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 ≤50% 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 temperature-sensitive phenotype is the result of a defect affecting mRNA synthesis.