|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1999 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1998 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1997 : ¥1,200,000 (Direct Cost : ¥1,200,000)
We previously produced a monoclonal antibody, Th-10a mAb, that recognizes a 95 kDa mouse nuclear protein (Np95). Np95 was stained with the Th10a mAb specifically in the S-phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells showed a constantly high level for Np95 accumulation irrespective of cell stages during the cell cycle. To gain further insights into the physiological roles of Np95, we isolated the cDNA encoding Np95 by immunoscreening a λgt-11 cDNA expression library with the Th-10a mAb. Sequencing of the whole 3.5kb cDNA revealed that Np95 is a novel nuclear protein with an open reading frame (ORF) consisting of 782 amino acids. The ORF contains a leucine zipper motif, a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site and retinoblastoma protein (Rb)-binding motifs "LXCXE" and "IXCXE". Np95 was strongly expressed in the testis, spleen and thymus, lung tissues, but not in the brain, liver and skeletal muscles. We also demonstrated that NP95 was localized in S-phase nuclei as dot-like foci. Double immunostaining of NP95 and proliferating cell nuclear antigen (PCNA) showed that NP95 was co-localized with PCNA. Construction of three-dimensional images indicated that NP95 was localized with PCNA in replication sites in a somewhat distinct temporal manner. During meiosis, NP95 was present not only in proliferating spermatogonia but also in meiotic spermatocytes and differentiating spermatids which were not proliferating. During the couse of experiments to generate a knockout mouse for NP95, we found that NP95 (-/-) embryonic stem cells had increased sensitivity to gamma-irradiation compared to the sensitivities of NP95 (+/-) and wild type embryonic stem cells. We propose that NP95 is involved in both mitotic and meiotic progression,presumably in a post-replication fashion and is a novel cell-cycle regulator that is associated with DNA fidelity maintenance.