<Original Article> The Effect of Ageing on Mutagen-Induced SCE in Mouse Spleen Lymphocytes

To investigate the effects of ageing, spleen lymphocytes from young and old mice were cultured for the analysis of spontaneous and mutagen-induced sister chromatid exchange (SCE) frequencies. Mutagens used were Mitomycin C, nitrogen mustard, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, and ethyl methane sulfonate. All induced significantly increased SCE frequencies in a dose-dependent manner. However, no significant difference between young and old samples was observed. Although mitogen stimulation definitely induces age-related changes in mouse spleen lymphocytes, the present SCE assay found no age-related impairment in DNA repair. This suggests that SCE methods in in vitro culture systems such as those used in the present study may not be suitable for the evaluation of age-related changes in DNA repair capacity.journal articl

インデックス付き講義収録システムの開発と運用

名古屋大学法科大学院では，学生の講義復習を支援するため，インデックス付き講義収録システムを開発し，運用している．学生は講義中に，その内容が「わからない」等と考えた場合，Microsoft Office Word のアドイン・ソフトウェアを利用して講義ノート（MS-Word ファイル）にインデックスを付すことにより，講義後，インデックス付加時点からの講義映像を視聴できる．また，インデックスの統計情報から，教員・学生双方が，学生の講義理解度を確認できる．本報告では，本システムの概要および運用状況について述べる．Nagoya University Law School has developed and been managing an indexed digital video recording system for reviewing class instructions, “Friends in Need,” to support law students after hours. The video recording system enables students who use an add-in software for Microsoft Office Word, to index important parts of the class, during a lecture, which they want to replay after class. Students can always review the class efficiently by replaying indexed parts of the video. Statistical information of the indexing provides both teachers and students with a way to check comprehension of class topics.journal articl

Study of double charmonium production in e+e- annihilation at sqrt[s]≈10.6 GeV

journal articl

ゼンホウコウ イドウ ロボット ノ ゼンホウイ シカク ニ モトヅク ニンゲン トノ キョウチョウ

奈良先端科学技術大学院大学修士(工学)master thesi

3.2 ことばのワークショップを評価の観点からふりかえる

departmental bulletin pape

Mechanisms Employed by <em>Escherichia coli</em> to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V

<div><p><em>Escherichia coli</em> pol V (UmuD′₂C), the main translesion DNA polymerase, ensures continued nascent strand extension when the cellular replicase is blocked by unrepaired DNA lesions. Pol V is characterized by low sugar selectivity, which can be further reduced by a Y11A “steric-gate” substitution in UmuC that enables pol V to preferentially incorporate rNTPs over dNTPs <em>in vitro.</em> Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A <em>in vitro</em>, strains expressing <em>umuC</em>_Y11A exhibit low UV mutability and UV resistance. Here, we show that these phenotypes result from the concomitant dual actions of Ribonuclease HII (RNase HII) initiating removal of rNMPs from the nascent DNA strand and nucleotide excision repair (NER) removing UV lesions from the parental strand. In the absence of either repair pathway, UV resistance and mutagenesis conferred by <em>umuC</em>_Y11A is significantly enhanced, suggesting that the combined actions of RNase HII and NER lead to double-strand breaks that result in reduced cell viability. We present evidence that the Y11A-specific UV phenotype is tempered by pol IV <em>in vivo</em>. At physiological ratios of the two polymerases, pol IV inhibits pol V–catalyzed translesion synthesis (TLS) past UV lesions and significantly reduces the number of Y11A-incorporated rNTPs by limiting the length of the pol V–dependent TLS tract generated during lesion bypass <em>in vitro</em>. In a <em>recA730 lexA</em>(Def) Δ<em>umuDC</em> Δ<em>dinB</em> strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, <em>umuC</em>_Y11A-dependent spontaneous mutagenesis is only ∼7% of that observed with wild-type pol V, but increases to ∼39% of wild-type levels in an isogenic Δ<em>rnhB</em> strain and ∼72% of wild-type levels in a Δ<em>rnhA</em> Δ<em>rnhB</em> double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the <em>E. coli</em> genome, but at the cost of higher levels of cellular mutagenesis.</p> </div

Quantitative UV survival and mutagenesis assays.

