Unequal segregation of parental chromosomes in embryonic stem cell hybrids

Chromosome segregation was studied in 14 intra- and 20 inter-specific hybrid clones generated by fusion of Mus musculus embryonic stem (ES) cells with fibroblasts or splenocytes of DD/c mice or Mus caroli. As a control for in vitro evolution of tetraploid karyotype we used a set of hybrid clones obtained by fusion of ES cells (D3) with ES cells (TgTP6.3). Identification of the parental chromosomes in the clones was performed by microsatellite analysis and in situ hybridization with labeled species-specific probes. Both analyses have revealed three types of clones: (i) stable tetraploid, observed only for ES x ES cell hybrids; (ii) bilateral loss of chromosomes of both ES and somatic partners; (iii) unilateral segregation of chromosomes of the somatic partner. Observed unilateral segregation was extensive in ES-splenocyte cell hybrids, but lower in ES-fibroblast hybrid clones. Developmental state of the somatic partner is presumably responsible for directional chromosome loss. Nonrandom segregation implies that initial differences in the parental homologous chromosomes were not immediately equalized implying at least transient persistence of the differentiated epigenotype.     

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.     

Hybridization of somatic cells derived from mouse and Syrian hamster: Evolution of karyotype and enzyme studies

Somatic hybrids of drug-resistant mutant hamster and mouse cell lines have been isolated and propagated in long-term culture and have been studied in respect to karyotype and three enzymes. During the course of propagation the long-surviving hybrid clones show progressive loss of telocentric chromosomes associated in at least one case with loss of mouse enzyme. Hybrid clones showed hybrid molecules for malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6PGD) made up by recombination of parental subunits.This work was supported by National Institutes of Health Grant HD 00486.     

Visible and "cryptic" segregation of parental chromosomes in embryonic stem hybrid cells

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.     

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.     

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.     

Stability of the "two active X" phenotype in triploid somatic cells.

We examined triploid cells of XXY karyotype heterozygous for glucose 6 phosphate dehydrogenase (G6PD) electrophoretic variants with regard to the stability of their X chromosome phenotype. Clonal populations of cells derived from these human fibroblasts maintained a precise 1:2:1 ratio of A:heteropolymer:B isozymes throughout their life span, indicating stability of the two active X chromosomes in these cells. To determine the influence of the autosomal complement on X chromosome expression, we attempted to perturb the relationship. Fusion of these triploid cells with human diploid fibroblasts carrying a novel G6PD variant (B') resulted in heterokaryons exprssing a novel heteropolymer, presumably indicating that all three parental X chromosomes were active. However, no derepression of the inactive X chromosome was observed. Analysis of interspecific hybrids derived from triploid cells and mouse fibroblasts confirmed that activity of parental X chromosomes is maintained. Some human mouse hybrid clones, however, expressed only a single human G6PD isozyme, probably attributable to segregation of the pertinent X chromosome, but elimination of a relevant autosome cannot be excluded. The triploid cells transformed by SV40 showed alterations in LDH pattern and an approximately 10-20% decrease in chromosome number, but maintained the precise G6PD phenotype of the untransformed cell. These studies provide evidence for the stability of the X chromosome phenotype in triploid cells.     

Characteristics of the submicroscopic structure of hybrid clone cells obtained by the fusion of mouse hepatoma cells and mink fibroblasts and containing different numbers of mink chromosomes

Four hybrid clones obtained by fusion of mouse hepatoma cells with mink fibroblasts treated with polyethyleneglycol were studied morphologically and morphometrically using electron microscopy. The clones studied contained a double set of mouse chromosomes and different numbers of mink chromosomes. It is demonstrated that clones containing different mink chromosomes differ considerably from each other and from the parental cells in the manifestation of some morphological characters (form and type of cell growth, form of the nucleus, structure of mitochondria, distribution of membranes of the granular endoplasmic reticulum), as well as in some quantitative parametres of organelles (area of the cut of the cell and of the nucleus, a relative volume of the nucleus). The data obtained witness for the fact that some morphological traits characteristic of cells of a certain parental type may appear in hybrids independently of each other, and that the degree of their manifestation may depend on the number of chromosomes of one of the parents or, possibly, on one particular chromosome.     

Suppression of the transformed phenotype of hepatoma cells after hybridization with normal diploid fibroblasts

Hybrids were generated between mouse hepatoma cells which exhibit a transformed phenotype, and rat normal diploid fibroblasts. Most isolated hybrid clones contain a single set of chromosomes from each parent. Such clones grow to low saturation densities and are unable to grow or to form colonies in soft agar. The transformed phenotype of the parental hepatoma cells is thus suppressed in these hybrids. Suppression is very stable; however, subclones which have regained a transformed phenotype could be selected; these subclones show a significant reduction of their chromosome number. Amongst the hybrid clones isolated after fusion, a few are characterized by an excess of mouse chromosomes and a reduced number of rat chromosomes. Such clones exhibit a transformed phenotype. Our results show that, provided the hybrids contain an almost complete single set of chromosomes of each parent, spontaneous transformation behaves as a recessive trait in hybrids formed with normal diploid cells.     

