Yazarlar Matthias Peter ve bu yöntemler geliştirilmiştir Zürich ETH Biyokimya Enstitüsü&#39;nde yaptığı grup ederim. Bu çalışma, İsviçre Ulusal Bilim Vakfı tarafından desteklenmiştir.

1.. Mikroskopi Ölçümler <ol><li> Maya sentetik ortam 5 ml inoküle. </li><li> Gece boyunca 30 ° C&#39;de hücreler büyür. </li><li> Ertesi sabah, gece boyunca kültürün OD 600 ölçün. </li><li> 600 0.05 OD 5 ml sentetik kültür ortamı içinde bir gece boyunca seyreltin. </li><li> En az 4 saat boyunca 30 ° C&#39;de seyreltilmiş kültür büyütün. </li><li> Hazırlama 3 kat sentetik aracı madde içinde (0.6 M) NaCl solüsyonu konsantre edildi. </li><li> PBS, Concanavalin A (ConA) bir 1 mg / ml çözelti hazırlayın. </li><li> Süzüntü ~ iyi slayt içine ConA&#39;nın çözeltisi 200 ul. </li><li> 30 dakika bekletin. </li><li> ConA&#39;nın çözüm çıkarın. </li><li> De 150 ul H 2 O ekleyin. </li><li> H 2 O. kaldır </li><li> Hücre kültürü OD 600 ölçün. </li><li> 1.5 ml = 0.02 mikrotüp içinde OD 600 hücre kültürünün 300 ul hazırlayın. </li><li> 45 saniye için bir su banyosu içinde hücreleri sonikasyon. </li><li> Vortex kısaca microtube. </li><li> 45 saniye için bir su banyosu içinde hücreleri sonikasyon. </li><li> Kısaca Vortex Mikrotüp. </li><li> Oyuğuna hücre kültürünün 200 ul ekleyin. </li><li> Hücreler kuyunun dibine çöker izin 30 dakika bekleyin. </li><li> Mikroskop iyi slayt yerleştirin. </li><li> Kaydedilecek floresan kanal aydınlatma ayarlarını seçin. </li><li> (: Hücrelerinin uygun bir segmentasyon sağlamak için odak düzleminin altında 3 um biraz odak dışında biri) iki parlak bir alan görüntüler için aydınlatma ayarlarını seçin. </li><li> Time-lapse ölçümü için zaman aralıklarını seçin </li><li> Görüntü görüş alanları seçin. </li><li> Bir kaç kare alımı başlatabilirsiniz. </li><li> 0.6 M NaCl ortamında 100 ul eklemek için satın Pause. </li><li> Görüntüleme Devam. </li></ol> 2. Sitometrisi Ölçümler <ol><li> Maya sentetik ortam 5 ml inoküle. </li><li> Gece boyunca 30 ° C&#39;de büyür. </li><li> TedbirOD, gece boyunca kültürün 600 ertesi sabah. </li><li> 600 0.1 OD 5 ml sentetik bir ortam içinde bir gece boyunca kültür seyreltin. </li><li> OD 0,2-0,4 600 ulaşmak için en az 4 saat boyunca 30 ° C&#39;de büyür. </li><li> H2O içinde sikloheksimid çözelti 1 mg / ml hazırlanması </li><li> Hazırlama 3 kat sentetik aracı madde içinde (0.6 M) NaCl solüsyonu konsantre edildi. </li><li> Ölçülecek her zaman noktası için 0.6 M NaCl ortamın 100 ul bir 1.5 ml mikrotüp hazırlayın. </li><li> 0 zamanında, her bir mikrotüp hücre kültürü 200 ul ekleyin. </li><li> 30 ° C&#39;de çalkalayarak kuluçkalayın </li><li> T süresi de, bir mikrotüpe 30 ul sikloheksimid ekleyin. </li><li> Her istenilen zaman noktaları için adımı 2.11 tekrarlayın. </li><li> En az 45 dakika boyunca tüm mikrotüpler inkübe edin ve çalkalanarak 30 ° C&#39;de 3 saat kadar. </li><li> 400 ul PBS ile FACS tüpleri hazırlayın. </li><li> 1 dakika süre ile bir su banyosunda sonikasyon hücreleri. </li><li> Kısaca Vortex mikrotüpler. </li><li> Oğlum1 dakika boyunca bir su banyosu içinde hücreleri TARĐHĐ BELGE. </li><li> Kısaca Vortex mikrotüpler. </li><li> Her mikrotüpünden olarak karşılık gelen FACS tüpüne 100 ul hücre kültürü ekleyin. </li><li> Tespiti için 488 nm uyarma lazer ve 530/30 nm band geçiren filtre seçin. </li><li> Testi olmayan ifade ve tam onlar dedektör hassasiyet aralığında düşüş olmadığını doğrulamak için örnek ifade. </li><li> Alınan her bir örnek için 10,000 hücre ölçün. </li></ol>

