The overall goal of this project is to advance understanding of certain fundamental, molecular processes involved in the regulation of thyroid cellular function. Specifically, it is proposed to perform the following studies: 1. Studies on genes other than thyroglobulin that are regulated by TSH. a. Initial studies on TSH-induced genes already cloned from FRTL5 cells. i) nucleotide sequencing (in part). ii) determination of whether the increased levels of mRNA reflect increased transcription. iii) time-course of stimulation of cellular mRNA levels by TSH. b. In selected genes of interest it is planned to: i) sequence the entire structural (cDNA) gene; ii) characterize the gene in the FRTL5 genome; iii) localize and study the promoter area; iv) study the function of the gene product by enhancing or inhibiting the expression of the gene in thyroid cells. 2. Studies on the human thyroid peroxidase (TPO) gene. a. Identification and sequencing of the full-length human TPO cDNA gene. b. Characterization of the TPO gene in the human genome. c. Determination of TPO mRNA levels in normal and pathological human thyroid tissue. d. Study on the potential genetic basis for abnormal TPO activity in selected diseased thyroid glands. e. Study of the regulation of TPO mRNA expression in thyroid cells. 3. Studies on ras and myc proto-oncogene expression in the thyroid. a. Studies on the mechanism by which TSH activates ras gene expression. b. Examination of the role of the ras and myc proto-oncogenes (alone or together) on selected thyroid cellular differentiated functions. i) transfection of FRTL5 cells with plasmids containing the c-ras and c-myc genes. ii) transfection of FRTL5 cells with """"""""antisense"""""""" mRNA plasmid constructs designed to specifically inhibit the functions of these genes.
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