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

Abstract

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.

Characterization of the translational defect to fiber synthesis in monkey cells abortively infected with human adenovirus: role of ancillary leaders

“…The message was on authentic ribosomes as defined by their sensitivity to puromycin. In addition, the large amount of fiber mRNA migrating in the 80S region of the gradient appeared to be associated with ribosomes, since pronase treatment at 4°C (which disrupts ribonucleoprotein aggregates but not monosomes [25,59,68]) did not alter the distribution of the fiber mRNA (data not shown). Moreover, Anderson and Klessig (5) demonstrated that these ribosomes had initiated translation in vivo and were active in translational runoff in vitro.…”

Enhanced synthesis of the glucose/calcium‐regulated proteins in a hamster cell mutant deficient in transfer of oligosaccharide core to polypeptides

“…It was renamed H3, and H3.5 is a subclone of H 3 . The temperaturesensitive mutant H20 with a defect in RNA metabolism was subsequently isolated from WglA cells by ethyl methane sulfonate mutagenesis (Wong and Schemer, 1982). These cells were routinely maintained in Dulbec-CO'S modified Eagle's medium (DMEM) containing 10% cadet calf serum.…”