Transcription of 5S RNA by RNA polymerase III from genomic bovine DNA templates

Abstract

RNA polymerase III in an extract of HeLa cells transcribes RNA 5S in size from genomic bovine DNA template. This RNA represents a major fraction of the RNA synthesized. 5S RNA is not transcribed from bovine chromatin at equivalent concentrations and RNA the size of tRNA or tRNA precursor is not detected. At low UTP concentrations RNA slightly smaller in size than 5S is synthesized in addition to 5S RNA.

Transcription of genomic bovine and Xenopus laevis DNA species by RNA polymerase III in HeLa-cell cytosol extracts

“…species, similar results were obtained on transcription of bovine DNA obtained from fetal-calf thymus, calf liver and bovine lymphosarcoma (Furth, 1983). The size distribution of RNA species differed from RNA transcribed from a variety ofhuman DNA species (Furth, 1983) and from mouse DNA. RNA transcribed from genomic mouse DNA, either liver or thymus, included not only heterogeneous RNA, approx.…”

“…Genomic DNA was isolated by methods described previously or as described by Maniatis et al (1982). Xenopus laevis erythrocyte DNA was prepared from nuclei obtained as described by Bogenhagen et al (1982) and from tissue as described previously (Furth & Su, 1983). 5S rRNA was prepared from rat liver as previously described (Ackerman et al, 1979) and from calf thymus by the same procedure, slightly modified.…”

“…A bovine DNA library, in Charon 28, grown in Escherichia coli K802, was a gift of F. Rottman, Case Western Reserve, Cleveland, OH. U.S.A. Cytosol extracts were obtained from HeLa cells grown in monolayers (Wu, 1978;Weil et al, 1979;Furth & Su, 1983).…”

“…Comparison of the individual oligonucleotide signal intensities on the radioautographs of the fingerprint suggests that the gene and the pseudogene were transcribed with approximately equal efficiency. Transcription of genomic bovine DNA RNA synthesized was heterogeneous in size except for a discrete 120 n RNA (Furth & Su, 1983;Furth, 1985; Fig. 4, lane a).…”

“…Only a limited number of genes are transcribed by this polymerase, and in cloning these genes, 5' or 3' sequences required for, or that may inhibit, transcription in vivo may have been lost. Initial results on transcription of genomic bovine DNA by RNA polymerase III in HeLa-cell cytosol extract indicated that, in addition to large heterogeneous RNA (of about 180-650 n), significant amounts of 5S-sized RNA (120 n) were synthesized (Furth & Su, 1983;Furth, 1985). Further studies, reported here, indicate that most of the 120 n RNA transcribed from genomic bovine DNA was transcribed on one DNA fragment obtained by restrictionfrom one widely dispersed bovine repeat, although a small amount of 5S rRNA was transcribed.…”