Transfer of the marker for morphologically transformed phenotype by isolated metaphase chromosomes in hamster cells.
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Animals↗
Biomedical subjects
L Siminovitch
Publications and source records attributed to L Siminovitch.
The nature of conditionally lethal temperature-sensitive mutations in somatic cells.
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Amanitins↗
Evidence for variation in the number of functional gene copies at the AmaR locus in Chinese hamster cell lines.
The hypothesis of functional hemizygosity has been examined for the alpha-amanitin resistant (AmaR, a codominant marker) locus in a series of Chinese hamster cell lines. AmaR mutants were obtained from different cell lines, e.g., CHO, DHW, M3- 1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards alpha-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to alpha-amanitin inhibition, only about 50% of the activity is highly resistant in AmaR mutants of CHW and M3- 1 cell lines. The remaining activity in the latter cell lines shows alpha-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by alpha-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.
Amanitins↗
Genetic and biochemical characterization of mutants of CHO cells resistant to the protein synthesis inhibitor trichodermin.
Mutants resistant to the protein synthesis inhibitor trichodermin have been selected in Chinese hamster ovary (CHO) cells. The mutants vary in their stability from those which rapidly lose their resistance to others which are relatively stable after prolonged growth in nonselective medium. Protein synthesis in extracts from the latter class of mutants (Trir) is resistant to the inhibitory action of trichodermin as compared to similar extracts from wild-type cells. After dissociation into subunits, the ability of the 60S ribosomal subunits from Trir cells to function in a protein-synthesizing system is greatly diminished. This subunit also shows reduced binding of [acetyl-14C]TRICHODERMIN. The lesion in Trir mutants therefore seems to have affected this ribosomal subunit. Trir X Tris hybrids are sensitive to trichodermin indicating that the Trir mutation behaves recessively to Tris in hybrids. The Emtr and Trir markers segregate independently from hybrid cells showing that the Trir mutation is probably not linked to the Emtr locus, which as we have shown earlier affects the 40S ribosomal subunit.
Cell Line↗
Diphtheria-toxin-resistant mutants of CHO cells affected in protein synthesis: a novel phenotype.
Stable mutants highly resistant to the protein-synthesis-inhibitor diphtheria toxin have been selected in Chinese hamster ovary (CHO) cells. Protein synthesis in extracts of mutant cells is resistant to the inhibitory action of diphtheria toxin, indicating that the lesion has affected the protein-synthesis machinery. However, about 50% of the elongation factor-2 (EF-2) activity in the mutant cells can still be ADP-ribosylated by diphtheria toxin, and this remaining EF-2 activity is similar to that present in the wild-type cells. We suggest that this result is best explained by assuming that our CHO cells contain two functional copies of the EF-2 gene, and that only one of the copies is altered in the mutants. According to this view, the mutated allele produces EF-2 resistant to ADP-ribosylation which is capable of supporting cell growth in the presence of diphtheria toxin. Although the Dipr marker seems to act dominantly in the parental CHO cells, its behavior in Dipr X Dips hybrids (CHO X CHO) is recessive as measured by cell survival in presence of the toxin. This paradoxical behavior may be due to a gene dosage effect. Segregation studies from hybrids show that the Dipr marker segregates independently of the Emtr and Thgr markers indicating that the Dipr locus is not linked to either the Emtr locus or to the X chromosome.
Adenosine Diphosphate Sugars↗
Genetic and biochemical studies with the adenosine analogs toyocamycin and tubercidin: mutation at the adenosine kinase locus in Chinese hamster cells.
