Name: polysome profiling in leishmania, human cells and mouse testis
Uploaded: 2018-04-08T15:00:00
Description: Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

Forfatterne takke Ching Lee hjælp til lydoptagelse. Forskningen blev støttet med midler fra opstart fra Texas Tech University Health Sciences Center og af Center of Excellence for Translationel neurovidenskab og Therapeutics (CTNT) give PN-CTNT 2017-05 AKHRJDHW A.L.K.; en del af NIH grant R01AI099380 K.Z. James C. Huffman og Kristen R. Baca var CISER (Center for Integration af STEM Uddannelse & forskning) lærde og blev støttet af programmet.

Alle dyr behandlinger og håndtering af væv i undersøgelsen blev udført efter protokoller godkendt af institutionelle Animal Care og brug Udvalget på Texas Tech University Health Science Center i overensstemmelse med de nationale kontorer i Sundhed dyrevelfærd retningslinjer, protokolnummer 96005. Venligst ofre hvirveldyr og forberede væv ifølge retningslinjerne fra det institutionelle Animal Care og brug udvalg. Hvis mangler et sådant udvalg, henvises til National Institutes of Health dyrevelfærd retningslinjer...

<ol>
	<li>Schwanhausser, B., 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+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473 (7347), 337-342 (2011).</li><li>Capewell, P., 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=Regulation+of+Trypanosoma+brucei+Total+and+Polysomal+mRNA+during+Development+within+Its+Mammalian+Host.">Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host.</a> PLoS One. 8 (6), e67069 (2013).</li><li>Warner, J. R., Knopf, P. M., Rich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiple+ribosomal+structure+in+protein+synthesis.">A multiple ribosomal structure in protein synthesis.</a> Proceedings of the National Academy of Science, USA. 49, 122-129 (1963).</li><li>Piccirillo, C. A., Bjur, E., Topisirovic, I., Sonenberg, N., Larsson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+of+immune+responses:+from+transcripts+to+translatomes.">Translational control of immune responses: from transcripts to translatomes.</a> Nature Immunology. 15 (6), 503-511 (2014).</li><li>Ennis, H. L., Lubin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cycloheximide:+Aspects+of+Inhibition+of+Protein+Synthesis+in+Mammalian+Cells.">Cycloheximide: Aspects of Inhibition of Protein Synthesis in Mammalian Cells.</a> Science. 146 (3650), 1474-1476 (1964).</li><li>Masek, T., Valasek, L., Pospisek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+analysis+and+RNA+purification+from+sucrose+gradients.">Polysome analysis and RNA purification from sucrose gradients.</a> Methods in Molecular Biology. 703, 293-309 (2011).</li><li>Gandin, V., 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=Polysome+fractionation+and+analysis+of+mammalian+translatomes+on+a+genome-wide+scale.">Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.</a> Journal of Visualized Experiments. (87), (2014).</li><li>Zuccotti, P., Modelska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+Translatome+with+Polysome+Profiling.">Studying the Translatome with Polysome Profiling.</a> Methods in Molecular Biology. 1358, 59-69 (2016).</li><li>Chasse, H., Boulben, S., Costache, V., Cormier, P., Morales, J. <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+using+polysome+profiling.">Analysis of translation using polysome profiling.</a> Nucleic Acids Research. 45 (3), e15 (2017).</li><li>Arava, Y., 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=Genome-wide+analysis+of+mRNA+translation+profiles+in+Saccharomyces+cerevisiae.">Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae.</a> Proceedings of the National Academy of Science, USA. 100 (7), 3889-3894 (2003).</li><li>Gandin, V., 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=nanoCAGE+reveals+5'+UTR+features+that+define+specific+modes+of+translation+of+functionally+related+MTOR-sensitive+mRNAs.">nanoCAGE reveals 5' UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs.