Hybrids between irradiated and unirradiated mammalian cells: Survival and chromosome segregation

We have studied the effect of X or γ irradiation, of one parent of a cell hybrid, on hybrid viability and chromosome segregation. The hybrid types studied were mouse-Chinese hamster (which spontaneously lose a few hamster chromosomes) and Chinese hamster-human (which spontaneously lose most of the human complement). Preirradiation of the segregated and retained cell parent resulted in highly asymmetric hybrid survival curves; survival was greatly reduced when the retained parent was irradiated, especially for hamster-human fusions. Preirradiation of the parents of mouse-hamster hybrids modified both the direction and the extent of chromosome segregation, but no consistent effect on elimination was observed for hamster-human hybrids, and reversal of the direction of loss was never observed. These results are more consistent with the hypothesis that chromosome segregation from hybrids results from an intracellular chromosome selection, than with the hypothesis that cellular selection acts on randomly generated chromosome variants.     

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.     

Gene for human CD59 (likely Ly-6 homologue) is located on the short arm of chromosome 11

The CD59 (MEM-43) antigen, which probably is a human homologue of mouse Ly-6 antigens, is a broadly expressedM _r 18000–25000 human leucocyte surface glycoprotein recognized by monoclonal antibody MEM-43. Ten mouse-human T-lymphocyte hybrids, carrying all mouse chromosomes and a limited number of human chromosomes, were analyzed for expression of CD59 by indirect immunofluorescence and immunoblotting with MEM-43 antibody. Karyotypic analysis of the tested clones showed that the presence of human chromosome 11 correlated with the expression of CD59 in all clones tested. Three other human chromosome 11-encoded antigens, 4F2 (Trop-4), Leu 7 (HNK-1, CD57), and lymphocyte homing receptor, were expressed concordantly with CD59. A more exact localization of the gene for CD59 was obtained by the study of Chinese hamster-human cell hybrids containing short or long arm deletions of human chromosome 11. CD59 segregated with hybrids containing part of the short arm of human chromosome 11, but not with the hybrids containing the long arm. Based on these studies we assign the gene for CD59 to regionP14–p13 of the short arm of chromosome 11.     

Genes controlling gp25/30 cell-surface molecules map to chromosomes X and Y and escape X-inactivation.

The monoclonal antibody AbO13 defines a cell-surface antigen that is expressed on most cultured human cells, but not on rodent cells. AbO13 precipitates glycoproteins of 25,000 and 30,000 mol. wt. from lysates of [3H]glucosamine-labeled human cells. Results of the serological typing of a panel of 25 rodent-human somatic cell hybrid clones show that reactivity with AbO13 segregates with the human X and Y chromosomes. The presence of either of these chromosomes is sufficient for O13 expression on the hybrid cell surface. Analysis of hybrid clones containing human X chromosomes with karyotypically defined deletions permitted the regional assignment of the X-linked gene locus controlling the expression of O13 to Xp22-pter. In addition, AbO13 is reactive with Chinese hamster-human hybrids derived from fibroblasts of a 49,XXXXX individual that contained only inactivated copies of the human X chromosome. These results suggest that the X-linked locus determining the expression of O13 is not subject to X-inactivation.     

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.     

Assignment of the gene for acid beta-glucosidase to human chromosome 1.

The structural gene for human acid beta-glucosidase (GBA) has been assigned to chromosome 1 using somatic cell hybridization techniques for gene mapping. The human enzyme was detected in mouse RAG cell-human fibroblast cell hybrids by a sensitive double antibody immunoprecipitation assay using a mouse antihuman GBA antibody. No cross-reactivity between mouse beta-glucosidase and human GBA or neutral beta-glucosidase (GBN) was observed. Fifty-two primary, secondary, and tertiary manmouse hybrid lines, derived from three separate fusion experiments, were analyzed for human GBA and enzyme markers for the human chromosomes. Without exception, the presence of human GBA in these hybrid clones was correlated with the presence of human chromosome 1 or its enzymatic markers, phosphoglucomutase 1 (PGM1), and fumarate hydratase (FH). All other human chromosomes were eliminated by the independent segregation of GBA and their respective enzyme markers and/or chromosomes. Using a RAG X human fibroblast line with a mouse-human rearrangement of human chromosome 1, the locus for GBA was limited to the region 1p11 to 1qter.     

