Name: measuring mrna levels over time during the yeast s. cerevisiae hypoxic response
Uploaded: 2017-08-10T15:00:00
Description: Watch this Scientific Journal Video about Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response at JoVE.com

Vi takker Lewis-Sigler Institute for Integrativ genomforskning sekventering Core facilitet på Princeton University for teknisk rådgivning og RNA bibliotek forberedelse og sekventering. Dette arbejde blev støttet af tilskud fra Rowan Universitet og NIH NIGMS R15GM113187 til M.J.H.

1. inducerende hypoxi
<ol><li>En dag eller mere før hypoxi tidsforløb: forberede rugemaskinen, cell filtrering system, vakuum, gastank, kolber, propper, glas rør og slanger, som i Materialer tabel.</li>
	<li>Place N2 tank, inkubator, vakuum og filtrering system i umiddelbar nærhed, der gør hurtig behandling af celler.</li>
	<li>Forberede sterile flydende YPD medier (1% gær-ekstrakt, 2% pepton, 2% glukose) ved at blande komponenterne i en glasflaske og autoklavering.</li>
	<li>Plan layo...

<ol>
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I denne undersøgelse, mRNA niveau for alle gener blev målt under hypoxi i gær S. cerevisiae. Målet var at analysere hvordan global gen expression ændringer som følge af vækst i en kontrolleret nær iltfrit miljø. Blev taget flere skridt til at sikre, at metoden beskrevet her var omhyggeligt kontrolleret og reproducerbar. Første celler blev udsat for et præcist definerede hypoksiske miljø: 99,999% N2 i rig medier (YPD). Andre undersøgelser af hypoxi har lukket kolbe eller rør til luft <sup...

