| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
We have isolated a novel nuclear protein with a molecular mass of 49 kDa (TIP49) from rat liver. TIP49 showed structural resemblance to several bacterial RuvBs and also displayed Walker A and B motifs. Importantly, biochemical characterization of TIP49 yielded the following significant findings. 1) TIP49 possessed ATPase activity that was stimulated by single-stranded DNA and DNA helicase activity. 2) TIP49 specifically interacted with ATP. 3) TIP49 was abundantly expressed in testes and moderately in spleen, thymus, and lung. In mouse seminiferous tubules, the protein was restrictively observed in germ lineages from late pachytene spermatocytes to round spermatids. From these observations, we propose that TIP49 is a novel DNA helicase and may play a role in nuclear processes such as recombination and transcription.
Immunofluorescence analysis using anti-TIP49 antibody showed a typical dot-shaped nuclear staining pattern, suggesting that TIP49 is included in a macromolecular structure i
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n the nucleus. Moreover, glycerol gradient analysis demonstrated that TIP49 is present in a macromolecular complex in nuclear extract. Interestingly, we detected a high level of autoantibodies against TIP49 in sera of patients with autoimmune diseases such as polymyositis and autoimmune hepatitis. The anti-TIP49 antibodies can be useful probes for clinical diagnosis and for investigation of intranuclear structure. We also demonstrated that TIP49-related gene(s) in variety organisms from human to archaea. An extensive homology search revealed another class of TIP49-related gene in the eukaryotes, designated as TIP49b. TIP49a is just called as simply TIP49 in previous reports. A phylogenetical study suggested that archaeal TIP49 genes belong to the TIP49b ancestor but not the TIP49a one and that TIP49a evolved from TIP49b in accordance with divergence of archaea and eukarya.
Like TIP49a, TIP49b was abundantly expressed in the testis and thymus. Enzyme assays revealed that TIP49b was an single-stranded DNA stimulated ATPase and ATP-dependent DNA helicase. TIP49a and TIP49b bound to each other and were included in the same complex of 〜700 kDa in a cell. We found that TIP49b was an essential gene for the growth of S. cerevisiae, as is the TIP49a gene, suggesting that TIP49b does not complement the TIP49a function and vice versa. From these observation, we suggest that TIP49b plays an essential role in the cellular processes involved in DNA metabolism Less
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