</a> Genome Research. 26 (5), 636-648 (2016).</li><li>Bazzini, A. A., Lee, M. T., Giraldez, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling+shows+that+miR-430+reduces+translation+before+causing+mRNA+decay+in+zebrafish.">Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish.</a> Science. 336 (6078), 233-237 (2012).</li><li>Zanchin, N. I., Goldfarb, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nip7p+interacts+with+Nop8p,+an+essential+nucleolar+protein+required+for+60S+ribosome+biogenesis,+and+the+exosome+subunit+Rrp43p.">Nip7p interacts with Nop8p, an essential nucleolar protein required for 60S ribosome biogenesis, and the exosome subunit Rrp43p.</a> Molecular Cell Biology. 19 (2), 1518-1525 (1999).</li><li>Darnell, J. 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=FMRP+stalls+ribosomal+translocation+on+mRNAs+linked+to+synaptic+function+and+autism.">FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism.</a> Cell. 146 (2), 247-261 (2011).</li><li>Gkogkas, C. G., 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=Autism-related+deficits+via+dysregulated+eIF4E-dependent+translational+control.">Autism-related deficits via dysregulated eIF4E-dependent translational control.</a> Nature. 493 (7432), 371-377 (2013).</li><li>Robichaud, N., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+and+the+cancer+cell+response+to+stress.">Translational control and the cancer cell response to stress.</a> Curr Opin Cell Biol. 45, 102-109 (2017).</li><li>Gordon, B. S., Kelleher, A. R., Kimball, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+muscle+protein+synthesis+and+the+effects+of+catabolic+states.">Regulation of muscle protein synthesis and the effects of catabolic states.</a> International Journal of Biochemistry and Cell Biology. 45 (10), 2147-2157 (2013).</li><li>Ishimura, R., 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=RNA+function.+Ribosome+stalling+induced+by+mutation+of+a+CNS-specific+tRNA+causes+neurodegeneration.">RNA function. Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.</a> Science. 345 (6195), 455-459 (2014).</li><li>Petersen, C. P., Bordeleau, M. E., Pelletier, J., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+RNAs+repress+translation+after+initiation+in+mammalian+cells.">Short RNAs repress translation after initiation in mammalian cells.</a> Molecular Cell. 21 (4), 533-542 (2006).</li><li>Juntawong, P., Girke, T., Bazin, J., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+dynamics+revealed+by+genome-wide+profiling+of+ribosome+footprints+in+Arabidopsis.">Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis.</a> Proceedings of the National Academy of Science, USA. 111 (1), E203-E212 (2014).</li><li>Bunnik, E. 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=Polysome+profiling+reveals+translational+control+of+gene+expression+in+the+human+malaria+parasite+Plasmodium+falciparum.">Polysome profiling reveals translational control of gene expression in the human malaria parasite Plasmodium falciparum.</a> Genome Biology. 14 (11), R128 (2013).</li><li>De Gaudenzi, J. G., Noe, G., Campo, V. A., Frasch, A. C., Cassola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+regulation+in+trypanosomatids.">Gene expression regulation in trypanosomatids.</a> Essays in Biochemistry. 51, 31-46 (2011).</li><li>Alves, L. R., Goldenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+related+to+stress+response+and+differentiation+in+protozoa.">RNA-binding proteins related to stress response and differentiation in protozoa.</a> World Journal of Biological Chemistry. 7 (1), 78-87 (2016).</li><li>De Pablos, L. M., Ferreira, T. R., Walrad, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+differentiation+in+Leishmania+lifecycle+progression:+post-transcriptional+control+conducts+the+orchestra.">Developmental differentiation in Leishmania lifecycle progression: post-transcriptional control conducts the orchestra.</a> Current Opinions in Microbiology. 34, 82-89 (2016).