The pyrrolopyrimidine nucleosides toyocamycin and tubercidin show several unique features of growth inhibition in Chinese hamster ovary (CHO) cells. Stable mutants which are more than 600-fold resistant to these drugs are obtained in CHO cells at a strikingly high frequency of approximately 10(-3), in the absence of mutagenesis. The mutants resistant to toyocamycin (Toyr) and tubercidin (Tubr) exhibit similar cross-resistance patterns to the two selective drugs as well as to adenosine and 6-methyl mercaptopurine riboside, indicating that the same lesion is probably involved in all cases. The mutants examined were found to be deficient in the enzyme adenosine kinase (AK), indicating that the phosphorylation of these analogs is an essential first step in their toxic action. The above mutants (AK-) behaved recessively in cell hybrids, and segregation studies indicate that the AK locus is not linked to the X chromosome. The frequencies of similar Toyr mutants in other Chinese hamster lines, e.g., V79, CHW, M3-1, GM7, and CHO-K1, varied from similar to more than three logs less than that observed for CHO cells, indicating that various cell lines probably differ in the number of functional gene copies for this locus.
Adenosine Kinase↗
Mutants of CHO cells resistant to the protein synthesis inhibitor emetine: genetic and biochemical characterization of second-step mutants.
Second-step mutants highly resistant to the protein synthesis inhibitor emetine (Emt(RII) have been selected from emetine resistant (Emt(RI)) Chinese hamster ovary cells described earlier. The frequency of the Emt(RII) mutants was increased 50- to 75-fold after mutagenesis, and none of these highly resistant mutants could be selected in one step using wild-type cells. Like the Emt(RI) mutants, the increased resistance of Emt(RII) mutants results from another lesion in the polyribosomal fraction, as measured by the effects of emetine in fractionated extracts. As with the first-step mutants, the Emt(RII) isolates behave recessively in somatic cell hybrids. Segregation studies have shown that the Emt(R) lesions are not on the X chromosome, and in at least one isolate there is evidence that the Emt(RI) and Emt(RII) mutations may occur at different sites.
Animals↗
The relationship between transformation and somatic mutation in human and Chinese hamster cells.
The frequencies of transformations of primary human and Chinese hamster fibroblasts have been compared with the spontaneous and induced frequencies of mutation for resistance to thioguanine and ouabain, and for ability to use fructose, using the carcinogens benzo (alpha) pyrene and urethane. Whereas the rates and frequencies of mutation were similar in the two cell systems, transformations to morphologically altered cells was observed only in hamster cells. The frequency of this latter transformation event in hamster cells was abour 10(3) greater than the frequencies of mutation in these cells. The morphologically altered cells formed in the above transformation process cannot grow in agar (aga-) and do not produce tumors when injected into animals. The frequency of transition of these latter cells to aga+ cells which produce tumors in animals is similar to the mutation-like events.
Benzopyrenes↗
Evidence for functional hemizygosity at the Emtr locus in CHO cells through segregation analysis.
The hypothesis of functional hemizygostiy at the emetine-resistant (Emtr, a non-X-linked recessive marker) locus in Chinese hamster ovary (CHO) cells has been examined by segregation analysis. The frequencies and the rates of segregation of the Emtr and Thgr (thioguanine-resistant, an X-linked recessive mutation) markers were determined from hybrids constructed between an Emtr-Thgr CHO cell line and various other Chinese hamster lines (V79, M3-1, CHO, GM7S, CHW and CHL). Thgr segregants were obtained at similar frequencies (10(-2)-10(-3)) from all the hybrids. The frequency of segregation of the Emtr marker, however, was similar to that of Thgr only in the CHO x CHO hybrids and was much lower (10(-4)-10(-6)) than the CHO x other Chinese hamster hybrids. Similar results were obtained when the segregation rates for the two markers from various hybrids were determined. These results are consistent with the hypothesis that in CHO cells, the gene responsible for Emtr is present in a single (functional) copy, whereas two copies of this gene are present in other Chinese hamster lines examined.
Cell Line↗
Isolation and characterization of mutants of human diploid fibroblasts resistant to diphtheria toxin.
