Subcytoplasmic distribution of thyroglobulin mRNA in normal sheep thyroid

The thyroglobulin 33-S mRNA was isolated from sheep thyroid total polysomes. The 33-S RNA, twice purified on a 1% sodium dodecylsulfate/sucrose gradient, was 30-fold enriched in thyroglobulin messenger activity and was estimated as 50% pure by its messenger activity and 80% pure by the electrophoretic profile. It was used as template for complementary DNA synthesis and hybridized up to 85% of the DNA copy with pseudo-first-order kinetics. Back-hybridization kinetics showed that the purified mRNA corresponds to a major kinetic component with a base sequence complexity of 10000 nucleotides as determined by comparison to globin mRNA. Cross-reactivity of [3H]cDNA with liver RNA is less than 10%. Restriction endonuclease digestion of [3H]cDNA yielded a discrete band pattern. The distribution of thyroglobulin mRNA among free polysomes, membrane-bound polysomes and extrapolysomal pools was analyzed using hybridization to the specific [3H]cDNA probe. Free particles were recovered in the supernatant and membrane-bound particles in the pellet after a brief centrifugation of detergent-free homogenate (5 min at 27000 x g: procedure A; 12 min at 130000 x g: procedure B) with precautions taken to avoid cross-contamination. Using procedure A, 80% of thyroglobulin mRNA sequences were found in the membrane-bound fraction. Using procedure B, where contamination of free particles by membrane-bound particles was avoided by high-speed initial centrifugation and further isolation through a discontinuous sucrose gradient, 95-98% of thyroglobulin mRNA sequences were recovered in membrane-bound polysomes. In total polysomes, 89% of thyroglobulin mRNA sequences were in the polysomal area and shifted to ribosomal subunits after EDTA treatment.