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(PP354) Isolation of human radiation susceptibility gene, hNp95.
Muto, Masahiro1, Kubo, Eiko 1, Fujimori, Akira1, Nenoi, Mitsuru1, Daino, Kazuhiro1, Matsuda, Yoichi2, Kuroiwa, Asato2, Ukai, Hideki1, Tsuji, HIdeo1, Tatsumi, Kouichi*,1,3, 1 Research Center for Radiation Safety, Chiba, Chiba, JAPAN2 Laboratory of Cytogenetics and Division of Bioscience, Sapporo, Hokkaido, JAPAN3 Institute of Radiation Epidemiology, Tokyo, JAPAN
ABSTRACT- Murine nuclear protein NP95 has been shown to confer on embryonic stem (ES) cells resistance to DNA insults and replication arrests (Muto et al., JBC, 277: 34549-34555, 2002). It is apparently essential for entry from G1/G0 into S phase in dedifferentiated myotube cells but not in proliferating ES cells. By using the databases for expressed sequenced tags and a two-step PCR procedure we isolated the full-length human homologue of the murine Np95 cDNA, the hNp95, which consisted of 4,327 bp and contained a single open reading frame (ORF) encoding a polypeptide of 793 amino acids with 73.3% homology to NP95. The ORF of the hNP95 cDNA was identical with UHRF1 and only different by two amino acids and seven nucleotides from that of the inverted CCAAT box (ICB2) binding protein of 90 kDa (ICBP90) that had been reported to regulate the transcription of the topoisomerase IIa gene during the cell cycle (Hopfner et al., Gene 266, 15-23, 2001). Chromosome mapping assigned the hNp95 to human chromosome 19p13.3. The hNp95 gene consisted of 18 exons and 17 introns, spanning 60 kb. Several stable transformants from HEK293 and WI-38 cells that had been transfected with the antisense hNP95 cDNA were more sensitive to X-rays, UV-light and hydroxyurea than corresponding parental cells. Unlike the reported case with ICBP90, hNP95 expression did not affect the activities of topoisomerase IIa in HEK293 cells. Furthermore, the binding of hNP95 to the ICB2 element of the topoisomerase IIa gene promoter was non-specific. These findings collectively indicate that the hNp95 gene is the human functional orthologue of the murine Np95 gene.
Key words: X-rays, UV light, hydroxyurea, antisence cDNA
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