Molecular cloning of transcripts that accumulate during the late G1 phase in cultured mouse cells

Abstract

To identify previously undetected genes that might be involved in later stages of the transition from a quiescent state (GO) to the DNA synthetic phase (S) of murine cells, we set out to isolate cDNA clones derived from mRNAs that appear late in G1 phase in serum-stimulated cells. A λ-cDNA library was prepared using poly(A) + RNA from chemically transformed Balb/c 3T3 cells (BP/A31) that had been brought to quiescence and subsequently stimulated for 12 h with serum. From the first screening of approximately 21,000 recombinant phage plaques, about 100 clones were isolated that hybridized to a single-stranded cDNA pool derived from stimulated-cell RNA but not to DNAs made from resting-cell RNA. Eventually, six different clones were identified. The mRNAs from five of these genes increased gradually during the GO to S transition, in contrast to the “immediate-early” rise of c- myc mRNA or the later rise of thymidine kinase mRNA. These six clones were sequenced and compared to the GenBank database. Clones LG-80, LG-2, and LG-69 are highly homologous to β-actin, lactate dehydrogenase, and α-tubulin. Clones LG-5, LG-61, and LG-74 had no significant homologies to known sequences. A subtractive cDNA library was used to isolate two additional clones, Sub-S1 and Sub-S2; these have homologies to enolase and ribosomal protein L32. Additional studies that examine the function and regulation of these newly identified “late response” genes in the pre-DNA synthesis pathway are in progress.