Acides ribonucléiques messagers de la glande thyroïde

Abstract

Nuclear and cytoplasmic RNA from sheep thyroid slices in vitro labeled with [ 3 H]uridine or [ 32 P]phosphate from 5 min up to 9 h, have been obtained by different phenol extraction procedures. Early‐labeled RNA was only extracted by phenol containing 0.1 or 1% (w/v) sodium dodecylsulfate (SDS) at 0° or 60°, whereas stable nuclear or polysomal RNA was easily extracted by pherol at 0°. Time course of incorporation of label into subcellular particles has shown the early labeling of nuclear RNA and the transfer of radioactivity into the cytoplasm. Rapidly‐labeled RNA extracted from nuclei by phenol‐1% (w/v) SDS and analyzed by sedimentation was shown to sediment in three discrete classes 50–30S, 22 S and 13 S, different from ribosomal RNA. After 9 h of incubation the 22 S and 13 S labeled RNAs were still present in detectable amounts in the nuclei, indicating a somewhat low turnover rate for these components. The kinetics of incorporation of label into polysomes was studied in thyroid slices incubated from 15 min up to 5 h with [ 3 H]uridine or [ 32 P]phosphate. RNA was dissociated from polysomes by dodecylsulfate treatment and centrifuged on sucrose gradient. As for nuclei, the rapidlylabeled RNAs sediment in discrete size classes, 22 S and 13 S. After a long incubation time (5 h) the label was predominant in 28 S and 18 S ribosomal RNA but 28 S RNA was not completely labeled. Up to 30 min labeling a high molecular weight, early‐labeled RNA was present in purified polysomes. Its radioactivity declines after 60 min labeling. A part of the rapidly labeled polysomal RNA can be reversibly separated from rRNP by the action of EDTA (1.25 to 5 μ moles EDTA/mg RNP). It sediments in a broad zone (12–30 S) of high specific radioactivity. Increasing Mg ++ concentration results in the reassociation of rapidly‐labeled RNA to ribosomal sub‐particles. Buoyant densities of the polysomal RNP particles obtained by EDTA treatment of 32 P‐labeled polysomes and stabilized by means of formaldehyde fixation, were determined by banding in CsCI gradients. The bulk of the stable material banded at characteristic densities ( d = 1.55 and 1.48), but most of the rapidly‐labeled RNA banded at d = 1.38. The specific radioactivity of this material was the same as that of the 12–30 S RNA isolated by sucrose gradient centrifugation from the same EDTA‐treated polysomes. Preliminary double labeling experiments and computation of the RNA to protein ratio calculated from the RNA and protein density values indicate this material to be early‐labeled nucleoprotein particles with a high protein content.