Cell cycle‐dependent gene expression in V point‐arrested BALB/c‐3T3 cells

Density‐arrested BALB/c‐3T3 cells stimulated to proliferate in an amino acid‐deficient medium arrest in mid‐G 1 at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G 1 and enter S phase. As data presented here show that mRNA synthesis is needed for 2–3 hr after release of cells from the V point, after which inhibition of mRNA synthesis does not prevent entry into S phase, we used this mid‐G 1 arrest protocol to analyze gene expression in late G 1 . We found that although stimulation of cells in amino acid‐deficient medium did not inhibit the induction of genes expressed in early G 1 , genes normally expressed in late G 1 were expressed only after release from the V point. The expression of late G 1 genes in cells released from the V point was temporally similar, in respect to G 1 location, as was seen in stimulation of quiescent G o cells. As this protocol effectively divides gene expression into early (pre‐V point) and late (post‐V point) categories, it should be useful in studies of growth factor‐modulated events that regulate traverse of late G 1 and commitment to DNA synthesis. In addition, we used c‐ myb antisense oligonucleotides to show that c‐ myb expression, which occurs in late G 1 , is required for BALB/c‐3T3 fibroblasts to traverse late G 1 and initiate DNA synthesis. © 1993 Wiley‐Liss, Inc.