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.     

Levels of fos, ets2, and myb proto-oncogene RNAs correlate with segregation of chromosome 11 of normal cells and with suppression of tumorigenicity in human cell hybrids.

The tumorigenicity in nude mice of human carcinoma-derived D98AH2 (D98) cells is suppressed when cell hybrids are made by fusing these cells with normal human diploid cells. Selection for hybrids that have segregated chromosomes results in the recovery of tumorigenic segregants. These segregants have all lost at least one copy of chromosome 11 of the diploid cell parent. Earlier we found that the parental D98 cells had detectable levels of mRNA specific for 13 of 21 proto-oncogenes examined. To determine if transregulation of proto-oncogenes by genes of the normal cell occurs in such hybrids, the steady-state levels of mRNA specific to 22 proto-oncogenes in the parental cells were compared with those of nontumorigenic D98 X human diploid hybrids as well as with those of their tumorigenic segregants and with the cells of the resulting tumors. The only chromosome consistently segregated in the latter was chromosome 11 of the diploid cell. fos and ets2 RNA levels and the amount of fos protein were consistently elevated in the segregants compared with amounts in the original hybrids. An unexpected finding was the inverse relationship for myb RNA that was barely detected in the parental D98 cells but was at least 10-fold elevated in hybrids that did not have segregated chromosomes compared with those that did. These patterns were evident in RNAs prepared from both subconfluent and confluent cell cultures. The findings suggest that genes of the normal cell parent can affect proto-oncogene expression. Whether the genes affecting fos, ets2, and myb RNA levels are on chromosome 11 and whether these alterations are causally related to the tumorigenic phenotype of the hybrid remain to be determined.     

Suppression of Vesicular Stomatitis Virus Defective Intefering Particle Generation by a Function(s) Associated with Human Chromosome 16

Human-mouse somatic cell hybrids were made between adenine phosphoribosyltransferase-deficient mouse L cells and a strain of human primary fibroblasts and selected in medium containing alanosine and adenine (J. A. Tischfield and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A. 71:45-49, 1974). These hybrids were tested for the generation of defective interfering (DI) particles of vesicular stomatitis virus to determine whether or not a host gene controls the induction of DI particles. None of the seven independently arising hybrid clones tested generated detectable DI particles during 13 successive undiluted passages. In addition, the parental human cells also failed to generate DI particles. In contrast, the parental mouse cells generated a detectable level of DI particles during continuous passage. Thus, failure to generate DI particles appears to act in a dominant fashion in these hybrids. Human chromosome 16 and adenine phosphoribosyltransferase were present, as a direct consequence of the selection system, in all of the hybrid clones that failed to generate DI particles. It was the only human chromosome observed in the cells of every hybrid clone. This was verified by both isozyme and karyotype analyses. After hybrids were back-selected (with 2,6-diaminopurine) for loss of human adenine phosphoribosyltransferase and chromosome 16, they gained the ability to generate DI particles. Replication of DI particles already present in virus stocks, however, was normal in all of the hybrid clones and the parental human cells. This suggests that the induction, but not the replication, of DI particles is affected by the human genome and that a factor on human chromosome 16 seems to selectively suppress the mouse cell's ability to generate DI particles in the hybrids. These results support the idea that the induction of DI particles is controlled in part by host cell function(s), as suggested previously (C. Y. Kang and R. Allen, J. Virol. 25:202-206, 1978).     

Genetic control of tumorigenicity in interspecific mammalian cell hybrids.

The nature of genetic control of cellular malignancy was investigated by examining the tumorigenicity of a series of interspecific mouse-human cell hybrids in the athymic nude mouse. Two highly malignant but genetically distinct mouse cell lines, A9 and PG19, were hybridized with three normal human diploid fibroblast strains, and 19 independently arising hybrid clones were isolated. Each of these clones was capable of forming progressive lethal tumors in the nude mouse, and thus resembled the malignant parental mouse cells rather than the nonmalignant parental human cells. We failed to obtain any evidence for complete suppression of tumorigenicity in these cell hybrids. The absence of suppression was observed regardless of the extent and composition of the human chromosome complements retained in the hybrid clones; the results of detailed cytological and isoenzyme analyses would make it highly improbable that the observed lack of suppression was due to cellular selection in vivo for a more tumorigenic subpopulation in the injected hybrid cells. These data demonstrate that at least for the parental cell combinations used in this study, no human chromosome, when present singly in the mouse-human cell hybrids, can suppress the tumorigenic phenotype of the mouse cells. Our results are consistent with the view that the suppression of cellular malignancy previously demonstrated in intraspecific (mouse × mouse) somatic cell hybrids does not occur in interspecific (mouse-human) cell hybrids, or alternatively, genetic determinants located on two or more human chromosomes are required simultaneously to suppress the malignancy of the mouse cells in cell hybrids derived from malignant mouse cell and nonmalignant human cells.     

