Showing papers in "Somatic Cell and Molecular Genetics in 1975"

Production of mammalian somatic cell hybrids by means of polyethylene glycol treatment

Abstract: Polyethylene glycol (PEG) is known to promote fusion of plant protoplasts. Various adaptations of this treatment to mammalian, including human, cell cultures are reported here. PEG is very effective in producing hybrids capable of indefinite multiplication even in cases, such as early passage human skin fibroblasts and lymphocytes, known to be highly recalcitrant to other treatments.

The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells.

Abstract: The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells clone K1-BH4 was studied at many EMS doses including the minimally lethal range (0-100 microng/ml) as well as the exponential killing portion (100-800 microng/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10(-6)(8.73+3.45 X), where 0 less than or equal to X less than or equal to 800 microng/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.

Gene linkage analysis in the mouse by somatic cell hybridization: assignment of adenine phosphoribosyltransferase to chromosome 8 and alpha-galactosidase to the X chromosome.

Abstract: Somatic cell hybridization techniques were applied to gene linkage analysis in the laboratory mouse. Cells of an established line of Chinese hamster lung fibroblasts were fused with mouse embryo fibroblasts and with mouse peritoneal macrophages obtained from different inbred strains: From 3 hybridization experiments, 123 primary and secondary clones were isolated in HAT selective medium and 24 were back-selected in 8-azaguanine. Hybrid clones were characterized for the expression of 16 murine isozymes by starch, acrylamide, and Cellogel electrophoresis, and on the basis of segregation data, 3 syntenic associations could be made. Malate oxidoreductase decarboxylating (MOD) and mannose phosphate isomerase (MPI) segregated concordantly, confirming an established linkage relationship;adenine phosphoribosyltransferase (APRT) segregated concordantly with glutathione reductase (GR) which is known to be on chromosome 8;α-galactosidase was observed to be syntenic with hypoxanthine phosphoribosyltransferase (HPRT), and X-linked enzyme. All other isozymes examined segregated independently of one another.

The dose-response relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells

Abstract: Exposure of Chinese hamster ovary (CHO) cells clone K1BE4 to ultraviolet (UV) light at doses up to 86 ergs/mm2 did not significantly reduce cell survival, but UV doses of 86–648 ergs/mm2 produced an exponential cell killing. Observed mutation frequency to 6-thioguannine resistance induced by UV increases approximately in proportion to increasing doses up to 260 ergs/mm2 in a range of 5–648 ergs/mm2 examined. The pooled data of mutation frequency f(X) as a function of dose X from 0–260 ergs/mm2 is adequately described by f(X)=10−6 (13.6+2.04 X). That the UVinduced mutations to 6-thioguanine resistance affects the hypoxanthineguanine phosphoribosyltransferase (HGPRT) locus is supported by the observation that all randomly isolated drugresistant colonies contained highly reduced or undetectable HGPRT activity.

Fluorescence analysis of late DNA replication in human metaphase chromosomes

Abstract: Thymidine incorporated as a terminal pulse into chromosomes otherwise substituted with 5-bromodeoxyuridine can be detected by associated bright 33258 Hoechst fluorescence. The location of metaphase chromosome regions identified by this method as last to complete DNA synthesis is consistent with the results of autoradiographic analyses with tritiated thymidine. The very late-replicating regions correspond to a subset of those which appear as bands after chromosomes are stained by quinacrine or modified Giemsa techniques. The high resolution of the 33258 Hoechst fluorescence pattern within individual cells is especially useful for revealing variations in the order of terminal replication. Both homolog asynchrony and fluctuations in the distribution of bright 33258 Hoechst fluorescence within chromosomes from different cells are apparent and localized to individual bands. The results are consistent with the possibility that these bands constitute units of chromosome replication as well as structure.

Selection by [3H] amino acids of CHO-cell mutants with altered leucyl- and asparagyl-transfer RNA synthetases.

