Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth

A library of double-stranded cDNA was constructed from ts13 cells, a G₁-specific temperature-sensitive hamster cell line. The cDNAs, cloned into pBR322, were prepared from poly(A)⁺ mRNA isolated from ts13 cells 6 hr after serum stimulation at the permissive temperature of 34° C. Differential screening of the library with G₁-specific and G₀-specific single-stranded cDNA probes prepared from the same cells identified 5 cDNA clones whose sequences were preferentially expressed in G₁. Levels of RNA complementary to these clones were 3- to 6-fold higher in G₁ than in other phases of the cell cycle. When ts13 cells were grafted in G₁ at the restrictive temperature of 39.6°C, the levels of RNA complementary to p13-2A9 and p13-4F1 were as high as 10 times that found in a resting population, while the expression of sequences complementary to p13-2A8 did not significantly change from levels found in G₀. RNA and Southern gel blot analysis suggest that these cell-cycle-specific clones represent either low copy or moderately repetitive gene sequences. Results with another ts mutant of the cell cycle, tsAF8, which is a ts mutant of RNA polymerase II, showed that these cell-cycle-specific sequences have a rapid turnover. The use of G₁-specific ts mutants of the cell cycle provides an approach to determine which cell-cycle-dependent genes are most relevant to cell-cycle progression.