Partial purification and characterization of the mRNA complementing a temperature-sensitive S-phase cell cycle mutation

E36ts24 is a temperature-sensitive (ts) cell cycle mutant derived from the Chinese hamster lung cell line E36. At the restrictive temperature for growth (40.3°C), the mutant cells are arrested at early S phase. We have microinjected poly(A)⁺ RNA isolated from the wild-type E36 cells into the cytoplasm of E36ts24 cells arrested at early S phase after 24 hr of incubation at 40.3°C. The ts mutation was transiently complemented in a significant fraction of the microinjected cells as evidenced by the incorporation of [³H]thymidine assayed by autoradiography. Microinjection of mRNA fractionated by methylmercuric hydroxide/agarose gel showed that the mRNA capable of transiently complementing the mutation in E36ts24 contains about 940 nucleotides. Hence, it can code at most for a protein containing about 230 amino acids. We estimate that the partial purification by fractionation of the mRNA active in the transient complementation is on the order of 100- to 200-fold.