<p>A: Survival. Exponentially growing cells were exposed to various doses of UV-light and serial dilutions spread on LB plates containing spectinomycin. The number of viable colonies was determined after overnight incubation at 37°C. Error bars indicate the standard error of the mean. Consistent with the semi-quantitative UV-survival assay shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003030#pgen-1003030-g001" target="_blank">Figure 1</a>, UV-resistance of strains expressing Y11A_UmuC increased significantly in the Δ<i>rnhB</i> background, while there was no change in UV-survival of the strain harboring vector, pGB2, or expressing wild-type pol V in the <i>rnhB</i>^+/− strains. B: Mutagenesis. UV-induced mutagenesis was determined by exposing exponentially growing cells to 20 J/m² UV light. Cell viability was in the range of 85–90% survival for wild-type pol V and ∼60–70% for vector control, pGB2, and the Y11A mutant. The average number of His⁺ revertants per 10⁸ surviving cells ± standard error of the mean is indicated on the graph. The <i>rnhB</i>⁺ strains are indicated by navy-colored bars, while Δ<i>rnhB</i> strains are indicated by the gold-colored bars. As observed, the UmuC_Y11A-expressing cells exhibited an ∼9-fold increase in UV mutagenesis compared to the <i>rnhB</i>⁺ strain.</p

Effect of Δ<i>rnhA</i> and Δ<i>rnhB</i> on spontaneous mutagenesis in <i>recA730 lexA</i>(Def) Δ<i>umuDC</i> Δ<i>dinB</i> strains expressing pol V variants.

<p>Spontaneous mutagenesis was measured by assaying reversion of the <i>hisG4</i> ochre allele (leading to <i>histidine</i> prototophy) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003030#s4" target="_blank"><i>Materials and Methods</i></a>. The average number of His⁺ revertants per plate ± standard error of the mean is indicated in the table. Since the extent of mutagenesis promoted by wild-type pol V differed in the various strains, we have expressed the level of mutagenesis promoted by the variants as a percentage of wild-type mutagenesis. As clearly observed, <i>umuC</i>_Y11A-dependent mutagenesis increased in the Δ<i>rnhB</i> strain and was further elevated in the Δ<i>rnhA</i> Δ<i>rnhB</i> double mutant. In contrast, <i>umuC</i>_F10L gave consistently low levels of mutagenesis in all strains, and <i>umuC</i>_Y11F higher than wild-type levels in all strains.</p

Role of NER in strains expressing pol V variants.

<p>10 µl of 10-fold serial dilutions of overnight cultures were spotted onto the surface of rectangular LB agar plates and exposed to 40 J/m² 254 nM UV-light (Panel A), or 1 J/m² 254 nM UV-light (Panels B and C). Both unirradiated (−) and UV-irradiated (+) plates were incubated overnight at 37°C. In each panel, UV survival is shown for the <i>recA730 lexA</i>(Def) Δ<i>umuDC</i> Δ<i>dinB</i> strains either harboring pGB2 vector, or expressing pol V variants. Panel A (<i>uvr</i>⁺ strain) is reproduced from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003030#pgen-1003030-g001" target="_blank">Figure 1A</a> for direct comparison to the isogenic Δ<i>uvrA</i> (Panel B) and Δ<i>uvrC</i> (panel C) strains. The main observation of these experiments is that while the <i>uvr</i>⁻ strains are considerably more UV-sensitive than the isogenic <i>uvr</i>⁺ strain, the relative sensitivity of the strains expressing pol V variants changes in the <i>uvr</i>⁻ background, with UmuC_Y11A promoting an increase in UV-survival to a similar extent as wild-type pol V.</p

Effect of Δ<i>rnhA</i> and Δ<i>rnhB</i> on UV survival of <i>recA730 lexA</i>(Def) Δ<i>umuDC</i> Δ<i>dinB</i> strains expressing pol V variants.

<p>10 µl of 10-fold serial dilutions of overnight cultures were spotted onto the surface of rectangular LB agar plates and exposed to 40 J/m² 254 nM UV-light (panels A and C) and 20 J/m² 254 nM UV-light (panels B and D). Both unirradiated (−) and UV-irradiated (+) plates were incubated overnight at 37°C. In each panel, UV survival is shown for the <i>recA730 lexA</i>(Def) Δ<i>umuDC</i> Δ<i>dinB</i> strains either harboring pGB2 vector, or expressing pol V variants. The main observation of these experiments is that the UV-resistance of cells expressing <i>umuC</i>_Y11A increase dramatically in strains lacking <i>rnhB</i>, whereas survival of cells equipped with wild-type pol V, <i>umuC</i>_F10L, or <i>umuC</i>_Y11F is largely unaffected by the status of <i>rnhB</i>.</p