| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2003: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
In human genome, five genes --- RECQ1, BLM, WRN, RECQL4, and Q5 --- have been found to encode DNA helicases. These genes share homology with the RecQ family including E.coli RecQ, cerevisiae Sgs1, pombe Rqh1, so that they are called as human RecQ-type DNA helicases. As far three of them, WRN, BLM and RECQL4, have been identified in human genetic disorders, Werner (WS), Bloom (BS) and Rothmund-Thomson syndromes (RTS). These three genetic disorders are associated with cancer predisposition and/or premature aging. However, the relationship between the deficiency of DNA helicase and phenotype in human is not clarified.
In order to analyze the function and the role assignment of RecQ-typed DNA helicase genes in vivo, we established four different mutant mouse lines, RecQ1, Wrn, Recql4, and RecQ5, by targeted mutagenesis. We established three different types of Recql4 mutant mouse lines. 1)Since it is unknown whether defects in RTS patients are manifestations of hypomorphic or null mutations,
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we expected the complete inactivation of RTS might reveal novel functions of this protein. Therefore, at first, we targeted the 5' region of Recql4 to produce a null allele (Q4-N allele), which was expected to lose the majority of functional Recql4 gene product.
Homozygous mice for Recql4-null mutation, Recql4N/N, were embryonic lethality at E3.5 - E6.5. This result shows the Recql4 gene is essential for passing through peri-implantation stage of development in mice in spite of the recessive inheritance of RTS. 2)Since the Recql4N/N mouse was embryonic lethal, it was impossible to establish the mouse model of RTS. Therefore, we established the conditional Recql4 knockout mice as a next step. The loxP was inserted in both sides of exons 9-10 in conditional silent allele (Q4-S' allele) that is carrying out the code of the helicase domain (exons 9-15). 3)We also created Q4-T allele that is missing C-terminal region from the downstream of helicase domain of Recql4.
Both Recql4T/T and Recql4T/N mutant mice recapitulate the major phenotypes found in Type II RTS patients (growth deficiency, poikiloderma, early graying and hair loss, cataracts, and osteosarcoma). The premature features observed on these mutant mice were analyzed in detail. Moreover, we succeeded to make the PoAbs to N- and C-terminal region of Recql4 molecule. The reactivity of these PoAbs was confirmed not only by Western blot analysis using the partial expressed Recql4 protein in E.Coli but also by indirect immunofluorescence staining using the MEFs. Less
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