Somatic Cell Genetics最新文献

Temperature-sensitive (ts) mutant cells for cell reproduction were isolated from the Syrian hamster cell line BHK21/13 by multiple culturing in the presence of 5-fluoro-2'-deoxyuridine (FdU) at 37.5 degrees after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. A simple method for cell fusion was devised, which enabled us to perform complementation studies with a large number of ts mutants. By using the method we have analyzed 219 ts mutants and classified them into 18 complementation groups. Mutants that belonged to the same complementation groups tended to exhibit similar patterns of inhibition of DNA synthesis at 39.5 degrees; however, some mutants belonging to the same group showed somewhat different patterns, probably due to occurrence of different mutations in the same gene. Distribution of the ts mutants among the 18 complementation groups was uneven; more than 50% of the mutants examined were assigned to complementation group B and G. The mutations belonging to complementation group B and G were found to be linked to the X chromosome.

Characterized mouse lens epithelial cell cultures and their clonal isolates obtained from normal and cataractous mice display a finite life-span. The cells of cataractous origin have a decreased number of population doubling levels compared to the capacity of the normal cells to replicate in vitro. Hybrid cells derived from individual cell fusions of normal (CD-1, DBA/2) and cataractous (Catfr) lens epithelial cells have a mode of replication similar to that of normal cells. These results demonstrate that the abnormal in vitro replicative state of the cataractous cells has been modified by the addition of normal lens epithelial cell components.

A new clonal CBA mouse teratocarcinoma cell line called H6 grows rapidly without calcium in suspension culture. When attached to a gelatin-coated surface in the presence of calcium, differentiation to large flat cells, which secrete plasminogen activator, occurs after exposure to retinoic acid. Differentiation is terminal after about six cell divisions. H6 variants resistant to 6-thioguanine, 5-bromodeoxyuridine, ouabain, chloramphenicol, retinoic acid, or combinations thereof, have been isolated. Cell hybridizations were made between undifferentiated stem cells and the differentiated derivatives of H6. These experiments indicate that the undifferentiated cells can "rescue" cells in the initial stages of differentiation and that the signs of differentiation are soon lost in such hybrids. Hybridization without "rescue" may also occur, as suggested by small abortive colonies of large cells.

Dimethyl sulfoxide (DMSO) has many biological effects, which include enhancement of polyethylene glycol (PEG) -mediated cell fusion, induction of cell differentiation in erythroleukemia and other cell systems, and cryoprotection of cells from freezing damage. In this study, compounds which induce erythroleukemia cell differentiation were tested for their ability to enhance PEG-mediated cell fusion. It was found that many compounds which induce erythroleukemia cell differentiation also promote cell membrane fusion as well as protect cells against freezing damage. Hence, many inducers of erythroleukemia cell differentiation have direct and similar effects on cell membranes. This study also demonstrates previously unrecognized effects of cryoprotective agents and cell fusogens on the differentiated state of cultured cells.

Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m2, each of the mutants showed less than 10% of the incision rate of the parental AA8 cells. After 50 J/m2, the rate in AA8 was similar to that at 6 J/m2, but the rates in the mutants were significantly higher (approximately 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, these CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV.

The mechanism by which 5-bromodeoxyuridine (BrdU) inhibits cell differentiation is unresolved. The ability of deoxycytidine to reverse the inhibition of myogenesis produced by BrdU has been cited as evidence that the inhibition is not a direct result of the incorporation of BrdU into cellular DNA. In contrast to previous work, the present study demonstrates a direct correlation between the effects of deoxycytidine on myogenic cells and a reduction in the substitution of BrdU for thymidine in the DNA. Further-more, the reversal occurs at the same degree of BrdU substitution (20-30%) as is required to inhibit myogenesis when cells are grown in BrdU alone or with deoxycytidine in a medium that prevents the conversion of deoxycytidine to thymidine. The effects of deoxycytidine thus do not support a mechanism of action of BrdU in myogenic cells independent of its effects on DNA.

A method is presented for the detection of antigenic changes in heterokaryons during the first 24 h after fusion, as detected by antibody- and complement-mediated inhibition of 125IDU uptake. The method employs a variation of the HAT selection protocol, and a mathematical model which allows the reactions of a minority population of heterokaryons to be distinguished from those of the parental cells. This procedure is applied to fusions of clones of the teratocarcinoma-derived cell line TerC, which demonstrate very low levels of H-2b expression, with an L-cell derivative rich in H-2k antigens. Heterokaryons demonstrate an initial tendency for levels of H-2b and H-2k expression to converge, beginning within a few hours of fusion; however, H-2b expression never attains the levels of H-2k, even in long-term hybrids. These results are largely confirmed by quantitative absorptions, except that no diminution of H-2k expression is observed, suggesting that fusion with the TerC parents may transfer a resistance to complement damage to the L-cell parent.

A study has been made of the H-2 profiles of the AKR thymoma K36 and two series of somatic cell hybrids derived from it. Despite expressing good amounts of the H-2Dk product, the H-2Kk antigen was barely detectable in this tumor, approximately only 1% of that expressed by a comparable AKR lymphoma, 339. The isoelectric focusing profile of the H-2kk product immunoprecipitated from K36 was found to be identical to that from normal AKR lymphocytes. The phenotype of K36 therefore results from regulatory constraints. Fusion of K36 with normal K-2k lymphocytes resulted in hybrids expressing the H-2Kk product. Fusion of K36 with normal H-2b lymphocytes resulted in good expression of the H-2Dk and H-2Db alleles in addition to good expression of the H-2Kb products. The expression of the H-2Kk antigen remained marginal. The data suggest a suppressive mechanism which is trans-acting and dominant and specific for the H-2Kk allele.

Stable mutants of Dede and CHO cells, resistant to suppression of cholesterogenesis by oxygenated sterols, have been isolated in a single step. Luria-Delbrück fluctuation analysis indicated a random occurrence of resistant at a rate of 1 x 10(-7) mutations/cell/generation. Cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and growth of the mutant cells were coordinately resistant to oxygenated sterols in the culture medium, and this resistance was expressed as a dominant trait in somatic cell hybrids of the wild-type and mutant cells. The dominant resistance was employed in the selection of various cells hybrids. There was complete additivity of reductase activities in mixed lysates of inhibited wild-type and uninhibited mutant cells, indicating that cytosolic (in)activation factors were not causative of this resistance. We suggest that oxygenated sterols are (co)repressors in suppression of the synthesis of the reductase and that the resistance mutant phenotypes result from altered regulatory loci.

When populations of single Chinesee hamster ovary (CHO) cells were exposed to the mutagen ethyl methane sulfonate (EMS), allowed to grow into colonies, and stained for glucose-6-phosphate dehydrogenase (G6PD) activity, two types of unstained colonies were observed at a frequency of about on per thousand stained colonies. These negative-staining colonies consisted of (1) colonies uniformly deficient in staining activity (pure); and (2) colonies containing both stained and unstained sectors (mosaic) in various relative sizes and patterns. Unstained cells isolated from mosaic colonies were genetically stable and had significantly reduced or absent G6PD activity. Random cell aggregation or chromosome segregation from tetraploid cells is not a significant cause of the sectoring phenomenon. Also, mosaic colonies are not principally caused by mutation at one of two replicated G6PD genes and their subsequent segregation during division. The simplest explanation for this phenomenon is that EMS induces a mutational change in one of the two DNA strands and DNA replication then produces normal and mutant double-stranded DNAs which segregate into wild-type and G6PD-deficient cell types, producing a mosaic colony.