</li><li>Soto, 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=Cell-cycle-dependent+translation+of+histone+mRNAs+is+the+key+control+point+for+regulation+of+histone+biosynthesis+in+Leishmania+infantum.">Cell-cycle-dependent translation of histone mRNAs is the key control point for regulation of histone biosynthesis in Leishmania infantum.</a> Biochemical Journal. 379, 617-625 (2004).</li><li>McNicoll, F., 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=Distinct+3+'-untranslated+region+elements+regulate+stage-specific+mRNA+accumulation+and+translation+in+Leishmania.">Distinct 3 '-untranslated region elements regulate stage-specific mRNA accumulation and translation in Leishmania.</a> Journal of Biological Chemistry. 280 (42), 35238-35246 (2005).</li><li>Folgueira, 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=The+translational+efficiencies+of+the+two+Leishmania+infantum+HSP70+mRNAs,+differing+in+their+3+'-untranslated+regions,+are+affected+by+shifts+in+the+temperature+of+growth+through+different+mechanisms.">The translational efficiencies of the two Leishmania infantum HSP70 mRNAs, differing in their 3 '-untranslated regions, are affected by shifts in the temperature of growth through different mechanisms.</a> Journal of Biological Chemistry. 280 (42), 35172-35183 (2005).</li><li>Dumas, C., Chow, C., Muller, M., Papadopoulou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+class+of+developmentally+regulated+noncoding+RNAs+in+Leishmania.">A novel class of developmentally regulated noncoding RNAs in Leishmania.</a> Eukaryotic Cell. 5 (12), 2033-2046 (2006).</li><li>Kapler, G. M., Coburn, C. M., Beverley, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+the+human+parasite+Leishmania+major+delineates+a+30-kilobase+region+sufficient+for+extrachromosomal+replication+and+expression.">Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression.</a> Molecular Cell Biology. 10 (3), 1084-1094 (1990).</li><li>Karamyshev, A. L., Johnson, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+SecA+association+with+signal+sequences+in+ribosome-bound+nascent+chains:+a+potential+role+for+SecA+in+ribosome+targeting+to+the+bacterial+membrane.">Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane.</a> Journal of Biological Chemistry. 280 (45), 37930-37940 (2005).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nature Protocols. 3 (6), 1101-1108 (2008).</li><li>Panda, A. C., Martindale, J. L., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+Fractionation+to+Analyze+mRNA+Distribution+Profiles.">Polysome Fractionation to Analyze mRNA Distribution Profiles.</a> Bio Protocols. 7 (3), (2017).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. A Laboratory Manual. , (1989).</li><li>Patrick, A. E., Karamyshev, A. L., Millen, L., Thomas, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+CFTR+transmembrane+span+integration+by+disease-causing+mutations.">Alteration of CFTR transmembrane span integration by disease-causing mutations.</a> Molecular Biology of the Cell. 22 (23), 4461-4471 (2011).</li><li>Kleizen, B., van Vlijmen, T., de Jonge, H. R., Braakman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+CFTR+is+predominantly+cotranslational.">Folding of CFTR is predominantly cotranslational.</a> Molecular Cell. 20 (2), 277-287 (2005).</li><li>van den Elzen, A. M., Schuller, A., Green, R., Seraphin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dom34-Hbs1+mediated+dissociation+of+inactive+80S+ribosomes+promotes+restart+of+translation+after+stress.">Dom34-Hbs1 mediated dissociation of inactive 80S ribosomes promotes restart of translation after stress.</a> EMBO Journal. 33 (3), 265-276 (2014).</li><li>Morita, 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=mTOR+Controls+Mitochondrial+Dynamics+and+Cell+Survival+via+MTFP1.">mTOR Controls Mitochondrial Dynamics and Cell Survival via MTFP1.</a> Molecular Cell. 67 (6), 922-935 (2017).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324 (5924), 218-223 (2009).</li></ol>

