Huguette Brocas

Structure, expression and regulation of the thyroglobulin gene

de Recherche Interdisciplinaire and Service Chimie, FactP transcription control; cyclic AMP; congenital goitre; chromosome 8.

Improved synthesis of DBM paper.

Abstract A modified synthesis of DBM paper is described wich is simple and easily reproducible. The method previously published has been improved in order to optimize the reaction conditions of each step. The paper obtained in this way shows a very high binding capacity for denatured DNA.

Alternative splicing may be responsible for heterogeneity of thyroglobulin structure

During the cloning of the bovine thyroglobulin cDNA, the restriction map of one of the recombinant plasmids was in disagreement with that of the full-length double-stranded thyroglobulin cDNA. When compared to the bovine Tg mRNA sequence, this cDNA clone exhibits a 333-nucleotide deletion which corresponds precisely to 2 exons of the Tg gene. It is thus likely that alternative processing of the premessenger RNA is at the origin of the deletion. The presence of giant introns in the vicinity of the dispensable exons may also reflect some error level in the splicing mechanism. Together with previous results the alternative splicing described in this study indicates that alternative processing of the Tg transcripts may be at the origin of thyroglobulin isoforms.

Translation in Xenopus oocytes of thyroglobulin mRNA isolated by poly(U)-Sepharose affinity chromatography

Abstract Three poly(A)-rich RNAs (approx. 33S, 16S and 13S) have been isolated from membrane-bound thyroid polysomes by affinity chromatography on Sepharose-poly(U). The 33-S RNA could also be characterized from thyroglobulin-synthesizing polysomes by hybridization of its poly(A) sequence with tritiated poly(U). When injected into Xenopus oocytes, the 33 and 16-S RNA promoted the synthesis of immunoprecipitable thyroglobulin. This suggests that thyroglobulin is composed of at least two different sized subunits.

Restriction mapping of synthetic thyroglobulin structural gene as a means of investigating thyroglobulin structure.

Bovine 33 S thyroglobulin mRNA was reverse transcribed into double-stranded DNA under conditions allowing the synthesis of a complete 8 kilobase pair copy. A physical map of the resulting synthetic thyroglobulin structural gene was constructed using six restriction endonucleases. The following conclusions could be drawn: (i) the polypeptide chains in thyroglobulin subunits are identical; (ii) thyroglobulin is composed of a major class of molecules sharing the same primary structure; (iii) there is no evidence for precise internal repetition in the structure of thyroglobulin subunits.

Molecular cloning of Pst I fragments from rat double stranded thyroglobulin complementary DNA

Abstract Thyroglobulin mRNA was prepared from thyroid glands of rats chronically treated with propylthiouracil. The double stranded complementary DNA was synthesized using Avian Myeloblastosis Virus reverse transcriptase and subjected to restriction with the endonuclease Pst I. The restriction fragments were ligated into the unique Pst I site of the plasmid pBR 322 and the resulting recombinant DNA was used to transform E.coli to tetracyclin resistance. The colonies harboring recombinant plasmids (42 out of 1852) could be classified into three categories containing 320, 550 and 640 base pair inserts. One clone from each size class was selected and the ability of their plasmid DNA to bind functionaly active rat thyroglobulin mRNA was demonstrated in a positive translation assay. Altogether the three cloned DNA fragments represent about 20% of thyroglobulin structural gene sequence.

Proximity of thyroglobulin and c-myc genes on human chromosome 8.

The human thyroglobulin structural gene (TG) was mapped to the long arm of chromosome 8 by blot hydridization of a TG cDNA probe to DNA from 21 human × mouse somatic cell hybrids containing overlapping subsets of human chromosomes. In situ hybridization of the TG probe to metaphase chromosomes from a karyotypically normal human lymphoblastoid cell line, JS, localized the TG gene to within the region 8q23 → q24.3. Thus, the TG and c-myc genes map to the same chromosome band in normal human cells. In a human colon carcinoma cell line (COLO 320 DM) which contains amplified c-myc, the TG gene is not amplified and hence it lies outside the amplification domain.

The thyroglobulin gene resides on chromosome 8 in man and on chromosome 7 in the rat.

Human chromosomes were separated by a dual laser FACS sorter and their DNA hybridized with a thyroglobulin gene probe. A strong hybridization signal was obtained with DNA from chromosome 8. A panel of mouse-rat cell hybrids was used to determine the chromosomal localization of the rat thyroglobulin gene by the Southern blotting method. Comparison of the cytogenetic data with the hybridization signals obtained with the rat thyroglobulin probe allowed assignment of this gene to rat chromosome 7. It is concluded that the synteny relationship between the thyroglobulin gene and the c-myc oncogene has been conserved in rat and man.

Translation in Xenopus oocyte of messenger RNA coding for a beef heavy-chain thyroglobulin.

Abstract Poly(A)-rich RNA extracted from membrane-bound thyroid polysomes has been shown to contain two fractions showing thyroglobulin messenger activity: a heavy 33 S fraction and a 16–18 S fraction. When injected into Xenopus oocyte, the 33 S mRNA specifically promoted the synthesis of a polypeptide chain of about 185 000 daltons as revealed by electrophoresis in sodium dodecylsulphate—polyacrylamide gel after reduction and alkylation. Our results strongly support models of thyroglobulin structure involving the existence of heavy thyroglobulin subunits.

Integrity of the thyroglobulin locus in tricho-rhino-phalangeal syndrome II

SummaryThe thyroglobulin gene has been mapped to chromosome band 8q24 by several investigators. This is the band implicated in the causation of Langer-Giedion syndrome (tricho-rhino-phalangeal syndrome II). We have examined a restriction fragment length polymorphism at the thyroglobulin locus in a patient with Langer-Giedion syndrome and 8q deletion in order to: (1) localize the Langer-Giedion deletion more precisely, (2) define the relative map positions of the thyroglobulin gene and the Langer-Giedion locus. The results indicate that the locus of the thyroglobulin gene is intact in the patient with an interstitial deletion of proximal band 8q24.1 which confirms its more distal localization reported earlier by Bergé-Lefranc et al. (1985). It also assigns the critical region for the causation of Langer-Giedion syndrome to the proximal part of band 8q24, viz. 8q24.11→q24.13.