<ol>
	<li>Gasch, A. P., Spellman, P. 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=Genomic+expression+programs+in+the+response+of+yeast+cells+to+environmental+changes.">Genomic expression programs in the response of yeast cells to environmental changes.</a> Molecular biology of the cell. 11 (12), 4241-4257 (2000).</li><li>de Nadal, E., Zapater, M., Alepuz, P. M., Sumoy, L., Mas, G., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAPK+Hog1+recruits+Rpd3+histone+deacetylase+to+activate+osmoresponsive+genes.">The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes.</a> Nature. 427 (6972), 370-374 (2004).</li><li>Ghaemmaghami, S., Huh, W. -. K., 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+analysis+of+protein+expression+in+yeast.">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li><li>Wu, J. -. Q., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+cytokinesis+proteins+globally+and+locally+in+fission+yeast.">Counting cytokinesis proteins globally and locally in fission yeast.</a> Science. 310 (5746), 310-314 (2005).</li><li>Colman-Lerner, A., Gordon, A., 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=Regulated+cell-to-cell+variation+in+a+cell-fate+decision+system.">Regulated cell-to-cell variation in a cell-fate decision system.</a> Nature. 437 (7059), 699-706 (2005).</li><li>Acar, M., Becskei, A., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+cellular+memory+by+reducing+stochastic+transitions.">Enhancement of cellular memory by reducing stochastic transitions.</a> Nature. 435 (7039), 228-232 (2005).</li><li>Pelet, S., Rudolf, F., Nadal-Ribelles, M., de Nadal, E., Posas, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+activation+of+the+HOG+MAPK+pathway+regulates+bimodal+gene+expression.">Transient activation of the HOG MAPK pathway regulates bimodal gene expression.</a> Science. 332 (6030), 732-735 (2011).</li><li>de Nadal, E., Casadome, L., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+MEF2-like+transcription+factor+Smp1+by+the+stress-activated+Hog1+mitogen-activated+protein+kinase.">Targeting the MEF2-like transcription factor Smp1 by the stress-activated Hog1 mitogen-activated protein kinase.</a> Molecular and Cellular Biology. 23 (1), 229-237 (2003).</li><li>Elowitz, M. B., Levine, A. J., Siggia, E. D., Swain, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li><li>Pelet, S., Dechant, R., Lee, S. S., van Drogen, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+image+analysis+platform+to+quantify+signal+transduction+in+single+cells.">An integrated image analysis platform to quantify signal transduction in single cells.</a> Integrative biology : quantitative biosciences from nano to macro. 4 (10), 1274-1282 (2012).</li><li>Carpenter, A. E., Jones, T. 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=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome biology. 7 (10), R100 (2006).</li><li>Chernomoretz, A., Bush, A., Yu, R., Gordon, A., Colman-Lerner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Cell-ID+1.4+with+R+for+microscope-based+cytometry.">Using Cell-ID 1.4 with R for microscope-based cytometry.</a> Current protocols in molecular biology. Chapter 14, Unit 14.18 (2008).</li><li>Dimopoulos, S., Mayer, C., Rudolf, F., Stelling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Single+Cell+Segmentation+in+a+Variety+of+Microscopy+Images.">Automatic Single Cell Segmentation in a Variety of Microscopy Images.</a> Current protocols in molecular biology. , (2012).</li><li>Miyawaki, A., Nagai, T., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+protein+fluorophore+formation+and+engineering.">Mechanisms of protein fluorophore formation and engineering.</a> Current Opinion in Chemical Biology. 7 (5), 557-562 (2003).</li><li>Hersen, P., McClean, M. 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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Mikroskopla inceleme ConA ile kuyunun tedavisi, hücrelerin uygun bir görüntüleme sağlamak için önemli bir adımdır. ConA PBS (5 mg / mi) içinde düşük bir çözünürlüğe sahip olduğundan, filtreleme işlemi sağlar: filtre çözelti içinde mevcut olan ve görüntüleme müdahale büyük agregaların çıkarılması. Hücreler, işlemden geçirilen yüzeye bağlı olarak güçlü bir şekilde tutunur ve uyaran çözeltisinin eklenmesinin uyaran önce ve sonra, bir sürekli izleme...

