| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Flag-tagged XAB2 was expressed in HeLa cells and affinity-purified with anti-Flag antibody. Flag-XAB2 was co-purified with at least 20 polypeptides. Mass spectrum analysis of them suggested that XAB2 might reside in complexes involved in cell-cycle control and pre-mRNA processing. Further purification of the complex revealed that XAB2 was tightly associated with 5 proteins. Two of five are orthologs of yeast proteins involved in splicing in Saccharomyces cerevisiae. One is a cyclophilin with RNA-binding motifs. The remainders are currently unidentified proteins. These results, combined with the fact that XAB2 was co-immunoprecipitated with RNA polymerase II large subunit, suggested that XAB2 might be a component of "RNA factory" involving transcription-coupled repair, pre-mRNA processing, cell-cycle control as well as transcription. To study the functions of XAB2 in vivo, we generated two mutation alleles in embryonic stem cells using gene-targeting technique. One is a null allele that
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has a deletion of the region containing promoter and exon 1-4 of XAB2 gene. The other is a carboxy-terminal deletion (cd) allele carrying a stop codon in exon 15 of the XAB2 gene. Both types of heterozygous cells and mice are apparently normal. However, when intercrossing heterozygotes, neither homozygous XAB2 (-/-) nor XAB2 (cd/cd) mice were found in the offspring and 13.5-day embryos. Analyses of the 3.5-day embryos obtained from heterozygous intercrosses showed that XAB2 (-/-) or XAB2 (cd/cd) morula were found in a ratio expected for a Mendelian distribution, whereas neither XAB2 (-/-) nor XAB2 (cd/cd) blastocysts were detected despite the presence of wildtype and heterozygous blastocysts. Furthermore, attempts to disrupt the remaining wildtype XAB2 allele in XAB2 (+/cd) ES cells failed, suggesting that the XAB2 gene is essential for growth of ES cells. These results, consistent with the involvement of XAB2 in transcription, indicate the essential function of XAB2 in mammals and in cellular viability. Less
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