Mange organismer reagere på hypoxi, eller lav O2, ved at ændre gen expression 1,2,3. Dette svar hjælper celler klare med manglen på en kritisk for aerob respiration og flere biosyntetiske reaktioner substrat, men også med en skiftende redox stat 4. Flere microarray undersøgelser udført i S. cerevisiae vis at mRNA niveauer af hundredvis af gener ændre svar på hypoxi 5,6,7,8,9 , 10 , 11 , 12. for nylig, RNA-seq blev brugt til at karakterisere gen expression ændringer over tid under hypoxi 13. Her, de eksperimentelle detaljer præsenteres og diskuteres.
Hypoxi kan opnås på forskellige måder, hver producerer et andet niveau af O2. Her, hypoxi blev etableret af konstant strømmende ultra høj renhed N2 i flasker, der sænker [O2] opløst straks med reproducerbare kinetik 10. Det er muligt, at der er nogle O2 molekyler findes der bidrager til metabolisme og gen udtryk men dette miljø betragtes meget tæt på anaerobe. I mangel af O2, kan gærceller ikke biosynthesize hæm, ergosterol og umættede fedtsyrer 4,12,14. Således, tidligere undersøgelser har medtaget disse metabolitter, når de vokser gær uden ilt 5,10,15. Men mange hypoksiske svar er medieret af nedbrydningen af disse metabolitter og dermed efterfyldning dem vender hypoksiske gen expression svar 12,16. For at efterligne naturlige hypoxi, blev disse metabolitter ikke føjet til medierne. I den korte tid at celler blev udsat for hypoxi uden tilstedeværelsen af disse væsentlige metabolitter, var der ingen mærkbar stigning i celledød (data ikke vist) eller en langvarig stress respons 13.
Svaret er også afhængig af stammen og dens genotype. Specielt vigtigt er alleler af de kendte regulatorer af hypoksiske svar 2. S288C stamme baggrund er meget eftertragtede, således at resultaterne kan sammenlignes med de andre genomiske undersøgelser udført med denne stamme. S288C indeholder imidlertid en delvis tab af funktion allel af HAP1 genet 17, en transcriptional regulator kritisk til det hypoksiske svar. Denne allel blev repareret i S288C ved hjælp af en vildtype kopi fra Σ1278b stamme baggrund 11.
Genekspression er meget afhængige af den cellulære miljø. Derfor, når du udfører genome-wide mRNA analyse, er det vigtigt at opretholde en konstant miljø samtidig med varierende en anden parameter som tid, stimulus eller genotype. For at opnå meget reproducerbare resultater, overveje disse tre fremgangsmåder for undersøgelsen og alle dens biologiske eller tekniske replikater. Først, den samme experimenter(s) skal foretage undersøgelsen, da tekniske fremgangsmåder kan variere på tværs af eksperimentatorer. Andet, det samme parti af ingredienser skal bruges i vækst medier, som hvert parti har en lidt anderledes sammensætning, der kan påvirke genekspression. For det tredje for at minimere cellecyklus effekter, bør hvert tidspunkt bestå af asynkrone celler i midten log fase af vækst (1-2 x 107 celler/mL).
Når kendetegner en kompleks reaktion som gen expression svar på hypoxi, er et tidsforløb fordelagtige for bestemmelse af kinetik af forskellige arrangementer. Bestemt tidspunkt punkter bør vælges der vil fange de store forandringer af respons. I denne undersøgelse, blev tidspunkter mellem 0 og 4 h observeret, fordi tidligere eksperimenter revealed omfattende ændringer i genekspression under denne periode 13.
For at måle globale genekspression var RNA-seq brugt 18,19. Denne metode bruger next generation sequencing til at bestemme den relative forekomst af hvert gen udskrift. I forhold til DNA microarray analyse, udstiller RNA-seq højere følsomhed (at opdage mindre rigelige afskrifter), et større dynamikområde (til at måle større fold ændringer) og overlegen reproducerbarhed (til præcist følger genekspression over tid). Mest cellulære RNA er typisk ribosomale RNA så mange metoder er blevet udviklet for at berige for specifikke RNA arter 20. Her, poly-T perler blev brugt til at rense poly-A-holdige mRNA udskrifter, selv om de forskellige kommercielt - tilgængelige rRNA udtynding kits kunne også være effektiv i mRNA berigelse.
Her, var S. cerevisiae gen expression svar på hypoxi karakteriseret. Celler blev udsat for iltsvind og derefter stikprøven på otte tiden point (0, 5, 10, 30, 60, 120, 180 og 240 min). At bekræfte reproducerbarhed og identificere statistisk ændrede udskrifter blev tre biologiske replikater udført. RNA blev udvundet ved mekaniske forstyrrelser og kolonne rensning, og derefter behandlet for RNA-seq analyse. Efter sekvensering rørledningen er beskrevet og programmering scripts er forudsat at give nøjagtige replikering af analyserne udføres. Specifikt, blev Trimmomatic 21, TopHat2 22, HTseq 23, R statistiske miljø 24og DESeq2 pakke 25 brugt til at behandle RNA-seq data og identificere 607 gener, der ændrer sig væsentligt i løbet hypoxi. Principal komponent analyse (PCA) og genekspression af replikater angivet reproducerbarhed af teknikken. Clustering og heatmaps afsløret vidtrækkende udtryk kinetik, mens gen ontologi (GO) Analysen viste, at mange cellulære processer, som aerob respiration, er beriget med ilt-regulerede gener sæt.

