Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5′-GTAC and leave a 3′-TA overhang (5′-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure

Notes on the age determination, ovariole changes and gonotrophic cycle of Simulium ochraceum in Guatemala

金沢大学留学生センター室内および野外個体の解剖観察をもとに, S. ochraceumの羽化から吸血までの最短期間を2日, 吸血から産卵までを5日, 産卵後まもなく2回目の吸血をすると仮定すると, 吸血-産卵-吸血(gonotrophic cycle)の最短期間は5日と考えられた。調査4地点(1977年10月∿12月)の平均産卵経験率はS. ochraceum 48.7%, S. callidum 46.9%, S. metallicum 41.2%, S. mexicanum 38.9%であった。産卵経験率およびsac-like relic率は日変化を示し, 午後に高い傾向を示した。以上のことからS. ochraceumの日生存率は0.866と計算され, 羽化後1回目の吸血でO. volvulusのMf.を取り込んだとして, これが感染型に発育し, 伝播可能になる吸血後9日目の生存率は27.3%と推定された。 Of the S. ochraceum in Guatemala, the shortest duration from emergence to blood-feeding, from blood-feeding to oviposition (both at 22℃) and from oviposition to next blood-feeding (under the field condition) were presumed to be 2 days, 5 days and 0 day, respectively. One gonotrophic cycle of this species was to be 5 days in the shortest case. The average parous rate observed in the blackflies collected at four locations from October to December, 1977 were 48.7% for S. ochraceum, 41.2% for S. metallicum, 46.9% for S. callidum and 38.9% for S. mexicanum. The parous rate showed diurnal change, those of which captured in the afternoon being somewhat higher. The survival rate of S. ochraceum for 9 days after blood-feeding when this species become infective, was estimated at 27.3%