<meta charset="utf8"></head><body>熱ストレスシロイヌナズナにおける並進制御の解析

<ol>
	<li>Picard, F., Loubi&#232;re, P., Girbal, L., Cocaign-Bousquet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+translation+regulation+in+the+stress+response.">The significance of translation regulation in the stress response.</a> BMC genomics. 14, 1-11 (2013).</li><li>Yuan, S., Zhou, G., Xu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+machinery:+the+basis+of+translational+control.">Translation machinery: the basis of translational control.</a> J Genet Genom. 51 (4), 367-378 (2024).</li><li>Dever, T. E., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elongation,+termination,+and+recycling+phases+of+translation+in+eukaryotes.">The elongation, termination, and recycling phases of translation in eukaryotes.</a> Cold Spring Harb Perspect Biol. 4 (7), a013706 (2012).</li><li>Jackson, R. J., Hellen, C. U., Pestova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+translation+initiation+and+principles+of+its+regulation.">The mechanism of eukaryotic translation initiation and principles of its regulation.</a> Nat Rev Mol Cell Biol. 11 (2), 113-127 (2010).</li><li>Dennis, M. D., Browning, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+phosphorylation+of+plant+translation+initiation+factors+by+Arabidopsis+thaliana+CK2+holoenzymes.">Differential phosphorylation of plant translation initiation factors by Arabidopsis thaliana CK2 holoenzymes.</a> J Biol Chem. 284 (31), 20602-20614 (2009).</li><li>Dennis, M. D., Person, M. D., Browning, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+plant+translation+initiation+factors+by+CK2+enhances+the+in+vitro+interaction+of+multifactor+complex+components.">Phosphorylation of plant translation initiation factors by CK2 enhances the in vitro interaction of multifactor complex components.</a> J Biol Chem. 284 (31), 20615-20628 (2009).</li><li>Chen, G. H., Liu, M. J., Xiong, Y., Sheen, J., Wu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TOR+and+RPS6+transmit+light+signals+to+enhance+protein+translation+in+deetiolating+Arabidopsis+seedlings.">TOR and RPS6 transmit light signals to enhance protein translation in deetiolating Arabidopsis seedlings.</a> Proc Natl Acad Sci. 115 (50), 12823-12828 (2018).</li><li>Chang, H. H., Huang, L. C., Browning, K. S., Huq, E., Cheng, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phosphorylation+of+carboxyl-terminal+eIF2&#945;+by+SPA+kinases+contributes+to+enhanced+translation+efficiency+during+photomorphogenesis.">The phosphorylation of carboxyl-terminal eIF2&#945; by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis.</a> Nat Comm. 15 (1), 3467 (2024).</li><li>Liu, M. J., Wu, S. H., Chen, H. M., Wu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+translational+control+contributes+to+the+regulation+of+Arabidopsis+photomorphogenesis.">Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis.</a> Mol Sys Biol. 8 (1), 566 (2012).</li><li>Des Marais, D. L., Juenger, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropy,+plasticity,+and+the+evolution+of+plant+abiotic+stress+tolerance.">Pleiotropy, plasticity, and the evolution of plant abiotic stress tolerance.</a> Ann New York Acad Sci. 1206 (1), 56-79 (2010).</li><li>Mittler, R., Zandalinas, S. I., Fichman, Y., Van Breusegem, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+signalling+in+plant+stress+responses.">Reactive oxygen species signalling in plant stress responses.</a> Nat Rev Mol Cell Biol. 23 (10), 663-679 (2022).</li><li>Shulaev, V., Cortes, D., Miller, G., Mittler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+for+plant+stress+response.">Metabolomics for plant stress response.</a> Physio Plantarum. 132 (2), 199-208 (2008).</li><li>Sun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+heat+stress+and+drought+stress+in+pearl+millet+based+on+Pacbio+full-length+transcriptome+sequencing.">Transcriptome analysis of heat stress and drought stress in pearl millet based on Pacbio full-length transcriptome sequencing.</a> BMC Plant Biology. 20, 1-15 (2020).</li><li>Wasternack, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+jasmonates+in+plant+stress+responses+and+development-applied+aspects.">Action of jasmonates in plant stress responses and development-applied aspects.</a> Biotechnol Adv. 32, 31-39 (2014).</li><li>Chen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+translational+induction+during+NLR-mediated+immunity+in+plants+is+dynamically+regulated+by+CDC123,+an+ATP-sensitive+protein.">Global translational induction during NLR-mediated immunity in plants is dynamically regulated by CDC123, an ATP-sensitive protein.</a> Cell Host Microbe. 31 (3), 334-342 (2023).</li><li>Cho, H. Y., Chou, M. Y., Ho, H. Y., Chen, W. C., Shih, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethylene+modulates+translation+dynamics+in+Arabidopsis+under+submergence+via+GCN2+and+EIN2.">Ethylene modulates translation dynamics in Arabidopsis under submergence via GCN2 and EIN2.</a> Sci Adv. 8 (22), eabm7863 (2022).</li><li>Wen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+TaHSFA6e+modulates+heat+shock+protein-mediated+translational+regulation+in+response+to+heat+stress+in+wheat.">Alternative splicing of TaHSFA6e modulates heat shock protein-mediated translational regulation in response to heat stress in wheat.</a> New Phytol. 239 (6), 2235-2247 (2023).</li><li>Maruri-L&#243;pez, I., Figueroa, N. E., Hern&#225;ndez-S&#225;nchez, I. E., Chodasiewicz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stress+granules:+trends+and+beyond.">Plant stress granules: trends and beyond.</a> Front Plant Sci. 12, 722643 (2021).