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			<td height="126" style="height:126px;width:292px;">Enclosed dry incubator</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">MaxQ 4000</td>
			<td style="width:380px;">Set at 30&#176;C. Modify the door to allow entry of one Tygon tube. Alternatively, use the New Brunswick G25 incubator, which contains a tube port. Do not use an open-air water shaker, as condensation will collect in the tubes between flasks, possibly cross-contaminating cultures.</td>
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			<td height="63" style="height:63px;width:292px;">Micro-analysis Filter Holder</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:119px;">XX1002530</td>
			<td style="width:380px;">25mm diameter, stainless steel support, no. 5 perforated silicone stopper mounts in standard 125mL filtering flask</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:292px;">Strong vacuum</td>
			<td style="width:183px;">Edwards</td>
			<td style="width:119px;">E-LAB 2</td>
			<td style="width:380px;">The &#8220;house&#8221; vacuum may be too weak. Alternatively, use an electric-power portable vacuum pump like the one listed here.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">1000-mL flask</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">To act as vacuum trap.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:292px;">~2-foot lengths of Heavy Wall Vacuum Tubing, inner diameter 3/8 in, outer diameter 7/8 in</td>
			<td style="width:183px;">Tygon</td>
			<td style="width:119px;">38TT</td>
			<td style="width:380px;">Two pieces: the first connects vacuum to trap, and the second connects trap to filter system.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">High-pressure N2 gas tank</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">99.999% purity, &#62;1000psi, with a regulator and gas flow controller</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Autoclaved 500mL flask</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">Opening covered with aluminum foil. One for each yeast strain.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Autoclaved 250mL flasks</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">Openings covered with aluminum foil. One for each time point plus two for water traps.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Flask stoppers (size 6, two holes with 5mm diameter)</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">Sterilized with 70% ethanol. One for each flask.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:292px;">Glass tubing, length 9 cm or 17 cm, inner diameter 2 mm, outer diameter 5 mm</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">Sterilized with 70% ethanol. Two tubes for each flask. Place into the holes of each stopper. See Figure 1 for placement of 9- vs 17- cm tubes.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">~25-cm lengths of plastic tubing, inner diameter 5 mm</td>
			<td style="width:183px;">Tygon</td>
			<td style="width:119px;">E-3603</td>
			<td style="width:380px;">One piece for each flask. Sterilized with 70% ethanol.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Sterile filter discs</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:119px;">HAWP02500</td>
			<td style="width:380px;">25mm diameter, 0.45 &#181;m pore size, one for each time point</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:292px;">Sterile dH2O (~100mL)</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">1-mL cuvettes</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">For measuring OD600 (i.e., cell concentration)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">50-mL sterile centrifuge tubes</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">One for each time point</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Clean and sterile tweezers</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">liquid nitrogen</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">For freezing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">acid-washed beads</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">G8772</td>
			<td style="width:380px;">Keep at 4&#176;C for lysing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Qiagen RNeasy Mini Kit</td>
			<td style="width:183px;">Qiagen</td>
			<td align="right" style="width:119px;">74104</td>
			<td style="width:380px;">For RNA column purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Qiagen RLT buffer</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">Prepare by adding 10&#181;L of &#946;-Mercaptoethanol per 1mL of RLT buffer, keep at 4C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-mL collection tubes</td>
			<td style="width:183px;">Qiagen</td>
			<td style="width:119px;"></td>
			<td style="width:380px;">included in the Qiagen Rneasy Mini Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Buffer RPE</td>
			<td style="width:183px;">Qiagen</td>
			<td style="width:119px;"></td>
			<td style="width:380px;">included in the Qiagen Rneasy Mini Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Buffer RW1</td>
			<td style="width:183px;">Qiagen</td>
			<td style="width:119px;"></td>
			<td style="width:380px;">included in the Qiagen Rneasy Mini Kit</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:292px;">DNase I stock and working solutions</td>