Assignment of the human gene for hexosaminidase B to chromosome 5

Mouse-human somatic cell hybrids between different mouse and human cells were studied for the expression of human hexosaminidases A and B activities. The expression of human hexosaminidase B in the hybrids was found to segregate concordantly with the presence of the human chromosome 5. Mouse-human hybrid clones containing either the human chromosomes 5 and 7 only or the human chromosome 7 only were also included in this study. Expression of human hexosaminidase B activity was detected only in those clones containing human chromosome 5. These results indicate that the gene(s) for human hexosaminidase B is located on chromosome 5. No hexosaminidase A activity was detected in clones which contained either human chromosomes 5 and 7 or chromosome 7.     

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)]     

Genetic linkage studies of the human glycosphingolipid β-galactosidases

The genetic linkage relationships of the human glycosphingolipid beta-galactosidases were determined using human--mouse somatic cell hybrids. A new method was devised for the estimation of human galactosylceramide, lactosylceramide, and GMI-ganglioside beta-galactosidase activities in the presence of their mouse counterparts, which takes advantage of the reproducible specific activity of lysosomal hydrolases under a given set of culture conditions and is based on differences in both pH optima and sensitivity to chloride ion. Human and mouse chromosomes were identified by their characteristic banding patterns obtained after quinacrine staining, and the optimum glycolipid beta-galactosidase activity was determined for three different substrates. A ratio was defined for each activity which was the specific activity at the human pH optimum divided by the specific activity at the mouse pH optimum. Linear regression analysis was used to test for concordant segregation between pH ratios for each enzyme and the frequency of occurrence of different human chromosomes in the man--mouse somatic hybrid clones. The results obtained from two independent series of hybrid clones indicated that human beta-galactosidase activities consistently segregated with human chromosome 12 in these somatic cell hybrids.     

Differential staining of interspecific chromosomes in somatic cell hybrids by alkaline Giemsa stain.

Staining of chromosome preparations of Chinese hamster-human hybrid cells and mouse-chimpanzee hybrids with alkaline Giemsa has yielded color differentiation of the interspecific chromosomes. Bicolor chromosomes, indicating apparent translocations also are observed for each of these hybrids. The specific color differences observed provide a rapid means of recognizing and aiding in the identification of the interspecific chromosomes and apparent translocations in these somatic cell hybrids.     

Investigations on the chromosomal localizations of the human and chimpanzee interferon genes: possible role of chromosomes 9 and 13

Analysis of a great number of independent hamster-human and mouse-chimpanzee somatic cell hybrid clones confirms the role of chromosome 9 as carrying one or more primate beta interferon genes. The presence of chromosome 13 in producing hybrids and its absence in all non producing clones must be kept in mind for future studies. The strong negative regulation of interferon production in the parental hamster cells also affects the human gene product. The UV irradiation target for these regulatory genes is significantly greater than the structural genes responsible for interferon production.     

A radiation hybrid map of the proximal short arm of the human X chromosome spanning incontinentia pigmenti 1 (IP1) translocation breakpoints.

Radiation hybrid mapping was used in combination with physical mapping techniques to order and estimate distances between 14 loci in the proximal region of the short arm of the human X chromosome. A panel of radiation hybrids containing human X-chromosomal fragments was generated from a Chinese hamster-human cell hybrid containing an X chromosome as its only human DNA. Sixty-seven radiation hybrids were screened by Southern hybridization with sets of probes that mapped to the region Xp11.4-Xcen to generate a radiation hybrid map of the area. A physical map of 14 loci was constructed based on the segregation of the loci in the hybrid clones. Using pulsed-field gel electrophoresis (PFGE) analyses and a somatic cell hybrid mapping panel containing naturally occurring X; autosome translocations, the order of the 14 loci was verified and the loci nearest to the X-chromosomal translocation breakpoints associated with the disease incontinentia pigmenti 1 (IP1) were identified. The radiation hybrid panel will be useful as a mapping resource for determining the location, order, and distances between other genes and polymorphic loci in this region as well as for generating additional region-specific DNA markers.     

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.     

Chromosome segregation from cell hybrids. VII. Reverse segregation from karyoplast hybrids suggests control by cytoplasmic factors.

Using human and Chinese hamster established lines as cell parents, we constructed hamster-human cell hybrids and human cell - hamster karyoplast hybrids. The cell hybrids retained one or two sets of hamster chromosomes and lost most of the human chromosomes. The karyoplast hybrids, however, retained a full set of human chromosomes and lost most of the Chinese hamster chromosomes. This reverse segregation pattern implies that cytoplasmic factors are major determinants of the direction of chromosome segregation.     

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.     