</li><li>Hamada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granule+formation+is+induced+by+a+threshold+temperature+rather+than+a+temperature+difference+in+Arabidopsis.">Stress granule formation is induced by a threshold temperature rather than a temperature difference in Arabidopsis.</a> J Cell Sci. 131 (16), jcs216051 (2018).</li><li>Jang, G. J., Jang, J. C., Wu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+functions+of+stress+granules+and+processing+bodies+in+plants.">Dynamics and functions of stress granules and processing bodies in plants.</a> Plants. 9 (9), 1122 (2020).</li><li>Ostberg, S., Schewe, J., Childers, K., Frieler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+crop+yields+and+their+variability+at+different+levels+of+global+warming.">Changes in crop yields and their variability at different levels of global warming.</a> Earth Sys Dyn. 9 (2), 479-496 (2018).</li><li>Wen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+TaHSFA6e+modulates+heat+shock+protein-mediated+translational+regulation+in+response+to+heat+stress+in+wheat.">Alternative splicing of TaHSFA6e modulates heat shock protein-mediated translational regulation in response to heat stress in wheat.</a> New Phytol. 239 (6), 2235-2247 (2023).</li><li>Coudert, L., Adjibade, P., Mazroui, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+translation+initiation+during+stress+conditions+by+polysome+profiling.">Analysis of translation initiation during stress conditions by polysome profiling.</a> J Vis Exp. (87), e51164 (2014).</li><li>Hsieh, E. J., Cheng, M. C., Lin, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+an+abiotic+stress-inducible+transcription+factor+AtERF53+in+Arabidopsis+thaliana.">Functional characterization of an abiotic stress-inducible transcription factor AtERF53 in Arabidopsis thaliana.</a> Plant Mol Biol. 82, 223-237 (2013).</li><li>Cheng, M. C., Liao, P. M., Kuo, W. W., Lin, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+ETHYLENE+RESPONSE+FACTOR1+regulates+abiotic+stress-responsive+gene+expression+by+binding+to+different+cis-acting+elements+in+response+to+different+stress+signals.">The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals.</a> Plant Physiol. 162 (3), 1566-1582 (2013).</li><li>Van den Broeck, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+network+to+phenotype:+the+dynamic+wiring+of+an+Arabidopsis+transcriptional+network+induced+by+osmotic+stress.">From network to phenotype: the dynamic wiring of an Arabidopsis transcriptional network induced by osmotic stress.</a> Mol Sys Biol. 13 (12), 961 (2017).</li><li>Dieterich, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=detection+and+identification+of+newly+synthesized+proteomes+with+bioorthogonal+non-canonical+amino-acid+tagging.">detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging.</a> Nat Protoc. 2 (3), 532-540 (2007).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+profiling+of+de+novo+protein+synthesis+in+starvation-induced+autophagy+using+bioorthogonal+noncanonical+amino+acid+tagging.">Proteomic profiling of de novo protein synthesis in starvation-induced autophagy using bioorthogonal noncanonical amino acid tagging.</a> Acad Press. 588, 41-59 (2017).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL eppendorf tube</td>
			<td>Labcon</td>
			<td>3012-870-000-9</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>13.2 mL centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Honeywell</td>
			<td>32712</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Honeywell</td>
			<td>32211</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Cycloheximide (CHX)</td>
			<td>Sigma-Aldrich</td>
			<td>SI-C7698</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate (DEPC)</td>
			<td>Sigma-Aldrich</td>
			<td>D5758</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>32221</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>GeneChip Eukaryotic Poly-A RNA Control Kit</td>
			<td>Invitrogen</td>
			<td>900433</td>
			<td>Normalization</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Honeywell</td>
			<td>15523</td>
			<td>Normalization</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>SI-H3149</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>HiScript III RT SuperMix for qPCR kit</td>
			<td>Vazyme</td>
			<td>R323-01</td>
			<td>Normalization</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>J.T.Baker</td>
			<td>&#160;3040-01</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>SI-M8266</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>MS basal medium</td>
			<td>Phyto</td>
			<td>M524</td>
			<td>Plant culture</td>
		</tr>
		<tr>
			<td>Peak Chart Syringe Pump</td>
			<td>Brandel</td>
			<td>SYN4007LS</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Polyoxyethylene-10-Tridecyl-Ether (PTE)</td>
			<td>Sigma-Aldrich</td>
			<td>P2393</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>RNasin</td>
			<td>Promega</td>
			<td>N251B</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate (DOC)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SI-D6750</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S5391</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>SYBR Green Supermix</td>
			<td>Bio-Rad</td>
			<td>BP170-8882</td>
			<td>Normalization</td>
		</tr>
		<tr>
			<td>TRI reagent</td>
			<td>MRC</td>
			<td>TR118</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>J.T.Baker</td>
			<td>&#160;4109-06</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima L-100K</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>UV/VISDETECTOR</td>
			<td>Brandel</td>
			<td>UA-6</td>
			<td>Polysome profile</td>
		</tr>
	</tbody>
</table>

