The long term goal of this research project is to elucidate the cellular processes involved in thyroid hormone (TH) action. Specifically, this proposal addresses the molecular mechanisms which regulate TH metabolism in developing vertebrate species. In the fetus, as in the adult, specific iodothyronine deiodinases convert thyroxine and other thyroid hormones into active and inactive metabolites. Recently, a cDNA which encodes the type I rat liver 5'- deiodinase (5'D) was isolated and its sequence determined. Thus, studies at a molecular level aimed at investigating the mechanisms by which deiodinases are regulated during development can now be undertaken. Two animal models will be employed: the rat, which is traditionally used as an experimental model system for studying mammalian TH economy, and the frog, which offers the major advantage that its larval form is free swimming and thus readily amenable to experimental manipulation.
Specific Aim 1 is to isolate and characterize cDNAs for the 5'D and 5D present in tadpoles.
In Specific Aim 2 and #4 these tadpole and rat cDNAs will be used as molecular probes to characterize the pretranslational regulation of the deiodinase enzymes at various stages of development by quantitating mRNA levels and by in situ hybridization. The role of TH and other factors in regulating 5'D and 5D mRNA levels will be investigated in rat fetuses and tadpoles (Specific Aim 3) and in cultured fetal rat hepatocytes and tadpole tail tips (Specific Aim 5). The effect of TH on 5'D and 5D gene transcription will then be determined using a transcription assay (Specific Aim 6).
Specific Aim #7 is to isolate from frog and rat genomic libraries the genes which encode the 5'D and 5D and their associated DNA flanking regions.
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