Intrachromosomal gene mapping in man: the gene for tryptophyl-tRNA synthetase maps in region q21 leads to qter of chromosome 14

A gene for tryptophanyl-tRNA synthetase (EC 6.1.1.2), the enzyme which attaches tryptophan to its tRNA, has previously been assigned to human chromosome 14 by analysis of man-mouse somatic cell hybrids. We report here a method for the electrophoretic separation of Chinese hamster and human tryptophanyl-tRNA synthetases and its application to a series of independently derived Chinese hamster-human hybrids in which part of the human chromosome 14 has been translocated to the human X chromosome. When this derivative der (X),t(X;14) (Xqter leads to Xp22::14q21 leads to 14qter) chromosome carrying the human gene for hypoxanthine-guanine phosphoribosyltransferase was selected for and against in cell hybrid lines by the appropriate selective conditions, the human tryptophanyl-tRNA synthetase activity was found to segregate concordantly. These results provide additional confirmation for the assignment of the tryptophanyl-tRNA synthetase gene to human chromosome 14 and define its intrachromosomal location in the region 14q21 leads to 14qter. Our findings indicate that the genes for tryptophanyl-tRNA synthetase and for ribosomal RNA are not closely linked on chromosome 14.     

Assignment of genes to the human X chromosome by the two-dimensional electrophoretic analysis of total cell proteins from rodent-human somatic cell hybrids.

The technique of two-dimensional (2-D) gel electrophoresis was sued to identify five human X-linked gene products in crude cell extracts of mouse-human and Chinese hamster-human somatic cell hybrids. The human origin of these five polypeptides was demonstrated by their comigration with human fibroblast proteins and their failure to comigrate with polypeptides in extracts from the mouse or hamster parental cells. All five polypeptides were present in extracts of rodent-human hybrids that contained a human X chromosome, but were not found in extracts of cells that lacked a human X chromosome. Chromosome analysis of the hybrid clones revealed that the human X chromosome is both necessary and sufficient for the expression of the five polypeptides, designated pX-24, pX-27, pX-37, pX-40, and pX-56. pX-56 can be identified as the human X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) (E.C.1.1.1.49), while polypeptides pX-24, pX-27, pX-37 and pX-40 have molecular properties unlike those of known human X-linked gene products. pX-24 appears to be a membrane-bound protein that maps to the distal portion of the long arm of the human X chromosome, while pX-27, pX-37, and pX-40 are soluble proteins that map to the proximal long arm or to the short arm of the human X chromosome. 2-D gel electrophoretic analysis of extracts from somatic cell hybrids provides a general method for identifying polypeptides in crude cell extracts coded for by any specific chromosome and can be used to study primary gene products not previously amenable to genetic analysis.     

Radiation hybrid map of 13 loci on the long arm of chromosome 5.

Radiation hybrid mapping was used in conjunction with a natural deletion mapping panel to predict the order of and distance between 13 loci in the distal portion of the long arm of human chromosome 5. A panel of irradiation hybrids containing fragments of 5q was generated from an HPRT+ Chinese hamster-human cell hybrid containing a derivative chromosome 5 [der(5)t(4;5)(5qter----5p15.1::4p15.1----4pter)] as its only human DNA. One hundred nine radiation hybrids containing human DNA were screened with polymerase chain reaction primer sets representing nine genes encoding growth factors, growth factor receptors, or hormone receptors (IL3, IL4, IL5, CSF1R, FGFA, ADRB2, GRL, GABRA1, and DRD1) as well as four other loci (FER, SPARC, RPS14, and CD14) to generate a radiation hybrid map of the area 5q21-q35. A physical map predicting the order of and distance between the 13 loci was constructed based on segregation of the 13 loci in hybrid clones. The radiation hybrid panel will be useful as a mapping tool for determining the location and order of other genes and polymorphic loci in this region as well as for generating new DNA probes from specific regions.     

Agarose gel electrophoresis in isozyme separation and visualization

Isozyme analysis of rodent-human somatic cell hybrids has been used frequently to detect specific human chromosomes. The majority of these isozyme systems employs starch gels, the use of which can be laborious when screening large numbers of cell lines. We describe the development of two procedures to detect the long arms of human chromosomes 1 and 2 in Chinese hamster-human cell hybrids by a rapid and reproducible method using 1-mm-thick agarose gels. Detection of human chromosome 1q was accomplished by screening for human fumarate hydratase activity, whose gene has been mapped to 1q42.1. Detection of chromosome 2q was performed by screening for the isozyme isocitrate dehydrogenase 1, which has been localized to 2q32-qter. These systems provide a basis for the further development of procedures for detecting chromosome-specific isozyme markers in agarose gels.