Carole Hansen

cAMP-dependent binding of a trans-acting factor to the thyroglobulin promoter

We have investigated the interaction of a nuclear factor(s) with the promoter region of the thyroglobulin (Tg) gene, which is only expressed in differentiated thyroid cells under the positive control of the pituitary hormone thyrotropin (TSH) via a cAMP-dependent pathway. Using the mobility shift assay, we first demonstrated that a thyroid nuclear factor interacts with a short segment of 60 bp (-136 - -77) which is conserved among species in the regulatory region of the Tg gene. A specific binding site was then localized in a subfragment of 20 bp located between -126 bp and -107 bp relative to the transcription initiation site. The corresponding nuclear factor is absent in a tissue which does not express the Tg gene. This factor differs from previously identified factors shown to mediate a direct cAMP response since the observed binding is neither competed out by the cAMP responsive element (CRE) nor by the activator protein 2 (AP2) binding site. This trans-acting factor represents a new candidate intermediate in the regulation of transcription by a cAMP dependent mechanism.

Normal and Defective Expression of the Thyroglobulin Gene

Molecular studies of the thyroglobulin (Tg) gene have progressed significantly in recent years. Cloning and sequencing the complete bovine Tg cDNA led to the knowledge of the primary structure of the Tg subunit. This large polypeptidic chain displays a repetitive structure, especially in its amino-terminal half, and bears a striking homology with the acetylcholinesterase molecule of Torpedo californica in its carboxy-terminal portion. The four specific domains known to be involved in the formation of the thyroid hormones have been assigned to both terminal parts of the polypeptide, a location which could play a role in the process leading to hormone release. The very large (greater than 250 kb) Tg gene has been localized on the long arm of chromosome 8 in man, in close linkage with the c-myc oncogene. The study of its structure allowed the characterization of the molecular defect responsible for a congenital flaw in Tg gene expression in a herd of South-African cattle. This work led to the unexpected finding that the Tg pre-mRNA undergoes alternative splicing in normal animals, too. A DNA segment involved in the transcriptional control of Tg gene expression by cAMP has been identified by transfecting primary cultured thyrocytes with recombinant genes.

Preparation of single-stranded deoxyribonucleic acid probes using an immobilized template

A new method for the easy preparation of specific single-stranded DNA fragments is presented. Recombinant M13 DNA containing the strand complementary to the sequence of interest is made partially double-stranded by elongating a conventional M13 sequencing primer. Following linearization by enzymatic digestion downstream from the insert (relative to priming site), this DNA is coupled to diazotized paper through its single-stranded (vector) portion. Subsequent denaturation of the double-stranded region generates an immobilized template strand. Successive runs of primed syntheses of the (desired) complementary strand can be realized using the same template. The copies are easily isolated by release upon denaturation. DNA probes prepared by this method have proven to be valuable tools for gene analysis.