Evidence for an indirect effect of radiation on mammalian chromosomes : I. Indirectly-induced chromosome loss from somatic cell hybrids

Mouse cells UV-irradiated with doses of 0–72 J/m² were fused with unirradiated Chinese hamster cells, and the chromosome constitutions of cell hybrids were examined. The number of mouse chromosomes retained by hybrids decreased with UV dose, and, unexpectedly, the number of hamster chromosomes also decreased in a dose-dependent manner. It is suggested that some component contributed by the irradiated mouse parent cell has indirectly induced damage and loss of hamster chromosomes.     

THE RIBOSOMAL RNA OF HAMSTER-MOUSE HYBRID CELLS

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

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.     

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.     

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

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

Chinese hamster X mouse hybrid cells segregating mouse chromosomes and isozymes.

Hybrid cells are readily formed by fusing clonal Chinese hamster cells to fresh, noncultured, adult mouse spleen cells followed by isolation in selective medium. The vast majority of such hybrids retain Chinese hamster chromosomes and isozymes while segregating mouse chromosomes and isozymes. The growth, plating efficiency, ease of karyology, and rapid segregation of mouse markers allows linkage tests in primary clones. Analysis of 13 isozymes showed 12 to be asyntenic and on epair (PGD-PGM2) to be syntenic This system will allow extensive somatic cell hybrid gene mapping in the mouse and permit a comparison of human and mouse linkage relationships.     

Patterns of chromosome segregation in chinese hamster × mouse cell hybrids between permanent cell lines and thymus cells

Hybrids between a tumorigenic Chinese hamster cell line (DC3F-aza) and normal mouse thymus cells very rapidly lost most of their mouse chromosomes, whereas hybrids between tumorigenic mouse cell lines (either Cl.1D of L cell line origin, or PCC4-aza1 teratocarcinoma cells) and normal Chinese hamster thymus cells lost most of their hamster chromosomes. From three such fusion experiments, 20 cell lines were developed which all followed the same evolution, namely, the elimination of the majority of the chromosomes contributed by the normal thymus cell. In some hybrids, the elimination process resulted in the total absence of intact chromosomes contributed by the thymus cell parent. Such hybrids were distinguished from revertant parental cells growing in the selective hybrids were distinguished from revertant parental cells growing in the selective medium by the presence of at least one enzyme in their cell extracts which displayed the electrophoretic mobility of the enzyme of the thymus cell parent. These observations, together with data from other reports, suggest that, as a rule, interspecific cell hybrids which develop upon fusion between normal diploid cells and tumorigenic cell lines maintain the chromosomes of the latter and eliminate preferentially many or most of the chromosomes contributed by the normal cell parents, independent of the respective species of the parental cells.     

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

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

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.     

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.     

Assignment of murine cellular Harvey ras gene to chromosome 7

Mouse-Chinese hamster somatic cell hybrids containing various combinations of mouse chromosomes were analyzed for the presence of the mouse c-Ha-ras (1) sequences after restriction endonuclease digestion and hybridization with a 32P-labeled Ha-ras specific probe according to the procedure of Southern (2). The presence of the mouse c-Ha-ras containing fragment was correlated with the presence of mouse chromosome 7 in the hybrids.     

Gene mapping in the Chinese hamster and conservation of syntenic groups and Q-band homologies between Chinese hamster and mouse chromosomes

Using Chinese hamster/mouse somatic cell hybrids segregating hamster chromosomes, we assigned 15 enzyme genes to six different Chinese hamster autosomes. Of the 15 loci, three genes, HK1, PEPC, and SORD, were newly assigned to chromosomes 1, 5, and 6, respectively, while ENO1, PGD, and PGM1 were assigned to the long arm of chromosome 2, in the segment 2q113----qter. The locations of the following loci were confirmed: ESD, NP, and PEPB on chromosome 1, ME1 and MPI on chromosome 4, AK1 on chromosome 6, and GPI and PEPD on chromosome 9. Comparative mapping of Chinese hamster and laboratory mouse chromosomes revealed conservation of syntenic groups and extensive banding homology between the Chinese hamster and mouse chromosomes on which homologous enzyme markers have been mapped.     

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.     

Conservation of the syntenic group enol - pgd - pgm in mammals: its assignment to chromosome 2 in the Chinese hamster

Hybrids between cells from mouse permanent lines and Chinese hamster thymus cells explanted from animals maintained mouse chromosomes and lost most hamster chromosomes. In twenty-seven hybrids examined for expression of enolase 1. phosphogluconate dehydrogenase, and phosphoglucomutase, the Chinese hamster forms of the three enzymes were either expressed together, or not expressed at all. Thus, the three genes eno1, pgd, and pgm appear syntenic in Chinese hamster as they are in man (chromosome 1p), and in mouse (chromosome 4). The three markers map on the Chinese hamster chromosome 2.     

