|Budget Amount *help
¥7,700,000 (Direct Cost : ¥7,700,000)
Fiscal Year 1997 : ¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1996 : ¥4,200,000 (Direct Cost : ¥4,200,000)
The functions and expressions of wild-type (w) and mutant (m) thyroid hormone receptors (TR) were analyzed and the following results were obtained.
1. Generation of the transgenic mice (TG) expressing the carboxyl (C)-terminal 11 amino acid-truncated TRbeta (betaF451X) or TR alpha1 which possesses the same C-terminal truncation as betaF451X (alphaF397X).
The expression vectors bearing the promotion region of the elongation factor gene and betaF451X-or alphaF397X-gene were constructed and microinjected into the pronucleus of fertilized mouse eggs. The eggs were transferred into the oviducts of pseudopregnant foster mothers and the existence of the transgene was examined in offsprings by PCR and Southern blot analysis. The productivity of betaF451X-TG was 17% (9/53 mice), which was average in various TG generation in our institute, while very low as only 3.4% in alphaF397X-TG (4/117 mice). Among four alphaF397X-TG,one was born dead and another died during growth. The two alphaF397X-TG,but
none in betaF451-TG,showed decrease in body weight gain. No apparent abnormality was noticed in either TG.The basal T4 and TSH levels were within normal limits. The study of learning and behavior showed the tendency of higher spontaneous motor activity in TG,especially female betaF451X-TG as compared with the litter mates. No other abnormality including learning ability was found. The results suggest that the reason no mutation has been found in TRalpha1 in patients with thyroid hormone resistance may be due to the lethal effect of mutant TRalpha1.
2. Measurement of the heterodimer formation (HD) ability between TR and retinoid X receptor (RXR) by a new assay system of yeast two hybrid system.
Expression of two fusion proteins of yeast DNA binding domain, GAL4, and ligand binding domain (LBD) of RXR (GAL-RXR), and TRbetaLBD and VP16 (VP-TR) in CV1 cells transactivate the GAL reporter gene. Since TR coexpressed competes with VP-TR in RXR binding, HD ability of w-and mTR can be assayd quantitatively and sensitively. The RXR binding activities of various mTRs were well paralleled with their dominant negative effects (DNE), suggesting the importance of HD in DNE.
3. Analysis of the silencing activity of betaF451X and alphaF397X.
Both betaF451X and alphaf397x have very strong and identical silencing activities. The binding activities with RXR and corepressors such as SMRT and N-CoR were significantly higher in betaF451X,but not in alphaF397X,compared with each wild-type TR,suggesting that the mechanism of silencing was not necessarily identical in both truncated TRs.
4. Functional analysis of amino (N) -terminal A/B region of TR/beta1.
Studies using deletion mutants demonstrated the transactivating function in region 32-51 amino acid and suppressing function in region 51-94 amino acid. interestingly, these regions may affect the HD ability.
5. Transcriptional interaction between TR and vitamin D receptor (VDR).
We found that TR and VDR interfered each other in transcriptional level only when in the presence of each ligand. The interaction was observed between the endogenous native receptors in osteosarcoma cells also. The experimental date strongly suggested the functional competition between TR and VDR in some coactivator (s). Less