Abstract: Efficient selection procedures, using [3H]amino acids as the selecting agent, were developed for isolating temperature-sensitive (TS) mutations in CHO cells affecting protein synthesis. After chemical mutagenesis, leucyl-tRNA synthetase mutants were obtained when [3H]leucine was used as the selecting agent in two independent experiments. These mutations seem to involve the same genetic locus as the TSH1 mutant described previously (1). A selection with [3H]valine, in which all amino acids except leucine were at low concentration in the selective medium, resulted in a new class of mutants with reduced asparagyl-tRNA synthetase activity. These results were consistent with the finding that all mutants were phenotypically dependent on the concentration of amino acid, specific to the altered synthetase, in the medium. Our observations suggest that although leucyl synthetase mutations are a relatively common class of TS mutations in CHO cells, the spectrum of mutants obtained can be at least partially manipulated through concentrations of amino acids in selective media. The asparagyl-synthetase mutation was shown to be recessive and to complement the leucyl-synthetase mutation in cell-cell hybrids.

Expression of differentiated functions in hepatoma cell hybrids: IX extinction and reexpression of liver-specific enzymes in rat hepatoma-Chinese hamster fibroblast hybrids

Abstract: Most of the hybrid clones derived from a cross of Chinese hamster fibroblasts (DON) with rat hepatoma cells (Faza 967) showed preferential loss of rat chromosomes. Two of the hybrid clones retained the rat chromosomes, and both showed extinction of 4 liverspecific enzymes: aldolase B, liver alcohol dehydrogenase, and the inducible enzymes tyrosine aminotransferase and alanine aminotransferase. Subcloning of 1 of these hybrids, which contained 2 sets of hepatoma chromosomes and 1 set of hamster chromosomes, permitted the isolation of some clones which reexpressed 1 or more of the liver-specific enzymes. Liver alcohol dehydrogenase was the most frequently reexpressed enzyme and aldolase B the least. Tyrosine aminotransferase inducibility was reexpressed independently of basal activity, and the enzyme produced by the reexpressing hybrid cells was precipitated by a specific antiserum. No correlation was detected between the presence or absence of the marker chromosomes (large metacentrics) of the hamster parent and the extinction and reexpression of the hepatic enzymes. The results reported confirm and extend to interspecific hybrids the observation of the stable and independent reexpression of tissue-specific enzymes.

Stable alterations at the cell membrane of Chinese hamster ovary cells resistant to the cytotoxicity of phytohemagglutinin

Abstract: Chinese hamster ovary (CHO) cells selected for resistance to the cytotoxicity of phytohemagglutin (PHA) have been found to exhibit stable alterations at their plasma membranes. The PHA-resistant (Pha R) cells bind markedly less125 I -PHA than do sensitive CHO cells and also exhibit an increased sensitivity to the cytotoxicity of concanavalin A, a lectin of different receptor specificity. Mutagenesis with ethylmethanesulfonate increases the proportion of Pha R cells 20- to 100-fold. PHA-resistant cells maintained for up to 8 months in continuous culture in the absence of the selective agent have retained the PhaR phenotype. These and other characteristics of the experimental system suggest that CHO cells selected for PHA resistance are authentic somatic cell mutants. The Pha marker appears to behave recessively in hybrids formed between PhaR and PhaS cells.

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: Biochemical analysis of eight mutants

Abstract: Purine biosynthesis was studied in 8 mutants of Chinese hamster cells which require purines for growth and in wild-type cells which do not show this nutritional requirement. Of these, 6 mutants, ade −B, ade−D, ade−E, ade−F, GAT−, and AT−, were shown to accumulate metabolic intermediates not accumulated by wild-type cells. These intermediates were shown to be compounds unique to the adenylic acid biosynthetic pathway by the following criteria: (a) their radioisotopic labeling properties, (b) their response to agents which specifically inhibit known enzymatic steps in the pathway, (c) their chromatographic properties, and (d) spectrophotometric analysis. Two mutants, ade−A and ade−C, accumulate no detectable compounds not accumulated by the wild type. These 2 mutants are believed to be defective in steps very early in the purine biosynthetic pathway. The sites of the defects in the other mutants are proposed, and the usefulness of these mutants is discussed.

Chinese Hamster Mouse Hybrid Cells Segregating Mouse Chromosomes and Isozymes

Abstract: 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.

Incorporation of isolated chromosomes and induction of hypoxanthine phosphoribosyltransferase in Chinese hamster cells.