Characteristics of somatic cell hybrids (mouse x Chinese hamster) with a different ratio of chromosome sets from the parent species. II. Thymidine kinase activity]

The activity of thymidine kinase (TK) was studied in series of somatic cell hybrids between the mouse cell line 3T3-4E (TK-) and Chinese hamster cells M-15-1 (HGPRT-). Four groups of hybrid lines with different ratio of parental chromosome sets have been investigated: 1) three lines containing one hamster and one mouse chromosome set (1 hs+1 ms); 2) one line with 2 hs+1 ms; 3) one line containing 3 hs+1 ms and 4) one line containing 1 hs+2 ms. Mixtures of extracts from the parental cells were shown to possess the expected TK activity. The calculation of the activity per cell revealed that the 1 hs+1 ms and 2 hs+1 ms hybrid lines possessed about 50% of the initial hamster cell TK activity. The decreased TK activity in these hybrids might be due either to a loss of hamster chromosomes or to some inhibitory effect of mouse genome in cells with the studied ratio of parental sets. The enzyme activity in the 3 hs+1 ms hybrid was as expected, about three times greater than that of hamster cells.     

Identification of a cellular receptor for mouse mammary tumor virus and mapping of its gene to chromosome 16

Pseudotypes of vesicular stomatitis virus (VSV) containing envelope glycoproteins provided by C3H mammary tumor virus (MTV) instead of the normal VSV G-proteins were prepared and used to assay the presence of an MTV receptor on cells. The assay was specific as demonstrated by competition studies with excess MTV particles and neutralization of the pseudotypes with anti-MTV serum or monoclonal antibodies directed against MTV gp52. The MTV receptor was abundantly present on mouse cells but hardly detectable on nonmurine cells, including the Chinese hamster cell line E36. Somatic cell hybrids between E36 cells and GRS/A spontaneous leukemia cells (GRSL cells) and between E36 and GRS/A primary mammary tumor cells were made. The hybrids retained all Chinese hamster chromosomes but segregated mouse chromosomes. From the analysis of the isoenzymes and chromosomes of the hybrid cell lines we conclude that the gene for the receptor (MTVR-1) is located on mouse chromosome 16.     

Fibroblast interferon in man is coded by two loci on separate chromosomes.

We have examined viral and poly(rl):poly(rC) induction of interferon synthesis in several human, mouse and Chinese hamster cell lines, and in hybrids derived from the fusion of such cells. We observed species and cell-type differences in inducer effectiveness and in the kinetics of interferon production. In some cases, parental characteristics are preserved in somatic cell hybrids, and in other cases, the expression of the donor phenotype is modulated by the epigenetic state of the recipient cell. Mapping studies in human/mouse and human/Chinese hamster hybrids indicate that there are at least two structural genes for human fibroblast interferon. Chromosomes 2 and 5 each contain genetic information for the synthesis of fibroblast interferon. Gene dosage experiments indicate that one gene is on the long arm of chromosome 2 and another is on the short arm of chromosome 5. Leukocyte interferon genes could not be mapped to these chromosomes, but this negative result could be influenced by the epigenetic state of the hybrid cells.     

Assignment of RAS proto-oncogenes in Chinese hamsters: implications for mammalian gene linkage conservation and neoplasia

HRAS and KRAS are the cellular homologs of the oncogenic transforming genes found in the Harvey strain of murine sarcoma virus and the Kirsten murine sarcoma virus, respectively. Phyla as diverse as insects, birds, and mammals possess distinct HRAS and KRAS sequences, suggesting that these genes are essential to metazoa. In this report, we used a clone panel of Chinese hamster X mouse C11D somatic cell hybrids segregating hamster chromosomes to map those genes. Southern filter hybridization analyses of the hybrids revealed that hamster HRAS and KRAS gene sequences are on chromosomes 3 and 8, respectively. These gene assignments are consistent with the conservation of autosomal gene linkage groups observed among hamsters, humans, and mice and may provide insight into specific chromosomal alterations that have been observed during the spontaneous neoplastic transformation of Chinese hamster fibroblasts in vitro.     

Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding

We have isolated chromosomes from Chinese hamster ovary cells arrested in mitosis with vinblastine and examined the interactions of their kinetochores with purified tubulin in vitro. The kinetochores nucleate microtubule (MT) growth with complex kinetics. After an initial lag phase, MTs are continuously nucleated with both plus and minus ends distally localized. This mixed polarity seems inconsistent with the formation of an ordered, homopolar kinetochore fiber in vivo. As isolated from vinblastine-arrested cells, kinetochores contain no bound tubulin. The kinetochores of chromosomes isolated from colcemid-arrested cells or of chromosomes incubated with tubulin in vitro are brightly stained after anti-tubulin immunofluorescence. This bound tubulin is probably not in the form of MTs. It is localized to the corona region by immunoelectron microscopy, where it may play a role in MT nucleation in vitro.     

Predominant loss of hamster chromosomes in hybrids of somatic cells from the Dzungarian hamster and mice]

By means of the application of UV-inactivated Sendai virus interspecific hybrids of Dzungarian hamsterXmouse somatic cells were obtained in HAT selective medium. Karyotypic changes in these hybrid somatic cells were recorded during a 13 months' period. In the beginning each hybrid somatic cell contained 1 chromosome set of Dzungarian hamster and 1 mouse chromosome set. It was observed that throughout 13 months' of cultivation the elimination of Dzungarian hamster chromosomes prevailed over that of mouse chromosomes.