Peter Zahradka

RNA polymerase II-directed gene transcription by rat skeletal muscle nuclear extracts☆

A cell-free transcription system was developed using nuclear extracts of rat skeletal muscle to examine the transcription of specific genes involved in ribosome biogenesis and histone synthesis. Isolation and purification of muscle tissue nuclei were required prior to obtaining a transcriptionally active extract. The transcriptional abilities of myoblast, myotube, and muscle tissue nuclear extracts were then compared using the adenovirus major late promoter as a reporter gene. Transcription of r-protein L32 and histone H4 gene templates remained high in all extracts while histone H3 gene transcription was reduced in both myotube and muscle tissue extracts. These data indicate that transcription of these genes in myotubes and muscle tissue nuclear extracts is similar. Therefore, the L6 myoblast system accurately reflects the ability of intact muscle tissue to transcribe the genes concerned with histone production and ribosome biogenesis.

Regulation of ribosome biogenesis in differentiated rat myotubes.

The rate of ribosome biogenesis is closely coupled with cell proliferation, representing a unique model system for studying gene regulation. Terminal differentiation of rat L6 myoblasts, an example of a rapidly proliferating population of cells being converted into a non-dividing syncytial population, results in an 80% decline in the rate of ribosome accumulation. Ribosome production during myogenesis is regulated by a down-shift in the rate of rRNA accumulation, controlled at the level of transcription by specific trans-acting factors. The synthesis of both ribosomal proteins and 5S rRNA remains unchanged in myotubes, however, resulting in an over-production of these precursors. The excess molecules are rapidly degraded, preventing the accumulation of a static pool of ribosome components.

Down-regulation of histone H3 and H4 gene transcription in differentiated L6 myotubes

The core histone mRNA levels in terminally differentiated L6 myotubes decrease to less than 5% of the amount present in proliferating myoblasts in parallel with the cessation of DNA synthesis (Bird, RC., Jacobs, F.A., Stein, G., Stein, J. and Sells, B.H. (1985) Biochim. Biophys. Acta 824, 209-217). The role of gene transcription in the down-shift of histone mRNA levels was assessed using a cell-free system. The level of transcription from the differentiation-independent adenovirus major late promoter was directly related to the RNA polymerase II activity of myoblast and myotube nuclear extracts. In addition, both extracts actively transcribed the histone H4 gene template containing only the 5 proximal promoter region (-210 bp). In contrast, inclusion of the distal-proximal promoter region (-410 to -210 bp) in the template resulted in a 60% decrease in transcription by the myotube extract. A similar down-shift in transcription of the histone H3 gene template (containing 900 bp 5 of the initiation site) by myotube nuclear extracts was also observed. The decrease in histone mRNA levels in myotubes may therefore be controlled in part by a transcriptional mechanism involving a negative regulatory factor.

Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replication in vitro.

SummaryThe role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase α by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 µM) inhibitory to DNA polymerases β and γ, however, higher concentrations of ddTTP (200 µM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase ε (PCNA-independent DNA polymerase δ) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.