Stable mutants highly resistant to the protein synthesis inhibitor diphtheria toxin (dipr) have been selected in human diploid fibroblast cells at a frequency of 1-8 X 10(-6). Treatment of cells with mutagens, (e.g., ethylmethanesulfonate, nitrosoguanidine, and ICR-170), increased the frequencies of dipr mutants by 50- to 500-fold in different experiments, and the optimal expression time for dipr mutation was about 5 days. All mutants examined thus far have bred true, and no effects of cell density or cross feeding have been observed on the selection. Fluctuation analysis showed that the dipr mutation occurs in these fibroblasts at the rate of 5-6 X 10(-7) mutations per cell per generation. Protein synthesis in mutant extracts was resistant to diphtheria toxin, indicating that the dipr lesion in such mutants lies in the protein synthesis machinery. The characteristics of the dipr marker should make this system particularly useful for studies of quantitative mutagenesis in human diploid cells.
Cell Line↗
Mutants of CHO cells resistant to the protein synthesis inhibitors, cryptopleurine and tylocrebrine: genetic and biochemical evidence for common site of action of emetine, cryptopleurine, tylocrebine, and tubulosine.
Stable mutants resistant to the protein synthesis inhibitors cryptopleurine and tylocrebine can be isolated in Chinese hamster ovary (CHO) cells, in a single step. The frequency of occurrence of cryptopleurine (CryR) and tylocrebrine (TylR) resistant mutants in normal and mutagenized cell populations is similar to that observed for emetine resistant (EmtR) mutants. The CryR, TylR, and EmtR mutants exhibit strikingly similar cross-resistance to the three drugs used for selection, to tubulosine and also to two emetine derivatives cephaeline and dehydroemetine, based on assays of in vivo cytotoxicity and on assays of protein synthesis in cell-free extracts. The identity of cross-resistance patterns of the CryR, TylR, and EmtR mutants indicates that the resistance to all these compounds results from the same primary lesion, which in the case of EmtR cells has been shown to affect the 40S ribosomal subunit. This conclusion is strongly supported by the failure of EmtR, TylR, and CryR mutants to complement each other in somatic cell hybrids. Based on these results it is suggested that the above group of compounds possesses common structural determinants which are responsible for their activity. The above mutants, however, do not show any cross-resistance to other inhibitors of protein synthesis such as cycloheximide, trichodermin, anisomycin, pactamycin, and sparsomycin, either in vivo or in vitro, indicating that the site of action of these inhibitors is different from that of the emetine-like compounds.
Alkaloids↗
Complementation between mutants of CHO cells resistant to a variety of plant lectins.
Chinese hamster cell mutants resistant to the lectins PHA, WGA, RIC, LCA, and CON A were previously grouped into 8--10 distinct phenotypes on the basis of their unique patterns of lectin resistance and lectin-binding properties. All but one of these classes of lectin-resistant (LecR) mutants behave recessively in somatic cell hybrids. One ricin-resistant class (RicRII) behaves dominantly. Tests for complementation, by measuring the lectin-resistant properties of appropriate hybrids, show that seven distinct complimentation groups can be delineated among the phenotypically recessive mutants.
Cell Line↗
The molecular basis of emetine resistance in Chinese hamster ovary cells: alteration in the 40S ribosomal subunit.
The molecular basis of resistance to the protein synthesis inhibitor emetine has been examined in cell-free, protein-synthesizing extracts derived from normal and emetine-resistant (EmtR) mutants. We had earlier shown that protein synthesis in extracts of the mutant cells was resistant to the inhibitory action of emetin. When extracts from a wild-type and mutant cell line were fractionated into supernatant (S-100) and polyribosome fractions and mixed in different combinations, resistance to emetine was found to be associated with the mutant polyribosome fraction. Further fractionation of wild-type and mutant polyribosomes into 40S and 60S ribosomal subunits and mixing them in various combinations with an S-100 fraction from the wild-type cell indicates that resistance of mutant cells to emetine involves an alteration in the 40S ribosomal subunit. The behavior of EmtR has also been examined in somatic cell hybrids. Studies of EmtR X EmtS hybrid cell lines in vivo and in vitro show that EmtR is pheontypically recessive to EmtS, which is consistent with the ribosomal location of the genetic change.
Cell Line↗
Linkage of markers controlling consecutive biochemical steps in CHO cells as demonstrated by chromosome transfer.