			<td style="width:183px;">Qiagen</td>
			<td align="right" style="width:119px;">79254</td>
			<td style="width:380px;">The DNase I enzyme comes as lyophilized powder in a glass vial. Using a sterile needle and syringe, inject 550&#181;L of RNase-free water (provided in Qiagen kit) into the vial. Mix by gently inverting the bottle. To avoid denaturing the enzyme, do not vortex. Using a pipet, remove this stock solution from the vial and store in freezer (-20&#176;C) in single-use aliquots (80&#181;L each). The stock solution should not be thawed and refrozen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Buffer RDD</td>
			<td style="width:183px;">Qiagen</td>
			<td style="width:119px;"></td>
			<td style="width:380px;">included in the Qiagen DNase Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Ice cold 2-mL screw-cap tubes</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">For lysing cells during RNA extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">bead mill homogenizer</td>
			<td style="width:183px;">Biospec Mini-Beadbeater-24</td>
			<td align="right" style="width:119px;">112011</td>
			<td style="width:380px;">Keep in cold room</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Bacto Peptone</td>
			<td style="width:183px;">BD</td>
			<td style="width:119px;">DF0118</td>
			<td style="width:380px;">for liquid YPD media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Bacto Yeast Extract</td>
			<td style="width:183px;">BD</td>
			<td style="width:119px;">DF0886</td>
			<td style="width:380px;">for liquid YPD media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">glucose</td>
			<td style="width:183px;">Fisher</td>
			<td style="width:119px;">D16</td>
			<td style="width:380px;">for liquid YPD media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Qubit assay tubes</td>
			<td style="width:183px;">Thermo Fisher</td>
			<td style="width:119px;">Q32856</td>
			<td style="width:380px;">for measuring nucleic acid concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Quant-iTTM dsDNA BR Assay Kit</td>
			<td style="width:183px;">Thermo Fisher</td>
			<td style="width:119px;">Q32853</td>
			<td style="width:380px;">for measuring nucleic acid concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Quant-iTTM RNA Assay Kit</td>
			<td style="width:183px;">Thermo Fisher</td>
			<td style="width:119px;">Q32855</td>
			<td style="width:380px;">for measuring nucleic acid concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Qubit Fluorometer</td>
			<td style="width:183px;">Thermo Fisher</td>
			<td style="width:119px;">Q33216</td>
			<td style="width:380px;">for measuring nucleic acid concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Commercial electrophoresis system</td>
			<td style="width:183px;">Agilent</td>
			<td>Bioanalyzer 2100</td>
			<td style="width:380px;">for measuring nucleic acid quality</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Next-generation sequencer</td>
			<td style="width:183px;">Illumina</td>
			<td style="width:119px;">HiSeq 2500</td>
			<td style="width:380px;">for sequencing libraries</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">automated liquid handling system</td>
			<td style="width:183px;">Wafergen</td>
			<td style="width:119px;">Apollo 324</td>
			<td style="width:380px;">for creating sequencing libraries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">PrepX PolyA mRNA Isolation Kit</td>
			<td style="width:183px;">Wafergen</td>
			<td align="right" style="width:119px;">400047</td>
			<td style="width:380px;">for isolating mRNA from total RNA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">PrepX RNA SEQ for Illumina Library Kit</td>
			<td style="width:183px;">Wafergen</td>
			<td align="right" style="width:119px;">400039</td>
			<td style="width:380px;">for creating strand-specific sequencing libraries from total RNA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:292px;">Barcode Splitter</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">https://toolshed.g2.bx.psu.edu/repository?repository_id=7119c4f7a89efa57&#38;changeset_revision=e7b7cdc1834d</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Samtools, which includes the gzip command</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">http://www.htslib.org/download/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Trimmomatic</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">http://www.usadellab.org/cms/?page=trimmomatic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Bowtie2 (installed before TopHat)</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">http://bowtie-bio.sourceforge.net/bowtie2/index.shtml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">TopHat</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">https://ccb.jhu.edu/software/tophat/index.shtml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">HTSeq</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">http://www-huber.embl.de/HTSeq/doc/overview.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">R (installed before R Studio)</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">https://cran.rstudio.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">R Studio (free version)</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:380px;">https://www.rstudio.com/products/rstudio/download/</td>
		</tr>
	</tbody>
</table>