Polysome fraktionering af saccharose gradient kombineret med RNA og proteinanalyse af brøker er en kraftfuld metode til at analysere translationel status for individuelle mRNAs eller hele translatome samt roller af protein faktorer regulering translationel maskiner under normale fysiologiske eller sygdom tilstand. Polysomal profilering er en specielt velegnet teknik til at studere translationel forordning i organismer såsom trypanosomatids herunder Leishmania hvor transcriptional kontrol er stort set fraværen...

Regulering af genekspression i celler styres af transcriptional, posttranskriptionelle og posttranslationelle mekanismer. Fremskridt i dyb RNA sekventering tillade studiet af steady-state mRNA niveauer på en genom-plan på et hidtil uset niveau. De seneste resultater viste imidlertid, at steady-state mRNA niveau ikke altid korrelerer med protein produktion1,2. Skæbnen, en individuel udskrift er meget kompleks og afhænger af mange faktorer som interne/eksterne stimuli, stress, osv. Regulering af genekspression under proteinsyntesen giver et ekstra lag af udtryk kontrol nødvendige for en hurtig reaktion på ændrede vilkår. Polysome (eller "polyribosome") profilering, adskillelse og visualisering af aktivt oversætte ribosomer, er en kraftfuld metode til at undersøge regulering af proteinsyntesen. Selv om sine første eksperimentelle programmer dukkede op i 1960s3, er polysome profilering i øjeblikket en af de vigtigste teknikker i protein oversættelse undersøgelser4. Enkelt mRNAs kan oversættes af mere end én ribosomet fører til dannelsen af en polysome. Udskrifter kan være gået i stå på ribosomer med cycloheximide5 og mRNAs der indeholder forskellige antal polysomes kan adskilles ved at polysome fraktionering af saccharose gradient ultracentrifugering6,7 , 8 , 9. RNA analyse af polysomal brøker derefter tillader måling af ændringer i individuelle mRNAs translationel staterne på genome-wide skala og under forskellige fysiologiske tilstande4,7, 10. metoden har været også bruges til at afsløre rollerne af 5' UTR og 3' UTR sekvenser i kontrol af mRNA translatability11, undersøge miRNAs i translationel undertrykkelse12rolle, afdækker defekter i ribosomet Biogenese13 , og forstår rollen af ribosomet-associerede proteiner med menneskelige sygdomme14,15. I det sidste årti, er en voksende rolle for reguleringen af genekspression under oversættelse opstået der illustrerer dens betydning i menneskers sygdomme. Beviserne for Translationel kontrol i kræft, metaboliske og neurodegenerative sygdomme er overvældende15,16,17,18. For eksempel dysregulering af eIF4E-afhængige Translationel kontrol bidrager til autisme relateret underskud15 og FMRP er involveret i stalling af ribosomer på mRNAs knyttet til autisme14. Polysomal profilering er således et meget vigtigt redskab til at studere defekter i translationel forordning i flere sygdomme hos mennesker.
Proteinanalyse af polysomal brøker under forskellige fysiologiske tilstande dissekerer funktion af faktorer i forbindelse med ribosomer under oversættelse. Polysome profilering teknikken er blevet brugt i mange arter, herunder gær, pattedyrceller, planter og protozoer10,19,20,21. Protozo parasitter som Trypanosoma og Leishmania udviser begrænset transcriptional kontrol af genekspression. Deres genomer er organiseret i polycistronic gen klynger, der mangler promotor-regulerede transskription22. Udviklingsmæssige genekspression styres i stedet overvejende på niveauet af protein oversættelse og mRNA stabilitet i trypanosomatid arter23,24. Derfor er forståelse af Translationel kontrol i mangel af transkriptionel regulering særlig vigtigt for disse organismer. Polysomal profilering er et kraftfuldt værktøj til at studere posttranskriptionelle regulering af genekspression i Leishmania25,26,27,28.
De seneste fremskridt i påvisning af individuelle mRNAs niveauer af real time kvantitativ PCR (RT-qPCR) og fuld transkriptom af næste generation sequencing, samt proteomics-teknologier, bringer opløsning og fordelene ved polysomal profilering til et nyt niveau. Brugen af disse metoder kan udvides yderligere ved analyse af enkelte polysomal fraktioner af dyb RNA sekventering kombineret med proteom analyse at overvåge translationel status af celler på en genome-wide skala. Dette giver mulighed for identifikation af nye molekylære spillere regulering oversættelse under forskellige fysiologiske og patologiske betingelser. Vi præsenterer her, en universal polysome profilering protokol, der bruges på tre forskellige modeller: parasit Leishmania store, dyrkede humane celler og animalsk væv. Vi præsenterer rådgivning om forberedelsen af cellelysater fra forskellige organismer, optimering af gradientbetingelserne, valg af RNase hæmmere og anvendelsen af RT-qPCR, vestlige skamplet og RNA elektroforese til at analysere polysome fraktioner i denne undersøgelse.