Sinyal transdüksiyon cascades ile genellikle proteinlerin ekspresyonu son bulur. Bu ifade profilinin karakterizasyonu biyolojik yollarının işlevini anlamada önemli bir unsurdur. Up-regüle proteinler ve bunların aktivasyon dinamikleri spektrumunun tanımlanması, bu tür mikro-dizileri, kuzey ya da batı lekeleri blotlar 1-3 gibi çeşitli teknikler ile elde edilebilir. Bununla birlikte, bu teknikler arasında, hücreler bütün bir nüfusun ortalama tepki. Protein ekspresyonunun ince düzenleme anlamak için, tek hücre düzeyinde ölçümleri toplamak için tercih edilir. İdeal olarak, bu ölçümler de altta yatan yolun matematiksel modeller geliştirmek için uygun nicel veri sağlamalıdır. Mikroskopi ve akış sitometri ideal gibi nicel tek hücre ölçümlerini sunmak için uygundur iki teknik vardır. Bir floresan protein ile proteinlerin endojen etiketleme be ekspresyon seviyesi 4 ölçmek için kullanılır. Proteinin terminalinde büyük bir floresan yarımının eklenmesi işlevsel olmayan işlemek için Bununla birlikte, bu bir floresan yapısının ekspresyonunu tahrik eden bir promotere göre belirli ifade muhabir oluşturmak için genellikle daha fazla tercih edilir. Bu, raportör protein hücresel sistem için dışsal ve bu nedenle hücre içinde yer alıyor sinyal olaylarını etkilememektedir. Mikroskopi ve akış sitometrisi her ikisi yaygın olarak maya sinyal alanında kullanılmıştır. Örnek olarak, Colman-Lerner ve arkadaşları korelasyon, tek hücreler içinde, maya eşleşme sinyal iletim 5 gürültüyü ölçmek için mikroskopi ile spesifik ve yapısal olarak ifade edilen muhabir çiftleşme ifade Acar ve düzenleyici ağ incelemek için akış sitometrisi el arkadaşları GAL genlerin 6 ekspresyonunu kontrol etmektedir. Önceki bir çalışmada 7&#39;de, bir combinati kullandıkozmotik basıncı yüksek gliserol maya tomurcuklanma olarak (HOG) yolunun gelen ifade çıkışını incelemek için bu iki teknikleri üzerine. Bu, mitojen tarafından aktive edilen protein kinaz (MAPK) yolu hiper-osmotik stres tarafından tetiklenir. Bu, yaklaşık 300 genlerin ekspresyonu ile sonuçlanan bir transkripsiyon programı indükleme hücrenin çekirdeğine doğru Hog1 MAPK aktivasyonu ile sonuçlanır. Bu işlemi incelemek için, bir dört Venus floresan proteini (p-STL1 QV) ekspresyonunu tahrik STL1 promoteri (Hog1 aktivitesi 8&#39;e yanıt olarak, özellikle kaynaklı bir gen) dayanan bir sentezleme raportör mühendisliği vardı. Ölçümler, flüoresan raportör ifade nüfusun sadece bir kısmı ile orta gerilim seviyesinde (0.1 M NaCl) ile iki hücre popülasyonlarının varlığını ortaya sitometrisi akış. Biz daha bu davranışı araştırmak için mikroskobu kullandılar ve protein ifadesindeki bu gürültü içsel faktörlere 9 tabi olduğunu keşfetti. Biz could ayrıca Hog1 aktivite benzer bir düzeyde olan hücreler çarpıcı farklı ifade sonuçlarını görüntülemek verebilecek görüyoruz. Bu iki tekniğin kombinasyonu bize stres yanıtı genlerinin yavaş yeniden tek hücre düzeyinde 7 de ifade sonucunu nasıl etkilediğini göstermek için izin verdi. Bu yazıda, mikroskobu ile protein ifade miktarının bir örnek olarak hiper-ozmotik şok tarafından uyarılan p STL1-QV muhabiri ifadesini kullanın ve akım sitometri. 0.2 M NaCl strese maruz aynı gerilme hem tekniklerle incelenmiştir. Bu bize bu iki derece tamamlayıcı teknikler arasındaki bazı temel farklılıkları vurgulamak için izin verecektir.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Yeast Nitrogen Base</td><td>ForMedium</td><td>CYN6202</td><td></td></tr><tr><td>CSM amino-acid mix</td><td>ForMedium</td><td>DCS0011</td><td></td></tr><tr><td>Concanavalin A</td><td>GE Healthcare</td><td>17-0450-01</td><td></td></tr><tr><td>Cycloheximide</td><td>Fluka Aldrich Sigma</td><td>C7698</td><td>Toxic</td></tr><tr><td>ySP9</td><td></td><td></td><td>W303Mata leu2::LEU2-pSTL1-quadrupleV enus</td></tr><tr><td>pSP34</td><td></td><td></td><td>pRS305 pSTL1 (-800 -0) - quadruple V enus</td></tr><tr><td colspan="4">&lt;strong&gt;Material&lt;/strong&gt;</td></tr><tr><td>Microscope</td><td>Nikon</td><td>Ti-Eclipse</td><td></td></tr><tr><td>Microscope control software</td><td>Micro-manager</td><td>Ver 1.4.11</td><td></td></tr><tr><td>Incubation chamber</td><td>LIS</td><td>The Box</td><td></td></tr><tr><td>Fluorescence light source</td><td>Lumencor</td><td>SpectraX</td><td></td></tr><tr><td>Camera</td><td>Hamamatsu</td><td>Flash 4.0</td><td></td></tr><tr><td>Flow cytometer</td><td>BD</td><td>FACS calibur</td><td></td></tr><tr><td>Sonicator bath</td><td>Telesonic</td><td>TUC-150</td><td></td></tr><tr><td>8-well-slide </td><td>Thermo Lab-tek</td><td>155409</td><td></td></tr><tr><td>96-well-plate</td><td>Matrical bioscience</td><td>MGB096-1-2-LG</td><td></td></tr><tr><td>FACS tube</td><td>BD Falcon</td><td>352054</td><td></td></tr></tbody></table>