measuring mrna levels over time during the yeast s. cerevisiae hypoxic response

Komplekse ændringer i genekspression mægle typisk en stor del af en cellulær svar. Hvert gen kan ændre udtryk med unik kinetik, som genet er reguleret af de særlige timing af en af mange stimuli, signalering veje eller sekundære virkninger. For at fange hele genet blev udtryk svar på iltsvind i gær S. cerevisiae, RNA-seq analyse brugt til at overvåge mRNA niveauer af alle gener på bestemte tidspunkter efter udsættelse for hypoxi. Hypoxi blev etableret af voksende celler i ~ 100% N2 gas. Vigtigere, i modsætning til andre hypoksiske undersøgelser, blev ergosterol og umættede fedtsyrer ikke føjet til medierne fordi disse metabolitter påvirker genekspression. Tid point blev valgt i intervallet fra 0 - 4 h efter hypoxi fordi denne periode indfanger de store ændringer i genekspression. På hvert tidspunkt, medio log hypoksiske celler blev hurtigt filtreret og frosset, begrænsning af eksponering til O2 og samtidige ændringer i genekspression. Total RNA blev udvundet af celler og bruges til at berige for mRNA, som blev derefter omdannet til cDNA. Fra denne cDNA, multiplex biblioteker var lavet og otte eller flere prøver blev sekventeret i én vognbane af en næste-generations sequencer. Efter sekvensering rørledning er beskrevet, som omfatter kvalitet base trimning, læse kortlægning og fastsættelsen af antallet af læser per genet. DESeq2 inden for R statistiske miljø blev brugt til at identificere gener, der ændrer væsentligt ved et af punkterne hypoksiske tid. Analyse af tre biologiske replikater viste høj reproducerbarhed, gener af forskellige kinetik og et stort antal forventede O2-reguleret gener. Disse metoder kan bruges til at undersøge, hvordan cellerne i forskellige organismer reagere på hypoxi med tiden og tilpasset til at studere genekspression under andre cellulære svar.

Hypoxi tidsforløb og RNA-seq analyse blev udført uafhængigt tre gange. For at undersøge de tre replikater reproducerbarhed, blev genekspression data for alle gener analyseret ved hjælp af Principal komponent analyse (PCA). Figur 2 viser, hvordan prøverne ændres over de første to vigtigste komponenter, som tilsammen repræsenterer 58,9% af variabiliteten. Denne analyse viste, at hver gang kursus udviser lignende ændringer (som afbildet ved hver kurves...

Watch this Scientific Journal Video about Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response at JoVE.com

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Vi præsenterer her, en protokol, ved hjælp af RNA-seq for at overvåge mRNA niveauer over tid under det hypoksiske svar af S. cerevisiae celler. Denne metode kan tilpasses til at analysere genekspression under nogen cellulære reaktion.

Måling af mRNA niveauer Over tid under gær S. cerevisiae hypoksiske svar

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

Research

JoVE Journal

Genetics

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genom-bred kortlægning af protein-DNA-interaktioner med ChEC-seq in<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

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The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Kvantificering af oplysninger kodet af genet udtryk niveauer under levetid graduering Under bred vifte kosten begrænsning i C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

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The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

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Real-time analyse af transskription faktor bindende, transskription, Translation og omsætning skal vises Global begivenheder under aktivering af cellulære

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

Måling af motion niveauer i Drosophila melanogaster ved hjælp af den roterende motion kvantificering systemet (HENV)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Mating-based Overexpression Library Screening in Yeast

Parring-baserede overekspression bibliotek Screening i gær

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Saccharomyces cerevisiae Metaboliske mærkning med 4-thiouracil og kvantificering af nyligt syntetiserede mRNA som en Proxy for RNA Polymerase II aktivitet

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

Ved hjælp af in Vitro og i celler form at undersøge lille molekyle induceret Pre-mRNA strukturelle ændringer

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Kendetegner Histon posttranslationel modifikation ændringer i gær Neurodegenerative Proteinopathy modeller

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...