Genetic analysis of control of proliferation in fibroblastic cells in culture. I. Isolation and characterization of mutants temperature-sensitive for proliferation or survival of untransformed diploid rat cell line 3Y1

Abstract

Mutants temperature sensitive for proliferation or survival were isolated from an untransformed diploid clone of fibroblastic rat cells (3Y1), according to an isolation protocol that selected for mutants defective at 38.5 degrees C (selection temperature) in undergoing the transition from quiescent to proliferating state while maintaining viability at 38.5 degrees C. Of the 108 temperature-sensitive clones isolated, 32 were examined for survival in sparse cultures at 39.8 degrees C (nonpermissive temperature) and classified into four classes. Results of temperature shift-up experiments suggest that functions defective in 11 of the 32 mutants are necessary not only for changing from the quiescent to proliferating state but also for maintenance of the proliferating state. Of the 32 mutants, 17 were assigned to eight complementation groups. Results of the physiological characterization of the representative mutants of each of the eight complementation groups are presented.

Translation Initiation Requires Cell Division Cycle 123 (Cdc123) to Facilitate Biogenesis of the Eukaryotic Initiation Factor 2 (eIF2)

“…Cdc123 and its mammalian orthologs were originally described as cell proliferation factors required for the G1/S transition of the cell cycle (15,20). We propose that the observed cell cycle arrest in cdc123 mutants is a consequence of reduced translation initiation rates, because the G1/S transition is especially sensitive to defects in protein synthesis (39).…”

“…Cdc123 was first described in a rat fibroblast line carrying a temperature-sensitive allele which blocked the G1-S transition and prevented serum-induced cell cycle entry of quiescent cells (15,16). More recently, human CDC123 was described as a candidate oncogene in breast cancer (17), a candidate risk locus for type II diabetes (18) and a gene associated with lung function (19).…”

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

“…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).…”

Macromolecular Synthesis in a Set of Temperature-sensitive Mutants of Rat 3Y1 Fibroblasts under Proliferation-arresting and Proliferation-resuming Conditions

“…Temperature-sensitive (ts) mutants of somatic cells are useful for understanding the reproduction process and its control in animal cells in cult~re.2,3) Ohno et al 4 ) isolated a large number of ts mutants from a diploid rat fibroblast line 3YI,5,6) assigned them to one of 4 classes based on viability and proliferating ability at 39.8°C (a nonpermissive temperature), and briefly described the overall pattern of macromolecular synthesis in these mutants, when cells proliferating at 33.8°C (a permissive temperature) were exposed to 39.8°C. Class I mutants (3 complementation groups), which lose viability quickly at 39.8°C ("shortsurvival" mutants), are inhibited in all of their DNA, RNA, and protein syntheses at 39.8°C.…”