Kimihiko Sugaya

Proto‐oncogene of int‐3, a mouse Notch homologue, is expressed in endothelial cells during early embryogenesis

Background: Notch and its homologues are key regulatory receptors of the cell fate decision in various developmental processes. The int‐3 oncogene was originally identified as a frequent target in Mouse Mammary Tumour Virus (MMTV)‐induced mammary tumours and has been regarded as a Notch homologue, based on its similarity to the intracellular domain of Notch. Studies with int‐3 transgenic mice have suggested that the int‐3 transgene affects the differentiation capacity of stem cells and leads to neoplastic proliferation in epithelial cells. However, the exact nature and the in vivo expression pattern of the int‐3 proto‐oncogene are unknown. The function of gene products in embryogenesis is also not clear.

Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

Temperature-sensitive (ts) CHO-K1 mutant tsTM3 exhibits chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. Previously, complementation tests with other mutants showed that tsTM3 harbors a genetic defect in the ubiquitin-activating enzyme Uba1. Sequence comparison of the Uba1 gene between wild-type and mutant cells in this study revealed that the mutant phenotype is caused by a G-to-A transition that yields a Met-to-Ile substitution at position 256 in hamster Uba1. The ts defects in tsTM3 were complemented by expression of the wild-type Uba1 tagged with green fluorescent protein. Expression of the Uba1 primarily in the nucleus appeared to rescue tsTM3 cells. Incubation at 39°C resulted in a decrease of nuclear Uba1 in tsTM3 cells, suggesting that loss of Uba1 in the nucleus may lead to the ts defects. Analyses with the fluorescent ubiquitination-based cell cycle indicator revealed that loss of function of Uba1 leads to failure of the ubiquitin system in the nucleus. Incubation at 39°C caused an increase in endogenous geminin in tsTM3 cells. A ts mutation of Uba1 found in tsTM3 cells appears to be a novel mutation reflecting the important roles of Uba1 in nucleus.

Recombination in the class III region of the mouse major histocompatibility complex.

The sites of meiotic recombination in the class II region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. To search for hotspots in the class III region, we mapped combiantional break-points of 79 Ab: H2-D recombinants with 11 DNA markers; these included Tnx, the gene for an extracellular matrix protein, tenascin X, the Notch-related Int3 gene, and a microsatellite marker, D17Mit13, none of which had previously been mapped precisely. The results gave the gene order Eb-61.11-Int3-Tnx-Cyp21/C4-Bf-Hsp68c-D17Mit13-Tnfa/Tnfb-D. The crossover sites in 40 of the 79 recombinants were cofiend within the Eb/Int3:Tnx/Cyp21 interval. The result demonstrated that an unequal distribution of recombination is a general feature of the mouse MHC, suggesting the presence of a recombinational hotsopt within the Int3:Tnx interval.

Enlargement of speckles of SF2/ASF due to loss of function of Smu1 is characterized in the mammalian temperature-sensitive mutant

A temperature-sensitive (ts) CHO-K1 mutant cell line, tsTM18, exhibits chromosomal instability with decreased DNA synthesis at the nonpermissive temperature, 39oC. An amino acid substitution in Smu1 underlying the ts phenotypes of tsTM18 cells was identified previously. We also found a ts defect in splicing of the unc52/perlecan gene. In the present study, we have generated cell lines expressing Smu1 tagged with green fluorescent protein (GFP) to study the dynamics of Smu1 in living cells. The hybrids complement deficiencies in tsTM18 cells and allow them to grow normally at 39°C. GFP-tagged Smu1 is found in speckles in many discrete nucleoplasmic sites, and most of these also contained SF2/ASF. SF2/ASF is a member of the serine/arginine (SR)-rich splicing group of factors that are necessary for spliceosome assembly and can influence alternative splicing. SF2/ASF is also involved in the integrity of genome maintenance. In tsTM18 cells cultured at 39°C, the Smu1 ts defect appears to alter SF2/ASF localization, suggesting a physiological association of Smu1 with SF2/ASF. The significant decrease of Smu1 may lead the enlargement of speckles of SF2/ASF. These data show the importance of Smu1 as a regulator of splicing and genome maintenance.

Regeneration of Murine Hair Follicles is Inhibited by Low-Dose-Rate Gamma Irradiation.

To determine whether the effects of low-dose-rate gamma (γ) irradiation are identifiable in the regeneration of murine hair follicles, we irradiated whole bodies of C57BL/10JHir mice in the first telogen phase of the hair cycle with 137Cs γ-rays. The mice were examined for effects on hair follicles, including number, morphology, and pigmentation in the second anagen phase. Effects of γ-radiation on melanocyte stem cells were also investigated by the indirect immunolabeling of tyrosinase-related protein 2 (TRP2). Irradiated skin showed a decrease in hair follicle density and the induction of curved hair follicles along with the presence of white hairs and hypopigmented hair bulbs. There was a small, but not significant, change in the number of TRP2-positive melanocyte stem cells in the hair bulge region of the irradiated skin. These results suggest that low-dose rate γ-irradiation does not deplete melanocyte stem cells, but can damage stem cells and progenitors for both keratinocytes and melanocytes, thereby affecting the structure and pigmentation of regenerated hair follicles in the 2nd anagen phase.