| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
We have reported thyroglobulin (Tg) missense mutations, C1264R and C1996S, in Japanese patients, and recently we identified novel missense mutations, C1077R and G2375R in Japanese, and G233E and V1460I in Hispanics in the USA. We also analyzed G2320R mutation of Tg in hereditary hypothyloid rdw rats. These mutations cause misfolding of Tg, resulting in impaired intracellular transport of Tg and accumulation in endoplasmic reticulum. We analyzed the whole cell response to the misfolded Tg expression by profiling cDNA in G401 kidney cell line expressing the mutant Tgs. G401 kidney cells were transformed with expression vectors carrying C1264R and C1996S Tgs as well as normal Tg. Stable transformants with normal Tg secreted Tg into the medium, whereas those with mutant Tgs did not secrete Tgs. In the first experiment, Atlas cDNA arrays (Human 3.6, 3528 genes; Human stress, 234 genes) were hybridized with RI-labeled cDNA. The genes with signal difference of more than 2-fold were analyzed by quantitative RT-PCR. In the second experiment, cDNAs enriched in the cell lines with the mutant Tgs were cloned by a PCR-select cDNA subtraction kit. In the library of 1,500 clones, 244 clones which showed significant signal differences were identified by sequencing. The genes that were induced by the mutant Tgs were in the family of ribosomal proteins, molecular chaperones, proteasomes and transcription factors, and in the pathways associated with cell proliferation, apoptosis, and DNA replication. Functions of more than 30 genes identified in the PCR-select cDNA subtraction experiment were currently unknown.
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