translation efficiency test using polysome profiles under heat stress

<meta charset="utf8"></head><body>著者スポットライト:熱ストレス下での シロイヌナズ ナの翻訳調節を研究するためのポリソームプロファイリングプロトコル

ポリソームプロファイルを用いた熱ストレス下での翻訳効率試験

Translational control of different genes under heat stress is a critical step for plant adaptation to the environment. Assessing the translational ...

translation-efficiency-test-using-polysome-profiles-under-heat-stress

Research

JoVE Journal

Biology

Translation Efficiency Test Using Polysome Profiles Under Heat Stress

1.2K ビュー.  National Taiwan University. 私たちの研究では、植物の熱ストレス時の並進制御を調査しています。非ポリソーム結合RNAとポリソーム結合RNAを解析することで、植物が翻訳制御を通じてストレス耐性をどのように制御しているかを研究することを目指しています。そこで、ここでは、熱ストレス下でのシロイヌナズナの翻訳効率を調査するためのポリソームプロファイリン?...

ビデオ: 著者スポットライト:熱ストレス下での シロイヌナズ ナの翻訳調節を研究するためのポリソームプロファイリングプロトコル

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

ヒートショック法による大腸菌へのプラスミドDNAの形質転換

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

生物学

Psychophysiological Stress Assessment Using Biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.

Psychophysiological Stress Assessment Using Biofeedback

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

バイオフィードバックを用いた精神生理学的ストレス評価

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

マイクロ流体フローセルを使用して流れの下で血小板粘着と凝集

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

で遺伝子発現プロファイルを決定 C.エレガンスマイクロアレイとリアルタイムPCRを用いて

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

ポリソームプロファイリングによってストレス状態の間に翻訳開始の分析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

ポリソームプロファイリングによる哺乳動物細胞における選択的mRNAの翻訳の評価

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

工場ポリソームプロファイリングのための簡単​​な方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

リトマス試験を用いて細菌の比色検出

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of thermoregulation in birds across a variety of ecological circumstances. However, few studies characterize the contribution of the feathers alone to thermoregulation. Several previous studies have established methods for measuring the insulation value of animal pelts, but they require destructive sampling methods that are problematic for birds, whose feathers are not distributed evenly across the skin. More information is needed about 1) how the contribution of feathers to thermoregulation varies both across and within species and 2) how feather coats may change over space and time. Reported here is a method for rapidly and directly measuring the thermal performance of feather coats and the skin using dried whole skin specimens, without the need to destroy the skin specimen. This method isolates and measures the thermal gradient across a feather coat in a way that measurements of heat loss and metabolic cost in live birds, which use behavioral and physiological strategies to thermoregulate, cannot. The method employs a thermal camera, which allows the rapid collection of quantitative thermal data to measure heat loss from a stable source through the skin. This protocol can easily be applied to various research questions, is applicable to any avian taxa, and does not require destruction of the skin specimen. Finally, it will further the understanding of the importance of passive thermoregulation in birds by simplifying and accelerating the collection of quantitative data.

This protocol describes the quantification of heat transmission through a flat skinned avian specimen using a thermal camera and hot water bath. The method allows obtaining of quantitative, comparative data about the thermal performance of feather coats across species using dried flat skin specimens.

サーマルカメラを使用して鳥の羽のコートを通して熱損失を測定する

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of ...