1. When WI-38 human diploid fibroblasts form confluent monolayers, DNA synthesis and cell division almost completely cease. A change of medium causes these density-inhibited cells to proliferate and within 1h after the application of the stimulus there is an increase in template activity of the chromatin isolated from stimulated cells. 2. The number of binding sites for Escherichia coli RNA polymerase was determined on chromatin from WI-38 cells by two different methods, i.e. incorporation of [(3)H]UTP into RNA in the absence of reinitiation, and incorporation of [gamma-(32)P]GTP into chain termini. 3. Both methods indicate that the capacity of chromatin to bind E. coli RNA polymerase is increased in WI-38 cells stimulated to proliferate. 4. The increase in the number of binding sites for E. coli RNA polymerase parallels the increase in chromatin template activity and suggests that the latter reflects an increase in the number of initiation sites, rather than an increase in the rate of transcription.

Binding Sites↗

PubMed1974

Effect of 5-bromodeoxyuridine on chromatin transcription in confluent fibroblasts.

3T6 mouse fibroblasts were grown in 5-bromodeoxyuridine (BrdU) so that approximately 20% of the thymine residues in DNA were replaced by BrdU. BrdU replacement caused an alteration in the relative incorporation of labeled nucleotide precursors into RNA. The RNA synthesized by cells grown in BrdU has a lower proportion of adenine and a higher guanine complement. This was shown for (a) nascent RNA made in vivo by confluent monolayers of cells in culture; (b) RNA synthesized in vitro on a chromatin template with either homologous or heterologous RNA polymerase; and (c) RNA synthesized in vitro on a DNA template with a highly purified RNA polymerase. The product was completely digested by RNase. The relative decrease in the incorporation of adenine into RNA was reserved when BrdU-treated cells were allowed to proliferate in BrdU-free medium.

Adenosine↗

PubMed1974

Increased RNA synthesis in nuclear monolayers of WI-38 cells stimulated to proliferate.

Nuclear monolayers of WI-38 cells prepared by the method of Tsai and Green were used to determine RNA synthesis in isolated nuclei in situ. In nuclear monolayers, incorporation of [(3)H]UTP into RNA is dependent on the presence of the other three nucleotide triphosphate and is abolished by actinomycin D. The extent of RNA synthesis under these conditions was measured in density-inhibited WI-38 human diploid fibroblasts at various intervals after cell proliferation was stimulated by a change of medium.RNA synthesis increases 15 min after the nutritional change and reaches a peak at 18 hr, which is also the peak of DNA synthesis. Thereafter RNA synthesis declines. Essentially similar results are obtained whether the endogenous RNA polymerase or a bacterial polymerase is used. Replacement of the stimulating medium by conditioned medium stops the increase in RNA synthesis that occurs in cultures subject to continuous stimulation. Finally, RNA synthesis in nuclear monolayers, using the endogenous RNA polymerase, occurs by chain elongation only, while re-initiation occurs with the bacterial RNA polymerase.

Cell Division↗

PubMed1973