Acides ribonucléiques messagers de la glande thyroïde de mouton

Abstract

The messenger character of nuclear and polysomal rapidly‐labeled thyroid RNA has been established using stimulation of amino acid incorporation into trichloracetic‐insoluble material in the Escherichia coli system, mononucleotide composition of the early‐labeled material and tenacity of adsorption on columns of methylated serumalbumin adsorbed on Kieselguhr (MAK columns).A relation between the difficulty to extract nuclear or cytoplasmic thyroid RNA by phenol at 0°, their rapidity of labeling and their capacity to stimulate protein synthesis in vitro has been demonstrated.80% of the nRNA labeled for 15 min was tenaciously bound to MAK columns. After elution with a buffer containing sodium dodecylsulphate at 35–70°, it showed a DNA‐like base composition and sedimented in 4 discrete size classes: 30 S, 22 S, 13 S. The remaining 20% has been identified as preribosomal RNA. After a 9 h‐period of labeling the total nRNA fraction still contained labeled preribosomal RNA, rRNA and DNA‐like RNA.Most of the early‐labeled polysomal RNA has been identified as mRNA by tenacity of adsorption on MAK columns and base composition. Even after a 1 h‐pulse the labeled rRNA accounted only for 30–40% of the total RNA. Absence of preribosomal RNA in polysomes as shown by MAK column chromatography was considered as a good index of the absence of contamination of polysomes by nuclear material. Polysomes pulse‐labeled with [32P]orthophosphate and [14C]protein hydrolysate were dissociated by ethylenediaminetetraacetic acid and fixed with formaldehyde. The buoyant densities of the nucleoprotein particles were determined by banding in CsCI gradients. Under these conditions, the bulk of the material in ribosomes banded at characteristic densities (d= 1.57 and 1.52) but most of RNA with high specific radioactivity banded at a density approximately 1.38. This material contained [32P]RNA and [14C]protein, and was identified as an association of mRNA and protein by base composition of its RNA: (G + C)/(A + U) = 0.83.Purification of pulse‐labeled nuclear mRNA was carried out by the hot‐phenol fractionation procedure of Georgiev et al. [9,10]. Specific radioactivity, specific stimulating activity of protein synthesis in vitro and base composition of the five fractions obtained were determined. The RNAs extracted from nuclei between 55–65° and 65–85° were considerably enriched in mRNA. Sucrose‐density gradient sedimentation showed substantial polydispersity of the stable RNA of all the fractions. However, the main component of the RNA extracted by phenol between 65° and 85° culminated in a metabolically homogeneous 16 S peak. A definite correlation between pulse‐labeled nRNA isolated by the hot‐phenol procedure and pulse‐labeled polysomal RNA was noticed.As compared with 18 S and 28 S rRNA, the fractions of stable polysomal thyroid RNA which corresponded in sedimentation to the rapidly‐labeled RNA (30–40 S, 22 S, 13 S and 7 S) definitely stimulated polypeptide synthesis in vitro.The size of the monocistronic mRNA which codes for the peptide chains of thyroglobulin is discussed. If a molecular weight of 73,000 for the two different chains is assumed, their amino acid sequence might be encoded in the 13 S polysomal RNA. The functional significance of the 30–40 S and 22 S mRNA and the possible importance of polysomal messenger nucleoprotein particles are also discussed.