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

Abstract

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...

Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.

“…A 6-thioguanine (TG)-resistant clone deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (hprt-) was isolated from ts2O in Plasmids. p53A6.6 containing the whole polyomavirus genome cloned at the BamHI site in pAT153 was propagated and harvested from DH-1 (dam') bacteria as previously described (9,29).…”

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.

“…Moreover, this effect was observed even when the cells had been previously infected at 33°C, indicating that ongoing viral DNA synthesis was also affected. Since viral DNA synthesis would not be expected to be cell cycle dependent nine-free medium for 2 h, and incorporation into macromolecules was deterniined as previously described (9).…”

“…By contrast, few conditional DNA-defective mutants are available in mammalian cells, a fact that limits the use of genetic approaches to an understanding of their DNA metabolism (28). Dermody et al (9) have previously reported the isolation, utilizing a variety of selective and nonselective conditions, of a large collection of Chinese hamster cell mutants that are temperature sensitive for growth. Biochemical screening has identified a subset of nine mutants that were designated temperature-sensitive DNA (tsDNA) mutants, as they exhibited rapid inhibition of DNA synthesis but not protein synthesis upon shift of growing cultures from the permissive temperature (33°C) to the nonpermissive or restrictive temperature (39.5°C).…”

“…This finding, together with preliminary studies on correction of the temperature-sensitive growth defect in JB3-B by DNA-mediated gene transfer, raise the possibility that the primary defect is in a gene product other than that encoding either the Ml or M2 subunit of ribonucleotide reductase. (22 Ci/mmol), [5-3H] (9).…”

Regulation of the Multifunctional Ca2+/Calmodulin-dependent Protein Kinase II by the PP2C Phosphatase PPM1F in Fibroblasts

“…Chinese hamster fibroblast CHO-K1 was cultured in Dulbecco's modified Eagle's medium/F-12 medium (Invitrogen) and 10% fetal bovine serum as described previously (35). Mouse NIH 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 1 mM sodium pyruvate (Sigma) and 10% bovine calf serum (Sigma) as described previously (36).…”