32. Cloning of huNp95 : A Novel Human Homologue of Mouse Np95 Gene

Masahiro Muto, Yasuyoshi Kanaru, Eiko Kubo, Kouichi Tatsumu

Keywords: novel nuclear protein Np95, human homologue, PCNA, DNA replication, cell cycle

We previously produced a monoclonal antibody Th-1Oa mob, that recognizes a 95 kDa mouse nuclear protein (Np95). Np95 was stained with the Th1Oa mob specifically in the S-phase of normal mouse thymocytes. In contrast, mouse T cell lym phoma cells showed a constantly high level for Np95 accumulation irrespective of cell stages during the cell cycle. By immunoscreening a gt11 cDNA expression library with the Th-1Oa mAb, we isolated the cDNA encoding the mouse nuclear protein Np95 Sequencing of the whole 3.5 kb 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 motif s "LXCXE" and "IXCXE" Np95 was strongly expressed in the testis, spleen and thymus, lung tissues, but not in the brain, liver and skeletal muscles. Microscopieally Np95 appeared as foci in nuclei through late Gl to S phase of m5S cells, and these foci coincided with those for proliferating cell nuclear antigen (PCNA) in double immunostaining studies. These results collectively implicate this novel nuclear protein in cell cycle progression and/or DNA replication.

In this study, to identify the human homologue of mouse Np95, we carried out sequence database searching using the Blast algorithm. A tblastn search of the Genbank database using the mouse Np95 amino acid sequences revealed that several EST clones with the accession numbers, AA811O55 AA908902, AA3O6523, AA827671 and AA8112l7 have an amino acid sequence similarity with that of mouse Np95 in two parts. Using the sequence information, these cDNA fragments were amplified by PCR using 8 primers and a human testis cDNA library (GibcoBRL). Amplification of the 3' region was carried out by the two-step PCR procedure using the testis cDNA library. Since we could not amplify the 5' region, pMyr human testis cDNA 11 brary (Stratagene) and multiple testis cDNA (CH 1011; Origene technol. Inc.) were used to obtaine the 5' region. We finally isolated a 3295-bp cDNA which contains a single large open reading frame encoding a polypeptide of 793 amino acids. We also isolated three other isoforms of the homologue cDNA. To investigate the loealization on chromosome and the exon and intron of this gene, three bacterial artifi cial chromosome (BAC) clones which contain huNp95 gene were obtained by screening of BAC li braries of Roswell Park Cancer Institute and RPCI 11 human BAC library (Nihon Techno. Serv Japan).

Publication:
Uemura, T., Kubo, E., Kanari, Y., Ikemura, T Tatsumi, K. and Muto, M.: Cell Structure and Function, 10, 149-159, 2000.