Using the technology of metaphase chromosome transfer, evidence has been obtained in CHO cells that genes controlling enzymes in a common pathway in folate metabolism are closely linked. MtxRI and MtxRIII are co-dominant mutations which affect the structure and level of dihydrofolate reductase. Gat- is a glycine-, adenosine- and thymidine-requiring auxotrophic mutant with a lesion in folylpolyglutamate synthetase, an enzyme responsible for addition of glutamates to folate residues. GlyB- is an auxotrophic glycine-requiring mutant whose phenotype may be reversed by folinic acid. Using purified metaphase chromosomes, the MtxR genes were co-transferred into recipient cells with the auxotrophic markers, as demonstrated by the isolation of transferents when two of the phenotypes, either Mtx and Gat, or Mtx and GlyB, were selected at the same time. When recipient cells were selected for Gat+ or GlyB+ alone, the transferents carried the MtxR markers. The GlyA mutation, another glycine-requiring auxotrophic change, is not co-transferred with methotrexate resistance. Because of previous evidence that only a small fragment is involved in chromosomal transfer experiments, these results seem to provide the first indication that some genes which control enzymes on a common metabolic pathway in eucaryotes are closely linked or are at least syntenic.
Cell Line↗
Transfer of anchorage independence by isolated metaphase chromosomes in hamster cells.
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Agar↗
Transfer of codominant markers by isolated metaphase chromosomes in Chinese hamster ovary cells.
Codominant mutations to methotrexate and ouabain resistance in Chinese hamster ovary cells can be transferred to recipient Chinese hamster ovary cells by isolated metaphase chromosomes. For methotrexate, both the structural change and the increased activity of dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase EC 1.5.1.3), characteristic of the donor cells, are observed in the transferents (cells that carry and express functions derived by chromosome transfer). The transferents are unstable in the absence of selection although stable clones can be isolated. From results obtained by fractionation of chromosomes and transfer to recipients, the methotrexate and ouabain markers can be assigned to the middle and large size-classes of chromosomes, respectively. By fractionation and transfer of chromosomes, from transferents to new recipients, evidence has been obtained that chromosome integration is not restricted to a particular chromosomal site in the recipient.
Cell Cycle↗
Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells.
We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levls of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W.F., Davidson, S.V., and Siminovitch, L. (1976) Somatic Cell Genet.2,245-261). By selecting for increased resistance form these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In the report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.
Cell Line↗
Alpha-Amanitin resistance of RNA polymerase II in mutant Chinese hamster ovary cell lines.
A number of mutant Chinese hamster ovary (CHO) cell lines resistant to the cytotoxic action of alpha-amanitin have been isolated. The alpha-amanitin sensitivity of the different mutant cell lines varied widely, but correlated well with the alpha-amanitin sensitivity of the RNA polymerase II activity in each of these mutant cell lines. In comparison with the RNA polymerase II of wild-type cells, three mutants, Ama39, Ama6, and Amal, required respectively 2- to 3-fold, 8- to 10-fold, and about 800-fold higher concentrations of alpha-amanitin for inhibition of their polymerase II activity. Determination of the equilibrium dissociation constants (KD) for complexes between 0-[3H]methyl-demethyl-gamma-amanitin and RNA polymearse II indicated that differences in alpha-amanitin sensitivity were reflected in differences in the ability of the enzymes to bind amanitin. Hybrids formed by fusion of mutants with cells of wild-type sensitivity contained both mutant and wild-type polymerase II activities. Thus, each of the different alpha-amanitin resistance mutations was expressed co-dominantly. A test for complementation between two of these mutations by measurement of both the alpha-amanitin sensitivity and the [3H]amanitin binding by RNA polymerase II in Ama6 X Amal hybrid cells did not reveal any wild-type RNA polymerase II activity. These data provide evidence that the mutation to alpha-amanitin resistance involves structural changes in the gene coding for the alpha-amanitin binding subunit of RNA polymerase II. These changes appear to account for the alpha-amanitin-resistant phenotypes of these mutant cells.
Amanitins↗