Isolation of temperature-sensitive CHO-K1 cell mutants exhibiting chromosomal instability and reduced DNA synthesis at nonpermissive temperature

Abstract

Twenty-five temperature-sensitive (ts) mutants were isolated from Chinese hamster CHO-K1 cells after mutagenization with N-methyl-N'-nitro-N-nitrosoguanidine. Of 13 complementation groups identified, nine exhibited chromosomal instability at a nonpermissive temperature. They were classified into three major classes according to inducibility of sister chromatid exchange (SCE) and/or chromosomal aberration (CA): class 1 resulted in predominant SCEs, class 2 manifested both SCEs and CAs, and class 3 exhibited higher induction of CAs. Flow cytometric analysis of the mutants exhibiting chromosomal instability indicated that many of the mutants were arrested in the S or S to G2 phases of the cell cycle at the nonpermissive temperature, accompanied by a decrease in the rate of DNA synthesis. These results imply that ts defects are related to some points in DNA replication and might be responsible for the induction of SCEs and/or CAs at the nonpermissive temperature.

Vanadate Triggers the Transition from Chromosome Condensation to Decondensation in a Mitotic Mutant (tsTM13)

“…The method of isolation and the characteristics of tsTM13 cells have been described in detail [24]. Both the tsTM13 and wild-type CHO-K1 [281 cells free from mycoplasma contamination were routinely maintained as monolayers in Dulbecco's modified Eagle minimal essential medium supplemented with 10% fetal bovine serum (Gibco), 34.5 pg/ml of Lproline, and 60 pg/ml of kanamycin.…”

“…A temperaturesensitive Chinese hamster cell mutant tsTM13, has a defect in the M phase traverse and in chromosome decondensation [24]. The mutant exhibits a cell cycle arrest from anaphase to telophase and a lack of chromosome decondensation even in telophase when sister chromatids become segregated and cytokinesis is completed [25].…”

Different populations of RNA polymerase II in living mammalian cells

“…The mutation in tsTM4, which grows at 34 C but not at 39 C, has been mapped to RPB1 (Tsuji et al 1990, Sugaya et al 1997. The gene encoding wild-type human RPB1 was fused with another encoding GFP, and the construct expressed in tsTM4; the resulting GFPtagged polymerase (GFP-pol) complemented the defect at the restrictive temperature (39 C), and so enabled normal growth (Sugaya et al 2000).…”

Loss of Smu1 function de-represses DNA replication and over-activates ATR-dependent replication checkpoint

“…Tsuji and Sugaya showed that the temperature sensitive CHO-1 K1 mutant cell line (tsTM18) exhibited reduced DNA synthesis and cell cycle arrest at S and G2 phase at non-permissive temperature [12,13]. We think the contradiction is due to the different mutant forms of Smu1 studied in each work.…”

“…Smu1 is also involved in the genome stability maintenance of higher organisms. A temperature-sensitive (ts) CHO-K1 mutant cell line, tsTM18, was isolated in a screen to search for mutant cells that exhibit chromosomal instability at non-permissive temperatures [12]. Smu1 was found to be defective and responsible for the cause of abnormal splicing, increased chromosomal breakage, reduced DNA synthesis and accumulation of single-stranded DNA in tsTM18 cells [13].…”