Abstract: Evidence is presented for the uptake of radioactive-labeled isolated Chinese hamster chromosomes following incubation with Chinese hamster cells. Metaphases were found which contained radioactive labeled chromosomes in a very low frequency, and in some of the labeled chromosomes only one chromatid was labeled. Incubation of hypoxanthine phosphoribosyltransferase (HPRT)-deficient Chinese hamster cells with chromosomes isolated from HPRT+ Chinese hamster or human cells resulted in the appearance of HPRT+ cells. Clones derived from these cells were isolated in HA T medium. Cells in mitosis during incubation with the chromosomes yielded three times more HPRT+ clones than did cells in interphase. The intraspecies combination involving recipient cells and chromosomes from Chinese hamster origin yielded significantly higher numbers of HPRT+ clones than did the interspecies system using human chromosomes and Chinese hamster recipient cells (5×10−5 and 6×10−6 respectively). Electrophoresis of HPRT from Chinese hamster cells treated with human chromosomes revealed the pattern of the human enzyme.

Extinction of liver-specific functions in hybrids between differentiated and dedifferentiated rat hepatoma cells.

Abstract: A cross has been performed between dedifferentiated rat hepatoma cells and the differentiated cells from which they were derived. 10 hybrid clones, containing the complete chromosome sets of both parents, show extinction of 4 liver-specific enzymes: tyrosine aminotransferase (E.C. 2.6.1.5), alanine aminotransferase (E.C. 2.6.1.2), and the liver-specific isozymes of alcohol dehydrogenase (E.C. 1.1.1.1) and aldolase (E.C. 4.1.2.13). Moreover, the 4 hybrid clones examined do not produce albumin. The only function of the differentiated parent which is not extinguished in the hybrid cells is inducibility of the aminotransferases. For 3 of the hybrid clones, extinction of 3 of the 4 enzymes is incomplete, but these clones do not differ in modal chromosome number from those which show more complete extinction of the enzymes. Subcloning of several of the hybrids revealed that the phenotype of the hybrids is very stable; 4 subclones showing reexpression of intermediate levels of the enzymes are characterized. These results show that dedifferentiation of the parental cells is not due to the simple loss of some factor required for the maintenance of expression of differentiated functions, and suggest that dedifferentiation is due to the activation of some control mechanism, whose final effect is negative, and which may be a part of the epigenotype of the embryonic hepatocyte.

Genetics of somatic cell surface antigens. III. Further analysis of the AL marker.

Abstract: The AL antigen present on the surface of various human somatic cells, and on those hybrids of human and Chinese hamster ovary cells which have retained human chromosome number 11, has been resolved into at least two separate antigenic activities, a1 and a2. Specific antisera active against each antigen separately have been prepared. By treatment of the original AL+ hybrid with mutagenic agents and selection in particular antisera, stable clones are preparable whose phenotypic behavior corresponds to the antigenic compositions a1+ a2−, a1− a2+, and a1− a2−. The adsorption behavior of these variants for specific antisera is consistent with their phenotypic assignments.

Isolation of chloramphenicol-resistant variants from a human cell line

Abstract: Variant clones resistant to 40 μg/ml chloramphenicol were isolated from the human cell line VA 2-B after treatment with either ethyl methanesulfonate or N-methyl-N′-nitro-N-nitrosoguanidine. Among 17 clones analyzed, one variant, CAP-23, was investigated in detail. CAP-23 cells in the presence of 40 or 100 μg/ml chloramphenicol grew at essentially the same rate as cells in the absence of the drug; chloramphenicol resistance persisted even after 20 generations in the absence of the drug. No obvious morphological changes in mitochondria were observed by electron microscopy of thin sections of CAP-23 cells. In vivo mitochondrial protein synthesis in CAP-23 cells was inhibited little, if any, by chloramphenicol, and the variant showed partial cross resistance to mikamycin and carbomycin. In vitro protein synthesis in mitochondria isolated from CAP-23 cells showed, likewise, low levels of inhibition by chloramphenicol. This suggests that the drug resistance of the variant CAP-23 is due to altered mitochondria.

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

Abstract: 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-A 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.

Reactivation of chick erythrocyte nuclei after fusion with enucleated cells.