Genomic compatibility between two phyllotine rodent species evaluated through their hybrids

In order to investigate the genomic compatibility between allopatric rodent species, Phyllotis darwini and Phyllotis magister, we have studied several cytogenetic and reproductive features of their laboratory hybrids. Of thirty-one pairings between species, only five were successful, producing eleven newborns. Like parents, hybrids had 38 metacentric chromosomes, except for the subtelocentric Y chromosome inherited from P. magister. There was almost total C and G band correspondence between homeologous autosomes. However, parental sex chromosomes had different morphology, C and G bands. Ag-NOR bands appeared as small telomeric Ag+ regions, distributed in four chromosomal pairs of darwini, three of magister and four homeologous chromosomes of the hybrids. The three forms had similar indexes of NOR activity per cell, in spite of the variability in NOR expression which was always detected. Usually, only one member of parental homologous chromosomes showed AgNOR+; nevertheless, both homeologous chromosomes were active in many hybrid cells. The frequencies of cells that expressed their ribosomal genes in the two homologous or homeologous NOR chromosomes were similar in parental and hybrid cells. These results strongly suggest that ribosomal genes of both parental genomes would function codominantly in the hybrids. The gonad histological and morphometric analyses showed that hybrids conformed to Haldane's rule, since females were fertile and males were infertile. Our results indicate that P. darwini and P. magister genomes can function in relative harmony and compatibility when they are placed together in their laboratory generated hybrids, suggesting that these species have few genetic differences, probably because they have recently diverged.     

Somatic hybrids of normal and tumor Djzungarian hamster cells. I. Preferential elimination of chromosomes of the normal partner

Normal Djungarian hamster lymphoid cells were fused with SV40 transformed malignant fibroblasts. The resulting 11 hybrid clones were subjected to the chromosome analysis. The karyotype of hybrids proved to be unstable. In some cases the total tetraploid number of chromosomes in hybrids drastically decreased up to the near-diploid level close to that of the malignant parent cells. The G-band chromosome analysis showed that as a rule morphologically unchanged chromosomes were preferentially lost from the hybrid cells, the markers of the malignant partner being retained. On the basis of these data it is assumed than the hybrids between normal and tumour cells of Djungarian hamster preferentially lose the chromosomes of the normal parent cells during cultivation in vitro.     

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.     

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.     

Human intraspecific somatic cell hybrids: a genetic and karyotypic analysis of crosses between lymphocytes and D98/AH-2.

A number of human intraspecific hybrids were produced by fusing the 8-azaguanine-resistant cell line D98/AH-2 with PHA-stimulated lymphocytes from a normal human male, followed by selection in HAT medium. The parent cells differed in zymogram patterns for 4 enzyme systems. Hypoxanthine-guanine phophoribosyltransferase was missing in D98/AH-2 and was determined in the hybrids by the normal gene derived from the lymphocyte donor's X chromosome. The HL-A antigens of the lymphocyte donor as well as the W28 specificity from HeLa were easily recognized by a cytotoxicity assay on the hybrid cells, while D98/AH-2 itself was not killed in the normal way by any HL-4 typing sera. The initial hybrid karyotype in all lines was relatively stable, but slow loss of chromosomes occurred following extended growth in culture. The importance of the culture conditions for the rate of chromosome loss was demonstrated. The behavior of several chromosomes was followed in the hybrids and their derivatives. There was relatively nonspecific loss of small numbers of chromosomes, showing that loss of chromosomes from both the D98/AH-2 and the normal lymphocyte parent can occur. Cell lines resistant to 6-thioguanine were selected from the sensitive hybrids. Most had lost the lymphocyte donor's X chromosome, thereby losing the only active allele for HGPRT present in the initial hybrids. However, one line, DMR41, apparently retained the X chromosome and may have a mutated allele for HGPRT. Two lines that are the products of spontaneous segregation are also described. DM4CS and DM17A.     

Genetic processes in intergeneric cell hybrids Atropa + Nicotiana

Summary The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120–150 chromosomes including 50–70 of Atropa and 50–90 of Nicotiana. Other clones are polyploid and possess 200–250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.Dedicated to Professor G. Melchers with gratitude