8-Azaguanine Resistance in Mammalian Cells I. Hypoxanthine-Guanine Phosphoribosyltransferase

Gillin, Frances D.; Roufa, D J; Beaudet, Arthur L.; Caskey, C. Thomas

doi:10.1093/genetics/72.2.239

Cited by 264 publications

(7 citation statements)

References 17 publications

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“…Therefore, to obtain a better estimate of the efficiency of the mutagenesis, a selection for AG-resistant mutants was done using cells mutagenized by the same protocol as the cells used for the neurite-deficient screen. This screen produced AG-resistant mutants at a frequency of-10 -4 which is two orders of magnitude greater than the spontaneous frequency we observed and is similar to frequencies reported by others for AG-resistant mutants (Gillin et al, 1972;Sharp et al, t973). Thus, in EMS mutagenized cultures, induced mutations should outnumber spontaneous mutations by a factor of 100, and therefore the neurite-deficient clonal cell lines we have obtained are all likely to be EMS-induced rather than spontaneous mutants.…”

Section: Mutagenesis and Screensupporting

confidence: 90%

PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor.

Green¹,

Rydel²,

Connolly³

et al. 1986

The Journal of Cell Biology

274

163

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Abstract. Four mutant PC 12 pheochromocytoma cell lines that are nerve growth factor (NGF)-nonresponsive (PC12 n"r) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC 12 ""~ cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC 12 ~nr cells closely resembled non-NGFtreated PC 12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC 12 nnr lines differed from PC 12 cells in three ways in addition to failure of NGF response: (a) PC12 n"r cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC I2 cells. (b) The PC 12 nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC I2 cells possess both high-and low-affinity NGF binding sites. (c) The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC 12 n"r cells compared to PC I2 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC I2 TM cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity.

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Section: Mutagenesis and Screensupporting

confidence: 90%

PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor.

Green¹,

Rydel²,

Connolly³

et al. 1986

The Journal of Cell Biology

274

163

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“…The cells were washed and placed in culture. Treatment with EMS was according to the method of Gillin et al (20). 5 ml of tumor cells at 6 X 105 cells/ml were placed in small, sterile petri dishes and allowed to equilibrate to a 5% CO2 atmosphere in RPMI without fetal calf serum.…”

Section: Methodsmentioning

confidence: 99%

Selection of strongly immunogenic "tum-" variants from tumors at high frequency using 5-azacytidine.

Frost¹,

Liteplo²,

Donaghue³

et al. 1984

The Journal of Experimental Medicine

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Treatment of normally tumorigenic murine tumor cell lines in vitro with chemical mutagens followed by cloning of the surviving cells, results in the selection, at extraordinarily high frequencies (anywhere from <1% to >90%), of clones unable to grow progressively in normal syngeneic mice (1-7). Such clones, which are phenotypically stable in culture over a period of several weeks or months, have been designated by Boon and his colleagues (1-6) as "tum-" (nontumorigenic in normal hosts). ~ They have been derived using such standard mutagens as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and ethyl methanesulfonate (EMS), from at least eight different murine tumors of viral, chemical, or spontaneous origin (1-7). Because the turn-clones will grow readily in highly immunosuppressed recipients, e.g., X-irradiated or nude mice (1-8), the turn-phenotype appears to have an underlying immunological basis, a view confirmed by detailed in vitro studies of T cell-mediated cytotoxicity (CMC).These studies have also shown that each turn-clone derived from a particular mutagenized parent line possesses an individual tumor-specific antigen distinct from the new antigen found on any other turn-clone, i.e., there is a startling degree of tumor antigen polymorphism (1-7). Of considerable interest is that this holds true even for totally non-immunogenic tumors of spontaneous origin (5-7) and as such, has very broad and important implications for the field of tumor immunology, especially in relation to the controversies surrounding the antigenic and immunologic status of neoplastic cells (9-11). Nevertheless, very little is known about how mutagens can cause such drastic and heritable changes in the behavioral properties of tumors in vivo, and at such high frequencies.

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“…UCW 10 was mutagenized with ethylmethane sulfonate as described previously (7), then grown at 33*C in nonselective medium for 4 d . Cells were then distributed into 100-mm culture dishes at a density of 4 x 10' cells/dish in nonselective medium .…”

Section: Selection Of Chromate-resistant Mutantsmentioning

confidence: 99%

Linkage in cultured Chinese hamster cells of two genes, emtB and leuS, involved in protein synthesis and isolation of cell lines with mutations in three linked genes.

Wasmuth¹,

Chu²

1980

The Journal of Cell Biology

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We have determined via segregation analyses from appropriate hybrids that two genes involved in protein synthesis, one encoding for a ribosomal protein (emtB) and one encoding for leucyl-tRNA synthetase (IeuS), cosegregate at a very high frequency and are linked in both Chinese hamster ovary and lung cells. In contrast, the emtA locus, defined by a second complementation group of emetine-resistant mutants which also have alterations affecting protein synthesis and probably the ribosome, is not linked to IeuS . In addition, we have determined that a third gene, one that can be altered to give rise to chromate resistance, is syntenic with emtB and IeuS . We have selected cell lines with mutations in each of these three linked genes and have shown that the three loci cosegregate at a high frequency. Because the mutations in these three linked genes provide easily distinguishable phenotypes, these cell lines should provide a powerful tool for examining several important questions concerning mitotic recombination in somatic cells.The genetic and biochemical dissection of protein synthesis in mammalian cells has benefited greatly in recent years from the use of mutant methodology. A large number of cultured somatic cell mutants have been isolated with alterations in different components of the protein synthetic machinery (9,10,12,16,18,19). One goal of this laboratory is to expand the number of such mutants in cultured Chinese hamster cells and to characterize them at the molecular level, especially mutants with alterations affecting various components of the ribosome . A second goal is to use such mutants to begin constructing a genetic map of this group of functionally related genes.We have isolated and partially characterized three distinct types of Chinese hamster cell mutants that were selected as being resistant to the protein synthesis inhibitor, emetine (19; J Hill, L. Vock, and J. Wasmuth, manuscript in preparation) . Although the phenotypes of all three types of mutants are very similar, and all have alterations affecting protein synthesis directly, they clearly define three distinct complementation groups . For all three classes, the emetine-resistant phenotypes are recessive. We have designated the loci defined by these different complementation groups as emtA, emtB, and emtC . Two laboratories (4, 8) have obtained evidence that Chinese hamster ovary (CHO), but not Chinese hamster lung (CHL), cells are functionally hemizygous for a locus that can be altered

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8-Azaguanine Resistance in Mammalian Cells I. Hypoxanthine-Guanine Phosphoribosyltransferase

Cited by 264 publications

References 17 publications

PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor.

PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor.

Selection of strongly immunogenic "tum-" variants from tumors at high frequency using 5-azacytidine.

Linkage in cultured Chinese hamster cells of two genes, emtB and leuS, involved in protein synthesis and isolation of cell lines with mutations in three linked genes.

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