<table>
	<tbody>
		<tr>
			<td>Instruments:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient master</td>
			<td>Biocomp Instruments Inc.</td>
			<td>108</td>
		</tr>
		<tr>
			<td>Piston Gradient Fractionator</td>
			<td>Biocomp Instruments Inc.</td>
			<td>152</td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>Gilson, Inc.</td>
			<td>FC203B</td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop One</td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ts2-FL/Ts2</td>
		</tr>
		<tr>
			<td>2720 Thermal Cycler</td>
			<td>Applied Biosystems by Life Technologies</td>
			<td>4359659</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Panasonic Healthcare Co.</td>
			<td>MCO-170A1CUV</td>
		</tr>
		<tr>
			<td>HERATHERM incubator</td>
			<td>Thermo Scientific</td>
			<td>51028063</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet, class II, type A2</td>
			<td>NuAire Inc.</td>
			<td>NU-543-400</td>
		</tr>
		<tr>
			<td>Revco freezer</td>
			<td>Revco Technologies</td>
			<td>ULT1386-5-D35</td>
		</tr>
		<tr>
			<td>Beckman L8-M Ultracentifuge</td>
			<td>Beckman Coulter</td>
			<td>L8M-70</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Rotor SW41</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
		</tr>
		<tr>
			<td>Swing-bucket rotor</td>
			<td>Eppendorf</td>
			<td>A-4-62</td>
		</tr>
		<tr>
			<td>Fixed angle rotor</td>
			<td>Eppendorf</td>
			<td>F-45-30-11</td>
		</tr>
		<tr>
			<td>Quant Studio 12K Flex Real-Time PCR machine 285880228</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4470661</td>
		</tr>
		<tr>
			<td>TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0102</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>02-671-51B</td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triax software&#160;</td>
			<td>Biocomp Instruments Inc.</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides, dual chamber for cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
		</tr>
		<tr>
			<td>Open-top polyclear centrifuge tubes, (14 mm x 89 mm)</td>
			<td>Seton Scientific</td>
			<td>7030</td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>BD</td>
			<td>309646</td>
		</tr>
		<tr>
			<td>BD Syringe 3 mL23 Gauge 1 Inch Needle</td>
			<td>BD</td>
			<td>10020439</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 14 cm</td>
			<td>Thermo Scientific</td>
			<td>168381</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 9 cm</td>
			<td>Thermo Scientific</td>
			<td>172931</td>
		</tr>
		<tr>
			<td>Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPES</td>
			<td>Thermo Scientific</td>
			<td>569-0020</td>
		</tr>
		<tr>
			<td>BioLite 75 cm3 flasks</td>
			<td>Thermo Scientific</td>
			<td>130193</td>
		</tr>
		<tr>
			<td>Nunc 50 mL conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339653</td>
		</tr>
		<tr>
			<td>Chemicals:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol LS</td>
			<td>Ambion by Life Technologies</td>
			<td>10296028</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1378-5KG</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium-high glucose (DMEM)</td>
			<td>Sigma</td>
			<td>D6429-500ML</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F0926-50ML</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma</td>
			<td>D8537-500ML</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2x6H2O)</td>
			<td>Acros Organics</td>
			<td>AC413415000</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma</td>
			<td>P9541-500G</td>
		</tr>
		<tr>
			<td>Nonidet P 40 (NP-40)</td>
			<td>Fluka (Sigma-Aldrich)</td>
			<td>74385</td>
		</tr>
		<tr>
			<td>Recombinant Rnasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H3993-1MU</td>
		</tr>
		<tr>
			<td>cOmplete Mini EDTA-free protease inhibitors</td>
			<td>Roche Diagnostics</td>
			<td>11836170001</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Scientific</td>
			<td>R0551</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502-1L</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698-1G</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>194002</td>
		</tr>
		<tr>
			<td>Dithiotreitol (DTT)</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Fisher Scientific</td>
			<td>BP-1302-10</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium dehydrate (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>S316-212</td>
		</tr>
		<tr>
			<td>Optimem</td>
			<td>Life Technologies</td>
			<td>22600050</td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma</td>
			<td>P8833-100MG</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-3KG</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma</td>
			<td>T4049-100ML</td>
		</tr>
		<tr>
			<td>Hgh Capacity cDNA Reverse Transcriptase Kit</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4368814</td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4367659</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Scientific</td>
			<td>A144SI-212</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP26324</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Sigma</td>
			<td>221473-500G</td>
		</tr>
		<tr>
			<td>Anti-RPL11 antibody</td>
			<td>Abcam</td>
			<td>ab79352</td>
		</tr>
		<tr>
			<td>Ribosomal protein S6 (C-8) antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-74459</td>
		</tr>
		<tr>
			<td>1xM199</td>
			<td>Sigma</td>
			<td>M0393-10X1L</td>
		</tr>
		<tr>
			<td>Lithium cloride</td>
			<td>Sigma</td>
			<td>L-9650</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>D128-500</td>
		</tr>
		<tr>
			<td>Gel Loading Buffer II</td>
			<td>Thermo Scientific</td>
			<td>AM8546G</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Thermo Scientific</td>
			<td>16500-100</td>
		</tr>
		<tr>
			<td>Trichloracetic acid (TCA)</td>
			<td>Fisher Scientific</td>
			<td>A322-100</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS chemiluminescent substrate</td>
			<td>Thermo Scientific</td>
			<td>34580</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>BP531-500</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Sigma</td>
			<td>L5750-1KG</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626-5G</td>
		</tr>
		<tr>
			<td>RNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma</td>
			<td>A1852-1VL</td>
		</tr>
		<tr>
			<td>Cytosine 5&#39;-triphosphate disodium salt hydrate (CTP)</td>
			<td>Sigma</td>
			<td>C1506-250MG</td>
		</tr>
		<tr>
			<td>Uridine 5&#39;-triphosphate trisodium salt hydrate (UTP)</td>
			<td>Sigma</td>
			<td>U6625-100MG</td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate sodium salt hydrate (GTP)</td>
			<td>Sigma</td>
			<td>G8877-250MG</td>
		</tr>
		<tr>
			<td>SP6 RNA Polymerase</td>
			<td>NEB</td>
			<td>M0207S</td>
		</tr>
		<tr>
			<td>Pyrophoshatase</td>
			<td>Sigma</td>
			<td>I1643-500UN</td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266-1G</td>
		</tr>
	</tbody>
</table>

