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141.
Repeat-Length-Independent Broad-Spectrum Shuffling, a Novel Method of Generating a Random Chimera Library In Vivo
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Koichi Mori Takafumi Mukaihara Yoshiko Uesugi Masaki Iwabuchi Tadashi Hatanaka 《Applied microbiology》2005,71(2):754-760
We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL+ gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb+ background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained. 相似文献
142.
Hiroto Tsujikawa Kenta Sato Cao Wei Gul Saad Kazuya Sumikoshi Shugo Nakamura Tohru Terada Kentaro Shimizu 《Journal of structural and functional genomics》2016,17(2-3):39-49
We present a new method for predicting protein–ligand-binding sites based on protein three-dimensional structure and amino acid conservation. This method involves calculation of the van der Waals interaction energy between a protein and many probes placed on the protein surface and subsequent clustering of the probes with low interaction energies to identify the most energetically favorable locus. In addition, it uses amino acid conservation among homologous proteins. Ligand-binding sites were predicted by combining the interaction energy and the amino acid conservation score. The performance of our prediction method was evaluated using a non-redundant dataset of 348 ligand-bound and ligand-unbound protein structure pairs, constructed by filtering entries in a ligand-binding site structure database, LigASite. Ligand-bound structure prediction (bound prediction) indicated that 74.0 % of predicted ligand-binding sites overlapped with real ligand-binding sites by over 25 % of their volume. Ligand-unbound structure prediction (unbound prediction) indicated that 73.9 % of predicted ligand-binding residues overlapped with real ligand-binding residues. The amino acid conservation score improved the average prediction accuracy by 17.0 and 17.6 points for the bound and unbound predictions, respectively. These results demonstrate the effectiveness of the combined use of the interaction energy and amino acid conservation in the ligand-binding site prediction. 相似文献
143.
In vivo recombination efficiency of two site‐specific recombination systems,VCre/VloxP and SCre/SloxP,in medaka (Oryzias latipes)
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Kenta Kishimoto Manabu Nakayama Masato Kinoshita 《Development, growth & differentiation》2016,58(6):516-521
The present study delineates the in vivo efficiency of two site‐specific recombination systems, VCre/VloxP and SCre/SloxP, in medaka (Oryzias latipes). VCre, SCre, and Cre RNA was microinjected into fertilized medaka eggs belonging to three transgenic lines harboring VloxP, SloxP, and loxP cassette. VCre induced site‐specific recombination specifically at VloxP sequence and SCre at SloxP sequence without any cross‐reactivity. These findings provide two novel alternative recombination systems in vivo in addition to the existing Cre/loxP and Flp/FRT systems, thus enabling sophisticated gene expression in model organisms. 相似文献
144.
Mayuko Suzuki Nobuhiko Suminokura Kenta Tanami Shinpei Yoshitake Shingo Masuda Mitsutoshi Tomotsune Hiroshi Koizumi 《Ecological Research》2016,31(6):957-962
To clarify the effects of long-term warming on ecosystem matter cycling, we conducted an in situ 7-year experimental warming (2009–2015) using infrared heaters in a cool temperate semi-natural grassland in Japan. We measured plant aboveground biomass, soil total C and N, soil inorganic N (NH4 +-N and NO3 ?-N), and soil microbial biomass for 7 years (2009–2015). We also measured heterotrophic respiration for 2 years (2013–2014) and assessed net N mineralization and nitrification in 2015. We found that warming immediately increased plant aboveground biomass, but this effect ceased in 2013. However, the soil microbial biomass was continuously depressed by warming. Soil inorganic N concentrations in warmed plots substantially increased in the later years of the experiment (2013–2015) and the potential net N mineralization rate was also higher than in the earlier years. In contrast, heterotrophic respiration decreased with warming in 2013–2014. Our observations indicate that long-term warming has a contrasting effect on plants and soil microbes. In addition, the warming could have different effects on subterranean C and N cycling. To enhance the accuracy of estimation of future climate change, it is essential to continuously observe the warming effects on ecosystems and to focus on the change in subterranean C and N cycling. 相似文献
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Kenta Shinoda Dali Zong Elsa Callen Wei Wu Lavinia C. Dumitrache Frida Belinky Raj Chari Nancy Wong Momoko Ishikawa Andre Stanlie Trisha Multhaupt-Buell Nutan Sharma Laurie Ozelius Michelle Ehrlich Peter J. McKinnon André Nussenzweig 《Molecular cell》2021,81(12):2611-2624.e10
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Substantial advances have accrued over the last decade in the identification of the processes that contribute to lymphatic vascular development in health and disease. Identification of distinct regulatory milestones, from a variety of genetic models, has led to a stepwise chronology of lymphatic development. Several molecular species have been identified as important tissue biomarkers of lymphatic development and function. At present, vascular endothelial growth-factor receptor (VEGFR)-3/VEGF-C/VEGF-D signaling has proven useful in the identification of clinical lymphatic metastatic potential and the assessment of cancer prognosis. Similar biomarkers, to be utilized as surrogates for the assessment of inherited and acquired diseases of the lymphatic circulation, are actively sought, and will represent a signal advance in biomedical investigation. 相似文献
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