Nascent RNA molecules were labeled in vivo and elongated in vitro by incubation of the isolated nuclei in the presence bf mercurated nucleotides. The RNA molecules initiated and labeled in vivo and elongated in vitro were then selectively purified on a thiopropyl 6-B Sepharose affinity column. The procedure was shown to be free of artifacts since the addition of mercurated nucleotides and the retention on the affinity column is mediated by the endogenous RNA polymerase II (nucleoside triphosphate:RNA nucleotidyltransferase; EC 2.7.7.6), is sensitive to actinomycin D, and is dependent on the presence of all four ribonucleotide triphosphates. This general procedure was applied to the mapping of viral promoters late after adenovirus 2 infection of HeLa cells. RNA purified as described above was hybridized to restriction enzyme fragments attached-to nitroceliulose filters. The 5' ends of the nascent RNA chains are located in coordinates 9.5-17 for a rightward transcript, 0-25 for a leftward transcript, and possibly 60-70 for a second rightwa*d transcript. These locations clearly differ from locations of the eatly promoters and therefore suggest that the transition from early to late functions is controlled at the transcriptional level. Control of transcription in prokaryotes occurs at the level of initiation (1). In evkaryotes, a complex series of modifications, including poly(A) addition (2), processing (3), methylation and capping (3), and splicing (4), mark the transition between primary transcripts and mature mRNAs (5). Each of these steps can be controlled independently; in particular, the nature of the signals-controlling the initiation of the RNA chains that are later processed remains obscure.Human cells infected by adenovirus type 2 (Ad 2) have been used as a model system for the study of RNA metabolism in eukaryotes since many features of viral RNA synthesis appear similar tothe analogous cellular. processes (6). In this system the host DNA-dependent RNA polymerase II (nucleoside triphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) or a virus-modified RNA polymerase seems to be responsible for the synthesis of viral mRNA precursors (7-9). A strong initiation site (promoter) (16) and spun through a 25% glycerol (10 mM Tris/5 mM MgCl2/0.1 mM EDTA/25 mM thioglycerol) cushion. They were resuspended in the cushion buffer at a concentration of 6 X 108 nuclei per ml and used directly in the reactions.Reactions. Nuclei were incubated as described (8) except that the reaction volume was 1-10 ml and UTP or CTP was substituted with HgUTP (15) or HgCTP (17) at a final concentration of 0.4 mM.Preparation of RNA. The reactions incubated at 250 were terminated by the addition of purified DNase (18) (Worthington, DPFF) to a final concentration of 40 jg/ml for 15 min at 40, then by the addition of one volume of 8 M urea sodium dodecyl sulfate (NaDodSO4) adjusted to 0.5% and proteinase K.(Merck) to 100 Aig/ml. Incubation was continued at room temperature for 30-60 min. Then phenol/chloroform/isoamyl extraction, ...
Potassium iodide (KI) has been shown to impair thyroid protein biosynthesis both in vivo and in vitro. The present study was performed in order to clarify its mechanism of action. Ribonucleic acid (RNA) synthesis was studied in beef thyroid slices with either [32P] or [ 3H] \ x=req-\ uridine as labelled precursors. Both KI and thyroxine (T4) at 10\m=-\5 m significantly decreased RNA labelling under our conditions. In other experiments RNA degradation was examined in pulse-labelled and actinomycin D-treated slices. KI did not modify the degradation of the [ 3H] \ x=req-\ RNA thus indicating that it interferes with the biosynthesis rather than with the degradation of RNA. Taking the perchloric acid soluble radioactivity as a rough index of the precursor pool the present results would indicate an action at this level. Both KClO4 and methylmercapto-imidazole relieved the gland from the inhibitory action of KI, supporting the view that an intracellular and
Potassium iodide (KI) has been shown to have an antigoitrogenic action and to inhibit in vivo thyroid protein biosynthesis. Beef thyroid slices were used to clarify further the mechanism of action of KI. Incubations were performed in Krebs-Ringer-bicarbonate (KRB) buffer under 95% O2 and 5% CO2. KI caused a slight decrease in the uptake of [3H]leucine by the tissue. When labelled leucine incorporation into protein was measured it was found that 10−6 m KI caused a marked inhibition. Increasing concentrations of KI up to 10−3 m did not further increase this inhibition. This effect of KI was reduced by simultaneous addition of 0.5 mm KClO4 or 1 mm methylmercaptoimidazole (MMI). In several experiments it was found that equimolar amounts of thyroxine (T4) or triiodothyronine (T3) were more potent than KI in inhibiting thyroid protein biosynthesis. In double labelled studies KI decreased [3H] leucine incorporation into thyroid soluble proteins and into immunoprecipitable thyroglobulin (Tg) while it did not modify that of [14C]galactosamine. When tissue specificity was examined, KI failed to alter [3H] leucine incorporation into proteins either in the liver or in the submaxillary gland. The present results indicate that intracellular KI is necessary to exert its effect on protein synthesis, and that this effect is mediated through a organic form of iodine, probably iodothyronines. This action of KI is specific for the thyroid gland.
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