PMID: 7371633Feb 1, 1980Paper

Subcytoplasmic distribution of thyroglobulin mRNA in normal sheep thyroid

European Journal of Biochemistry
O ChabaudM Jacquet

Abstract

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,...Continue Reading

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