Two candidate G1 polypeptides in chick embryo fibroblasts

Resting cultures of primary chick embryo cells have been labeled with ³H-leucine for the first 2 or 3 hours after feeding basal medium or basal medium supplemented with 10% dialyzed serum. The labeling patterns of the ³H-polypeptides of the soluble cell fraction have been compared by fluorography of two-dimensional gels. Large and consistent differences are seen in only three of the more than 900 spots that can visualized. This report concerns two of the three spots. The ³H contents of the two polypeptides (41,000 daltons, pI 7.1, designated 41–7.1, and 34,000 daltons, pI 6.2, designated 34–6.2) are increased by serum by about ten-fold. The highly selective effect of serum on the labeling of the two spots does not appear to be an artifact related to the extractability, solubility, or state of aggregation of the polypeptides. The radio-intensities of both polypeptides decrease markedly when the cells are labeled later than 3 hours after “shift-up”. Drugs that inhibit RNA synthesis and are known to stop the progression of the chick cells through the G1 period, camptothecin, cordycepin and 5,6-dichloro-β-D-ribofuranosylbenzimidazole, depress, with great specificity, the enhanced labeling of polypeptide 41–7.1 in the stimulated cells, and all but camptothecin have a similar action on polypeptide 34–6.2. A high level of actinomycin D (10 μg/ml), but neither a low level of the drug (0.02 μg/ml) nor 5-fluorouridine prevents the increased labeling of the two polypeptides in serum-fed cells. That 5-fluorouridine enters the chick cells and is converted to its active form is shown by the inhibition of the processing of pre-ribosomal RNA. The observations with the RNA inhibitors are at least consistent with the conclusions that the enhanced labeling of the two sports results from increased rates of synthesis of the polypeptïdes that depend upon mRNA production but not on the formation of ribosomal RNAs, and that the polypeptides play a role in the regulation of DNA replication in the chick cells.