polysome profiling in leishmania, human cells and mouse testis

Ordentlig protein udtryk på det rigtige tidspunkt og i de rigtige mængder er grundlaget for normal cellefunktion og overlevelse i et hurtigt skiftende miljø. I lang tid, var gene expression undersøgelser domineret af forskning på det transkriptionel niveau. Men steady state niveauer af mRNAs korrelere ikke godt med protein produktion, og translatability af mRNAs varierer meget afhængigt af betingelserne. Nogle organismer, som parasit Leishmania, er protein udtryk reguleret for det meste på translationel niveau. Nylige undersøgelser påvist, at protein oversættelse dysregulering er forbundet med kræft, metabolisk, neurodegenerative og andre sygdomme hos mennesker. Polysome profilering er en kraftfuld metode til at studere protein oversættelse forordning. Det gør det muligt at måle individuelle mRNAs translationel status eller undersøge oversættelse på en genome-wide skala. Grundlaget for denne teknik er adskillelsen af polysomes, ribosomer, deres underenheder og gratis mRNAs under centrifugering af et cytoplasmatisk lysate gennem en saccharose gradient. Vi præsenterer her, en universal polysome profilering protokol brugt på tre forskellige modeller - parasit Leishmania store, dyrkede humane celler og animalsk væv. Leishmania celler vokse frit i suspension og dyrkede humane celler vokse i vedhængende éncellelag, mens musen testis repræsenterer en animalsk vævsprøve. Teknikken er således tilpasset til alle disse kilder. Protokol til analyse af polysomal brøker omfatter påvisning af individuelle mRNA niveauer af RT-qPCR, proteiner af vestlige skamplet og analyse af ribosomale RNA'er af elektroforese. Metoden kan udvides yderligere ved undersøgelse af mRNAs association med ribosomet på en transkriptom niveau af dyb RNA-FF. og analyse af ribosomet-associerede proteiner af masse spektroskopi af fraktioner. Metoden kan let justeres til andre biologiske modeller.

I denne undersøgelse, vi beskriver anvendelsen af den polysomal profilering teknik til tre forskellige kilder: parasitære Leishmania store, dyrkede humane celler og mus testis. Leishmania celler vokse frit i den flydende medier i suspension, dyrkede humane celler vokse i den vedhængende éncellelag på plader, og musen testis repræsenterer en vævsprøve. Metoden kan let justeres til andre typer af frit dyrkede celler i suspension, forskellige typer af væv eller fra...

Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Det overordnede mål med polysome profilering teknik er analyse af translationel aktivitet af individuelle mRNAs eller transkriptom mRNAs under proteinsyntesen. Metoden er vigtigt, at undersøgelser af protein syntese forordning, oversættelse aktivering og undertrykkelse i sundhed og flere sygdomme hos mennesker.

Polysome profilering i Leishmania, menneskelige celler og mus Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Research

JoVE Journal

Biochemistry

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Termostabilisering, Expression, Rensning, og Krystallisation af Human Serotonin Transporter Bundet til<em&gt; S</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Genome-wide kvantificering af oversættelse i spirende gær af ribosomet profilering

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Kalibrering-fri In Vitro kvantificering af Protein Homo-oligomerisering ved hjælp af kommercielle instrumentering og gratis, Open Source lysstyrke analyse Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

Antistatiske Microgravimetry at studere bindende dynamikken i Phospholipid bindende Protein Annexin A2 til fast-understøttet Lipid dobbeltlag ved hjælp af et Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Måling og fortolke ilt forbrug satser helt flyve hoved segmenter

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Nitropeptid profilering og identifikation illustreret af angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Complexome-profilering med høj opløsning ved Cryoslicing BN-MS-analyse

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Profilering af Ubiquitin og Ubiquitin-lignende afhængige posttranslationelle ændringer og identificering af væsentlige ændringer

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Fosforproteomic strategi for profilering osmotisk stress signalering i Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Ganglioside Extraction, Purification and Profiling

Gangliosidekstraktion, rensning og profilering

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...