Properties of ts Cl mouse L cells which exhibit temperature-sensitive DNA synthesis

Guttman, S.; Sheinin, Rose

doi:10.1016/0014-4827(79)90435-x

Cited by 21 publications

(8 citation statements)

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“…The conditions for the maintenance, growth, and temperature manipulation of the ts mouse L cells and their wild-type (WT-4) parent have been described [10][11][12]. The same is true for the BALB/c 3T3 mouse fibroblast [13] and its ts 2 derivative [14,15].…”

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“…Both gene products are required for semiconservative DNA replication, which occurs normally during the DNA-synthetic, or S, phase of the cell duplication cycle [3,5,11,12,24], Because they arrest at unique stages early in S phase, they are designated DNA'7S'\ The is 2 mouse fibroblast arrests at the Gi/S interface [28] upon temperature inactivation of a polypeptide which acts to effect S-phase entry, perhaps by facilitating interaction of DNA polymerase-a and DNA primase [27]. The ts2 cell is therefore DNA'1/ Gi'1 [3,5,28].…”

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Mouse Polyoma Virus and Adenovirus Replication in Mouse Cells Temperature-Sensitive in DNA Synthesis

1985

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Mouse adenovirus multiplies, apparently without impediment, in temperature-inactivated ts A1S9, ts C1 and ts 2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA-chain-elongation factor, and a protein required for traverse of the Gi/S interface, respectively, encoded in the ts A1S9, ts C1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus.

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Mouse Polyoma Virus and Adenovirus Replication in Mouse Cells Temperature-Sensitive in DNA Synthesis

1985

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“…(i) Asynchronous wild-type cells exposed to araC showed a shutoff in DNA synthesis within minutes, whereas RNA synthesis was not affected for at least 10 h. The same cells exposed to aamanitin (5 FLg/ml) had DNA synthesis depressed to -30% of the control value after 8 h and to 15% after 10 h. In the same cells total incorporation of [3H]uridine into RNA was still almost 100%o of control values 8 h after the addition of a-amanitin and was depressed by only 55% after 10 h (unpublished data). (ii) Several reports in the literature (11,41,45) It was noted above that 10 h after the addition of a-amanitin to wild-type cells the total incorporation of uridine began to be affected, and hence mRNA and rRNA synthesis appear to be coupled to some extent (see also below). A more precise comparison of the behavior of the mutant after a temperature shift with a-amanitintreated wild-type cells may not be warranted because of uncertainties related to (i) the rate of uptake of the drug and (ii) the rate of inactivation of the temperature-sensitive function.…”

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Temperature-Sensitive Chinese Hamster Fibroblast Mutant with a Defect in RNA Metabolism

Wong

Scheffler

1982

Mol. Cell. Biol.

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We describe a new temperature-sensitive mutant of Chinese hamster cell fibroblasts. After a shift to the nonpermissive temperature of 40.5°C, the rates of DNA, RNA, and protein synthesis declined rapidly (to n50% within 12 h) and the progression of unsynchronized cells through the cell cycle was affected. We believe that DNA synthesis came to a halt after a short time, because cells no longer entered the S phase. The decrease in protein synthesis at 40.5°C was shown to be a consequence of a decrease in the number of polysomes, whereas free 80S ribosomes accumulated. We concluded that the components of the protein biosynthetic machinery were intact (ribosomes and soluble factors), but synthesis was limited by a shortage of mRNA. The decline in mRNA production had a significant effect on the synthesis of proteins (e.g., heat shock proteins) translated from short-lived messages. We observed that both polyadenylated and nonpolyadenylated RNA syntheses declined at 40.5°C, whereas the synthesis of small RNAs (4 to 5S) was less reduced. The argument is made that the temperaturesensitive phenotype is the result of a defect affecting mRNA synthesis.Mutants with temperature-sensitive proteins provide one of the most powerful and simple opportunities for studying the role of a specific protein in metabolic pathways, biosynthetic reactions, cell proliferation, or regulation. Fur All manipulations of the cells at 40.5°C were performed in the same room to prevent significant fluctuations in the temperature.

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“…Whereas several temperature-sensitive mutants affected in progression through Gi of the cell cycle have been investigated within collections of mutants in Syrian hamster BHK cells (2,29), rat 3Y1 fibroblasts (31), and other cell lines (23,46), rarely have mutants been found to be defective in DNA synthesis. Temperature-sensitive mutants with biochemical defects in DNA synthesis have been identified in mouse cell lines such as L (5,10,43), BALB/3T3 (37,48), and FM3A (27). Those in the former two mouse cell lines may, however, be interrelated since they do not complement each other in cell hybrids (14).…”

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Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

Dermody¹,

Wójcik²,

Du³

et al. 1986

Mol. Cell. Biol.

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We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA-based on differential inhibition of macromolecular synthesis at the restrictive temperature (39°C) as assessed by incorporation of[3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39°C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33°C and subsequent shift to 39°C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39°C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.Temperature-sensitive mutants have been a powerful tool in understanding the molecular mechanism of many cellular processes. The paucity of such conditionally lethal variants has hampered severely the analysis of DNA replication in higher eucaryotes. For this discussion we wish to make a distinction between DNA replication which involves the totality of functions which are required for reproduction of a cellular (or viral) genome and the biochemistry of DNA synthesis itself. Whereas several temperature-sensitive mutants affected in progression through Gi of the cell cycle have been investigated within collections of mutants in Syrian hamster BHK cells (2, 29), rat 3Y1 fibroblasts (31), and other cell lines (23, 46), rarely have mutants been found to be defective in DNA synthesis. Temperature-sensitive mutants with biochemical defects in DNA synthesis have been identified in mouse cell lines such as L (5, 10, 43), BALB/3T3 (37,48), and FM3A (27). Those in the former two mouse cell lines may, however, be interrelated since they do not complement each other in cell hybrids (14). Methods of selection commonly utilized to isolate temperature-sensitive mutants have involved prolonged exposure to restrictive temperature, and the mutants in DNA synthesis described above typically lose viability under such conditions. Taken together, these results indicate limitations in the prevailing strategies used to isolate an...

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Properties of ts Cl mouse L cells which exhibit temperature-sensitive DNA synthesis

Cited by 21 publications

References 20 publications

Mouse Polyoma Virus and Adenovirus Replication in Mouse Cells Temperature-Sensitive in DNA Synthesis

Mouse Polyoma Virus and Adenovirus Replication in Mouse Cells Temperature-Sensitive in DNA Synthesis

Temperature-Sensitive Chinese Hamster Fibroblast Mutant with a Defect in RNA Metabolism

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

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