Thyroglobulin Messenger Ribonucleic Acid Translation in vitro

Abstract

Total polysomes were obtained by homogenization of sheep thyroid glands in Tris-C1-buffered sucrose, pH 7.5, treatment with 2 ' %; Triton X-100 and centrifugation of the magnesium-treated 27 000 x g supernatant through 1 M sucrose. Purification of thyroglobulin-specific polysomes was performed by indirect immunoprecipitation with rabbit antibodies to sheep thyroglobulin and donkey antibodies to rabbit immunoglobulin G. Thyroglobulin polysomes represented 30 yo of total polysomes. Synthesis of thyroglobulin peptides in a reticulocyte cell-free system, programmed with total RNA from immunoprecipitated polysomes, was 3 times higher than when total RNA from unfractionated polysomes was used. The size of the poly(A)-containing thyroglobulin mRNA was estimated by the capacity of RNA fractions obtained by sucrose density gradient centrifugation of total or immunopurified polysomal RNA to hybridize with 3H-labeled poly(U) and to stimulate thyroglobulin peptide synthesis in a reticulocyte lysate. 65 of the thyroglobulin messenger activity was located in a 33 -36-S RNA peak and 35 in zones corresponding to 30-S and 26-S RNA species.The thyroglobulin-specific immunoprecipitable peptides synthesized in the reticulocyte lysate from the 33 -36-S RNA were shown by polyacrylamide gel electrophoresis in denaturing conditions to contain a major component co-migrating with the constituent peptide chain of sheep thyroglobulin ( M , 330000). Other components migrated with M , of about 210000 and 120000.

Cloning of Four DNA Fragments Complementary to Human Thyroglobulin Messenger RNA

“…Compared to sheep thyroglobulin mRNA (Fig. I), a similar size (8500 bases [6] can be estimated. When tested in the reticulocyte lysate, this material promoted the synthesis of immunologically related thyroglobulin pcptides (Table 1).…”

“…Direct identification by polyacrylamide gel electrophoresis in denaturing conditions of the immunoprecipitable material as peptides of M , approximately 300000, as in the case of sheep thyroglobulin mRNA [6], proved more difficult with the human 33-S mRNA. Only peptides or M, 120000 and 1 50 000 have been characterized by dodecylsulfate/polyacrylamide gel electrophoresis of the immunoprecipitable peptides synthesized in the reticulocyte lysate both from the 33-S m R N h which served for the molecular cloning of cDNA and from the RNA obtained by filter hybridization selection (not illustrated).…”

“…Polysoma1 RNA was prepared as previously described [6] except that homogenization was performed in high-pH buffer (0.25 M sucrose, 0.2 M Tris/HCl pH 8.5, 50 mM KCI, 25 mM MgC12 and 1 mM dithiothreitol [13] containing 1.5 mg purified yeast RNA/ml [14]. Poly(A)-rich RNA was isolated by chromatography on oligo(dT)-cellulose (T3 Collaborative Research) and centrifuged in a 5-29'%, sucrose gradient in 10 mM Tris/HCl pH 7.4, 5 mM EDTA, 0.2';; sodium dodecylsulfate for 5 h at 38000 rev./min (SW 41 Beckman rotor) at 25°C.…”

“…Translation of RNA was performed in 11 pl of a mixture containing 10 pCi [3sSs]-methionine (New England Nuclear, specific activity 1200 Ci/ mmol) for 2.5 h at 30 "C. Total 35S incorporation was determined by precipitation with trichloroacetic acid. The amount of thyroglobulin-related peptides synthesized was determined by direct immunoprecipitation as previously described [6] using rabbit antiserum to human thyroglobulin. Antiserum was prepared by immunization of rabbits according to [32] using highly purified human thyroglobulin [3,9] ; 10 pl of antiserum was able to precipitate 3 pg human thyroglobulin.…”

“…Each subunit contains peptide chains of M, about 300000 [5,6] encoded in a 33-S mRNA of M I 2.8 x 106 [7,8]. Identity of the protomers was suggested by C-terminal analysis of the protein from several mammalian species including man [9] and more recently by restriction studies of the full-length double-stranded cDNA corresponding to bovine thyroglobulin inRNA [I 01.…”