Abstract: Inactivated Sendai virus was used to fuse nucleated chick erythrocytes with mouse L and A9 cells which had been enucleated by centrifugation in the presence of cytochalasin B. The enucleation step removed the nuclei from more than 99% of the cells. During the fusion step, chick erythrocyte nuclei were introduced into 20% of the enucleated mouse cytoplasms. This resulted in the formation of a large number of “reconstituted cells” where practically all the cytoplasm originated from the mouse cell while the nucleus was of chick origin. The chick erythrocyte nuclei appeared to become well integrated into the mouse cytoplasms since they increased dramatically in size and dry mass, formed nucleolus-like bodies, and resumed RNA synthesis. This, however, did not prevent a gradual decrease in the rate of protein synthesis in the cytoplasm after the removal of the mouse nucleus. Protein synthesis decayed at a similar rate in both reconstituted and enucleated cells. The majority of these “cells” died within 48 h and all of them within 5 days after enucleation/fusion. By contrast, the small number of L cells which failed to become enucleated multiplied rapidly. The results obtained suggest that the reactivation of the chick erythrocyte nuclei is not fast enough to rescue the enucleated mouse cytoplasms.

A human-mouse somatic hybrid line selected for human deoxycytidine deaminase.

Abstract: A new selective medium has been developed for cells containing the enzyme deoxycytidine deaminase. This medium contains hypoxanthine, aminopterin, and 5-methyldeoxycytidine (HAM medium). To survive in the presence of the aminopterin, the cells must utilize deoxycytidine deaminase to convert the 5-methyldeoxycytidine to thymidine. The cells must also have thymidine kinase and hypoxanthine phosphoribosyltransferase. A mouse cell line deficient in deoxycytidine deaminase has been isolated from a deoxycytidine kinase-deficient line, using 5-bromodeoxycytidine as the selective agent. A hybrid line between this double mutant and a human diploid fibroblast was isolated in HAM medium. The hybrid line contains the chromosomes expected of a human-mouse hybrid. The deoxycytidine deaminase isozyme patterns on cellogel show that the human-mouse hybrid cell line produces an enzyme with an electrophoretic mobility intermediate between that of the human and that of the mouse.

Replication of human chromosomes in human-mouse hybrids: Evidence that the timing of DNA synthesis is determined independently in each human chromosome

Abstract: The terminal phase of DNA replication was studied by autoradiography in hybrids between human lymphocytes and mouse fibroblasts. The hybrids contained on the average only 11 human chromosomes. It was found that the sequence of terminal DNA replication for the human chromosomes in the hybrids was the same as the sequence of terminal replication for the corresponding chromosomes in the human lymphocytes. Furthermore, it was shown that the maintenance of the normal terminal replication sequence of the human chromosomes in the hybrids was not dependent on the presence of any specific human chromosome. The results suggest that the timing of terminal DNA replication is determined independently in each human chromosome.

Synteny between the Pro+ marker and human glutamate oxaloacetate transaminase

Abstract: Chinese hamster ovary cells with a specific auxotrophy for proline were fused with human cells from a variety of sources and the resulting hybrids analyzed for human genetic markers. Of 63 hybrid clones examined, 27 possessed both proline and cytoplasmic glutamate oxaloacetate transaminase markers; 36 had neither; and no clones were found possessing one and not the other. These results constitute evidence that the proline and glutamate oxalocetate transaminase markers are syntenic. Evidence for absence of synteny between these and a variety of other human genes is presented. Biochemical tracer experiments established that the proline biosynthetic pathway through glutamate has been restored in the Pro+ hybrids.

Absence of Thy-1 antigen in L-cell X mouse lymphoma hybrids.

Abstract: Hybrid clones arising after inactivated Sendai virus mediated cell fusion between a bromodeoxyuridine-resistant derivative of the BALB/c mouse lymphoma cell line, S49, and a thioguanine-resistant derivative (A9) of the C3H mouse fibroblast cell line, L929, were selected in HAT medium. The responses of the clones to growth inhibitors as well as their chromosome numbers were consistent with properties expected of hybrids. Hybrid clones expressed the major histocompatibility (H-2) surface antigens of both parental types, i.e., H-2d of BALB/c and H-2k of C3H. The Thy-1.2 antigen, expressed on the surface of the lymphoma parent but not the fibroblast parent, was not detected on the hybrids.

Control of the expression of a herpes simplex virus thymidine kinase gene incorporated into thymidine kinase-deficient mouse cells.

