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-G1 at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G1 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-G1 arrest protocol to analyze gene expression in late G1. We found that although stimulation of cells in amino acid-deficient medium did not inhibit the induction of genes expressed in early G1, genes normally expressed in late G1 were expressed only after release from the V point. The expression of late G1 genes in cells released from the V point was temporally similar, in respect to G1 location, as was seen in stimulation of quiescent G0 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 G1 and commitment to DNA synthesis. In addition, we used c-myb antisense oligonucleotides to show that c-myb expression, which occurs in late G1, is required for BALB/c-3T3 fibroblasts to traverse late G1 and initiate DNA synthesis.
Olson
J E
JE
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Winston
J T
JT
Whitlock
J A
JA
Pledger
W J
WJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004277:DNA, Single-Stranded; D016193:G1 Phase / Q000235:genetics*; D015870:Gene Expression; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009857:Oncogenes; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics; D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger / Q000096:biosynthesis
D004277:DNA, Single-Stranded; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger