Cloning of Np95 gene which encodes a S-specific nuclear protein, and the sensitivity to radiation in Np95 deficient cells

我们之前生产了一种识别95 kDa小鼠核蛋白(Np95)的单抗Th-10a mAb。Np95与Th10a单抗在正常小鼠胸腺细胞的S期特异性染色。相比之下，小鼠T细胞淋巴瘤细胞显示出持续高水平的Np95积累，与细胞周期中的细胞阶段无关。为了进一步了解Np95的生理作用，我们用Th-10a单抗免疫筛选λGT-11c DNA表达文库，获得了Np95的编码基因。全长3.5kb的基因序列分析表明，Np95是一个新的核蛋白，其开放阅读框(ORF)由782个氨基酸组成。ORF包含一个亮氨酸拉链基序、一个锌指基序、一个潜在的ATP/GTP结合位点、一个可能的细胞周期蛋白A/E-CDK2磷酸化位点和视网膜母细胞瘤蛋白(RB)结合基序“LXCXE”和“IXCXE”。Np95在睾丸、脾、胸腺和肺组织中表达较强，而在脑、肝脏和骨骼肌中不表达。我们还证明了NP95定位于S期细胞核中，呈点状分布。NP95和增殖细胞核抗原双重免疫染色显示NP95与增殖细胞核抗原共定位。三维图像的构建表明，NP95与增殖细胞核抗原一起以某种不同的时间方式定位在复制部位。在减数分裂过程中，NP95不仅存在于增殖的精原细胞中，而且存在于减数分裂的精母细胞和分化的不增殖的精细胞中。在建立NP95基因敲除小鼠的实验过程中，我们发现与NP95(+/-)和野生型胚胎干细胞相比，NP95(-/-)胚胎干细胞对伽玛辐射的敏感性增加。我们认为NP95参与了有丝分裂和减数分裂的进程，可能是以复制后的方式进行的，是一种与DNA保真度维持相关的新型细胞周期调节因子。

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.