UV than the parental sensitive cells. However, their sensitivity increased with passages. The comparison of UV-sensitivity with data on karyotype analysis allowed to assume that the increase in sensitivity was correlated with the loss of particular chromosomes or chromosome regions. The results obtained indicated the existence of a polygenic control of UV-sensitivity, the multiple genes being assigned to different chromosomes. A reverse effect of ploidy was detected, i.e. a decrease in the resistance to the lethal action of UV-light in polyploids as compared to the parental clones.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

Patterns of late chromosomal DNA replication in unbalanced chinese hamster-mouse somatic cell hybrids

The chromosome late-replication patterns of five mouse × Chinese hamster somatic cell hybrids with reduced hamster complements were compared with those of the Chinese hamster parent cell, in order to determine whether the sequential order of chromosome replication is dependent on the presence of the whole chromosome set. In all hybrid clones the parental pattern could be recognized in a variable proportion of cells, although different chromosomes were missing in each clone. The results suggest that sequential order of replication is not the consequence of any sort of interaction between replication units (such as competition for limiting factors), and point to a considerable degree of autonomy in replication of individual chromosomes or chromosome parts.     

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.     

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.     

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.     

Unidirectional loss of human chromosomes in rat-human hybrids

Karyological analysis of 25 rat-human hybrid cell clones shows that only the human chromosomes are lost from the hybrids. Giemsa banding staining of the chromosomes of these hybrids allowed the identification of each human chromosome present in the hybrid cells.     

Complementation of repair gene mutations on the hemizygous chromosome 9 in CHO: a third repair gene on human chromosome 19

A human DNA repair gene, ERCC2 (Excision Repair Cross Complementing 2), was assigned to human chromosome 19 using hybrid clone panels in two different procedures. One set of cell hybrids was constructed by selecting for functional complementation of the DNA repair defect in mutant CHO UV5 after fusion with human lymphocytes. In the second analysis, DNAs from an independent hybrid panel were digested with restriction enzymes and analyzed by Southern blot hybridization using DNA probes for the three DNA repair genes that are located on human chromosome 19: ERCC1, ERCC2, and X-Ray Repair Cross Complementing 1 (XRCC1). The results from hybrids retaining different portions of this chromosome showed that ERCC2 is distal to XRCC1 and in the same region of the chromosome 19 long arm (q13.2-q13.3) as ERCC1, but on different MluI macrorestriction fragments. Similar experiments using a hybrid clone panel containing segregating Chinese hamster chromosomes revealed the hamster homologs of the three repair genes to be part of a highly conserved linkage group on Chinese hamster chromosome number 9. The known hemizygosity of hamster chromosome 9 in CHO cells can account for the high frequency at which genetically recessive mutations are recovered in these three genes in CHO cells. Thus, the conservation of linkage of the repair genes explains the seemingly disproportionate number of repair genes identified on human chromosome 19.     

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 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.     

The gene coding for a sphingolipid activator protein,SAP-1, is on human chromosome 10

Summary SAP-1 is a sphingolipid activator protein found in human tissues required for the enzymatic hydrolysis of GM1 ganglioside and sulfatide. It appears to be missing in patients who have a genetic lipidosis resembling juvenile metachromatic leukodystrophy. Using rabbit antibodies against human SAP-1 it could be visualized in extracts from cultured human skin fibroblasts after sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by electroblotting to nitrocellulose membrane and immunochemical staining (Western blotting). A series of 23 human-Chinese hamster ovary cell hybrids containing different human chromosomes were examined. The parent Chinese hamster ovary cells did not have a reacting protein in the region of human SAP-1. Only in the eight hybrid clones containing human chromosome 10 was a reacting protein identified. Other chromosomes were excluded by this method. Therefore the gene for SAP-1 and the genetic mutation resulting in a fatal lipidosis are located on human chromosome 10. Present address: Department of Pediatrics, Osaka University Medical School, Fukushima-Ku, Osaka, Japan     

Assignment of the gene for human sphingolipid activator protein-2 (SAP-2) to chromosome 10.

Sphingolipid activator protein-2 (SAP-2) has been found to stimulate the enzymatic hydrolysis of glucosylceramide, galactosylceramide, and sphingomyelin. When human skin fibroblast extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by electroblotting and immunochemical staining using monospecific antibodies against SAP-2, two or three major bands with estimated mol. wts. of 9,000-10,000 were found. These antibodies did not crossreact with purified SAP-1, another activating protein, or with extracts of CHO-K1 cells. A series of 22 human/Chinese hamster ovary cell hybrids containing different human chromosomes were examined by this method. All eight hybrid clones containing human chromosome 10 were found to have crossreacting protein in this region. Other chromosomes could be excluded by this method. From these results, we conclude that the gene coding for human SAP-2 is located on chromosome 10.     

Localization of human variable and constant region immunoglobulin heavy chain genes on subtelomeric band q32 of chromosome 14

Analysis of a group of human/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human variable region (VH), junctional (JH), and constant region (C epsilon) heavy chain immunoglobulin genes indicates that all of these IgH genes are localized on the subtelomeric (q32) band of chromosome 14. Somatic cell hybrids were isolated in selective medium after fusing human fibroblasts with hprt- Chinese hamster cells. The human parental cells contained two translocation chromosomes representing a reciprocal translocation between chromosomes X and 14. Only those hybrid cell lines retaining a complete human autosome 14 or the X/14 translocation chromosome (i.e. containing band 14q32) retained the human IgH genes. Retention of these genes did not correlate with the presence of the other translocation chromosome, 14/X. These results indicate that all human IgH genes (VH, JH, and CH) map to the same chromosomal band (14q32) which is commonly involved in reciprocal translocations with human chromosome 8 (8q24) in B-cell neoplasms.     

Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines.     

Density-dependent regulation of growth in somatic hybrids between normal Chinese hamster fibroblasts and V79-8 (G1) cells

The purpose of this work was to determine the relationship between the presence of a G1 period in the mitotic cycle and a cell's ability to respond to density-dependent regulation of growth (DDR). Somatic hybrids were obtained between normal fibroblasts from newborn Chinese hamsters, which show a strong response to DDR, and V79-8 Chinese hamster cells, which are insensitive to DDR. Two variant V79-8 sublines were used, one reported to lack a G1 period (G1-) and the other with a G1 period (G1+). Fourteen hybrid clones were isolated in selective medium and analysed for growth properties and cell cycle parameters; their hybrid nature was supported by chromosome counts. All hybrid clones, irrespective of whether a V79-8 G1- or G1+ cell was one of the parents, showed pronounced DDR and had G1 periods of various lengths. Previous experiments had shown the absence of G1 to be dominant in somatic hybrids between V79-8 G1- and G1+ cell lines. Our results may mean that the G1- property provided by V79-8 is unable to overcome the very long G1 of normal fibroblasts, or in cells that can be arrested in G1 in response to DDR, some function prevents the dominant effect of the G1- cell on at least part of the G1 period.