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PubMed1996

Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1.

Using the two-hybrid method, we isolated a Saccharomyces cerevisiae cDNA encoding a protein homologous to Schizosaccharomyces pombe protein Dis3sp, using as bait, human GTPase Ran. The DIS3 gene is essential for viability and complements S.pombe mutant dis3-54 which is defective in mitosis. Although Dis3sc has no homology to RanBP1, it bound directly to Ran and the S.cerevisiae Ran homologue Cnr1, but not to the S.cerevisiae RCC1 homologue Srm1. Upon binding to Ran with a 1:1 molar ratio, Dis3sc enhanced a nucleotide-releasing activity of RCC1 on Ran. In the presence of Dis3sc, the K(m) of RCC1 on Ran decreased by half, while the kcat was unchanged. In vivo, Dis3sp was present as oligomers of M(r) 670-200 kDa as previously reported, and the 200 kDa oligomer of Dis3sp was found to include Spi1 and Pim1, the S.pombe homologues of Ran and RCC1, respectively. Although the biological function of the heterotrimeric oligomer consisting of Dis3, Spi1 and Pim1 is unknown, our results indicate that Dis3 is a component of the RCC1-Ran pathway.

Amino Acid Sequence↗

PubMed1996

[Discovery of genetic polymorphism of human nucleases].

Research into the use of new genetic markers is difficult and costly, but it is necessary for more accurate criminal individualization and paternity testing as well as for analysis of genetic diseases. Recently, we discovered that human ribonuclease (RNase), deoxyribonuclease I (DNase I) and deoxyribonuclease II (DNase II) are characteristic markers showing genetic polymorphism and useful for forensic investigation. DNase I is particularly well suited to practical use, since it shows a well-balanced gene frequency, a high concentration in several body fluids (blood, sweat, urine, breast milk and semen) and tissues (pancreas, liver and kidney), stability against severe conditions (exposure of test samples to high temperature, high humidity and long-term storage), and easy and accurate detectability.

Exoribonucleases↗

PubMed1997

Isolation of murine and human homologues of the fission-yeast dis3+ gene encoding a mitotic-control protein and its overexpression in cancer cells with progressive phenotype.

To investigate genes involved in metastasis, we used a differential display method to compare the levels of gene expression in three cell lines derived from murine colon-adenocarcinoma 26 that show different metastatic potentials. The results, and subsequent Northern analyses, confirmed that one gene was expressed most strongly in NL17, the cell line with the highest experimentally metastatic potential to the lung; strongly in NL22, the line with moderately metastatic potential; and very weakly in NL4, which has no metastatic potential in recipient mice. Using this fragment as a probe, we isolated the murine cDNA as well as its human homologue and determined their DNA sequences. The cDNA sequences from both species contained open reading frames of 2874 nucleotides, encoding peptides of 958 amino acids with calculated molecular weights of approximately 109,000; the murine and human nucleotide sequences were 90% identical. The deduced amino acid sequences of these cDNAs revealed significant homology (45% identity) to the dis3+ gene product of Schizosaccharomyces pombe, a protein thought to be essential for mitotic control in the yeast. We therefore termed the murine and human genes hmc (homologue to the mitotic-control gene) and HMC, respectively. In 7 of 13 patients with colorectal cancers and liver metastases, expression of HMC was increased up to 38-fold in primary tumors and metastatic foci as compared to adjacent normal colorectal mucosa. An increase in expression of HMC, its novel product likely to belong to a structurally distinct family of mitotic-control proteins, may be associated with malignant phenotypes of some colorectal cancers.

Adenocarcinoma↗

PubMed1998

Functional analysis of the mouse Scn8a sodium channel.

The mouse Scn8a sodium channel and its ortholog Na6 in the rat are abundantly expressed in the CNS. Mutations in mouse Scn8a result in neurological disorders, including paralysis, ataxia, and dystonia. In addition, Scn8a has been observed to mediate unique persistent and resurgent currents in cerebellar Purkinje cells (Raman et al., 1997). To examine the functional characteristics of this channel, we constructed a full-length cDNA clone encoding the mouse Scn8a sodium channel and expressed it in Xenopus oocytes. The electrophysiological properties of the Scn8a channels were compared with those of the Rat1 and Rat2 sodium channels. Scn8a channels were sensitive to tetrodotoxin at a level comparable to that of Rat1 or Rat2. Scn8a channels inactivated more rapidly and showed differences in their voltage-dependent properties compared with Rat1 and Rat2 when only the alpha subunits were expressed. Coexpression of the beta1 and beta2 subunits modulated the properties of Scn8a channels, but to a lesser extent than for the Rat1 or Rat2 channels. Therefore, all three channels showed similar voltage dependence and inactivation kinetics in the presence of the beta subunits. Scn8a channels coexpressed with the beta subunits exhibited a persistent current that became larger with increasing depolarization, which was not observed for either Rat1 or Rat2 channels. The unique persistent current observed for Scn8a channels is consistent with the hypothesis that this channel is responsible for distinct sodium conductances underlying repetitive firing of action potentials in Purkinje neurons.

Amino Acid Sequence↗