Abstract: When thymidine kinase-deficient mouse cells “transformed” by inactivated herpes simplex virus and expressing the viral thymidine kinase (TK) are grown in nonselective medium, there is an exponential decay in the proportion of cells that continue to express the viral enzyme. However, the viral TK can be reactivated at a frequency of approximately 1 cell in 106in every population that has lost TK activity. When cells in which the viral TK has been reactivated are grown in nonselective medium, a decay in the expression of the viral enzyme occurs again at the same rate as in the initial transformed population. Studies on the reactivation of viral TK indicate that reappearance of the enzyme is not induced by the selective medium (HAT) used to detect cells in which the enzyme has reappeared. Furthermore, treatments known to induce latent viruses in other systems—eg, exposure of the cells to mutagens or cell fusion—do not affect the frequency with which viral TK is reactivated.

Globin synthesis in fibroblasts fused with erythroblasts. I. Avian fibroblasts.

Abstract: Heterokaryons of chick embryo erythroblasts fused with other avian fibroblasts were studied with regard to globin production. After the incorporation of radioactive amino acids, soluble proteins were separated on SDS-urea polyacrylamide gels. There was a striking increase in radioactivity above background in the globin region from lysates of fusion cultures when compared with fibroblast cultures. This was maximal at 24 hours after fusion, and then declined. Electrophoresis on acid-or alkaline-urea gels further identified the material as globin chains. Tryptic digestion and fingerprinting revealed methionine-labeled peptides characteristic of chick embryo erythroblast globin. An apparent stimulation of globin chain synthesis by heterokaryons compared to erythroblasts was found to be due to a difference in the specific activity of the precursor amino acid pools in the different cell types.

Human RNA transcripts in man-mouse somatic cell hybrids. II. Thermal denaturation studies and Cot analysis.

Abstract: The present report confirms an earlier finding that human RNA transcripts from a man-mouse somatic cell hybrid that has regularly retained only the human X chromosome can be identified by molecular hybridization on nitrocellulose filters. From thermal denaturation studies it is concluded that molecular hybrids between hybrid cell and human nucleic acids have higher T m's, and thus greater specificity, than those between mouse and human nucleic acids. Additional data, utilizing the kinetics of molecular hybridization in solution (Cot analysis), demonstrate the presence of “few gene copy” DNA sequences in the above-mentioned hybrid cells that are complementary to human (HeLa) HnRNA. Also shown is a fraction of hybrid cell HnRNA that is complementary to “few gene copy” DNA sequences of human DNA. Thus, the experiments reported may represent a specific assay for identifying reiterated and “few gene copy” DNA sequences of the human X chromosome and their complementary RNA. Theoretically this experimental approach may be extended to any chromosome. Some of the short-term and long-term perspectives of these types of studies are discussed.

Heterotopic chondrogenesis and osteogenesis induced by transformed cells: Use of nude mice as a model system

Abstract: A subline of HeLa cells was shown to induce cartilage and bone formation in congenitally athymic (nude) mice when injected intramuscularly. The chondrogenesis and osteogenesis observed was similar to that found when other inducing epithelia are injected intramuscularly into cortisonetreated mice. A tumorigenic SV40 T antigen-positive human-mouse somatic cell hybrid with human chromosomes C7 and C6 did not induce cartilage or bone formation under similar conditions. The nude mouse may thus provide a system in which to investigate experimental host-cell differentiation without the complications of immunosuppression.

Cell-mediated immunoselection against cell-surface antigens of somatic cell hybrids.

Abstract: Cytotoxic lymphoid cells derived from in vivo immunization of mice across H2 barriers were utilized in in vitro cytotoxicity assays. The target cells were somatic cell hybrids derived from parental cells differing at the H2 locus. The hybrid cells surviving cytotoxicity were grown to confluent populations and the H2antigens selected against were no longer demonstrable by indirect immunofluorescence. Comparative karyology of hybrid cells expressing both parental H2 types before immunoselection with hybrid cells surviving immunoselection revealed a decrease in the number of murine chromosomes number 17, suggesting that those cells surviving cytotoxicity had spontaneously lost these chromosomes prior to the selection event. The possibility of immunoconstruction of somatic cell hybrids on the basis of their cell-surface antigens is discussed.

Interaction of male and female germ cells of two mammalian species in culture.

Abstract: When viewed by scanning electron microscopy, the heads of mouse spermatozoa are smaller than those of the hamster. The vitelline microvilli of hamster eggs are longer than those of the mouse egg. Both these factors may contribute to the enhanced interaction of mouse spermatozoa and hamster eggs. Treating unfertilized mouse eggs with Newcastle disease virus causes the vitelline microvilli to elongate, thus improving the interaction between mouse eggs and hamster spermatozoa.