temporal quantification of mapk induced expression in single yeast cells

Tek hücre düzeyinde gen ifadesinin miktar nüfus düzeyinde gerçekleştirilen ölçümler gölgede yeni düzenleme mekanizmalarını ortaya çıkarır. İki yöntem mikroskopi dayalı ve akım sitometri gibi veriler elde edilebilir nasıl göstermek için sunulmaktadır. Mayada, ozmotik basıncı yüksek gliserol MAPK yolağının aktivasyonu ile uyarılan bir flüoresan raportör ekspresyonu, bir örnek olarak kullanılmıştır. Her bir yöntemin özel avantajları vurgulanır. Sitometrisi hücrelerin geniş bir sayısı (10,000) büyüklüğüne ve floresan proteinin yavaş olgunlaştırma kinetik bağımsız protein ifade dinamikleri doğrudan bir ölçümünü sağlar akış. Mikroskobu ile canlı hücrelerin Imaging az hücrelerinde raportör olgunlaşmış formunun ölçümü ile sınırlı aksine gereğidir. Ancak, bu teknikle üretilen veri setleri birden gazetecilere kombinasyonları ve mekansal ve zamansal bilgiler son derece zengin sayesinde olabilirtek tek hücrelerden elde. Bu iki ölçüm yöntemlerinin kombinasyonu sinyal yolları ile protein ifadesinin düzenlenmesinde yeni anlayışlar sunabilirsiniz.

Mikroskopla inceleme Sentezleme raportör p STL1-QV (ySP9 7) taşıyan maya hücreleri iyi slaydın tabanına bağlanmış ve mikroskop altında yerleştirildi. Hücreler görüntüleme oturumu sırasında doğrudan kuyuya stres ortamının eklenmesi ile uyarıldı. Bu bize yol indüksiyon öncesi hücrelerin birkaç görüntü elde ve stimülasyon sonrasında kendi kaderini takip sağlar. Bu durumda, hücreler, 10 dakika bir zaman aralığı ile ~ 2 saat boyunca takip edild...

Watch this Scientific Journal Video about Temporal Quantification of MAPK Induced Expression in Single Yeast Cells at JoVE.com

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Akış sitometrisi ve mikroskopiye dayanan iki yardımcı yöntem mayada bir MAPK yolağının aktivasyonu ile uyarılan gen ekspresyonunun dinamiklerinin tek hücre düzeyinde miktarının, ki olanak sunulmaktadır.

Tek Maya Hücreleri MAPK İsteyerek İfade zamansal Kantitasyonu

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

temporal-quantification-mapk-induced-expression-single-yeast

Research

JoVE Journal

Biology

8.5K Görüntüleme.  University of Lausanne.  Akış sitometrisi ve mikroskopiye dayanan iki yardımcı yöntem mayada bir MAPK yolağının aktivasyonu ile uyarılan gen ekspresyonunun dinamiklerinin tek hücre düzeyinde miktarının, ki olanak sunulmaktadır. 

Video: Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

S. cerevisiae yapışma ve Agar Invasion için Testi

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Biyoloji

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

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

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

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Maya S. cerevisiae hipoksik yanıt sırasında zamanla mRNA düzeyleri ölçme

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

Genetik

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

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

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

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabolik 4-thiouracil ve bir miktar ile etiketleme yeni mRNA RNA polimeraz II etkinlik için bir Proxy olarak sentezlenmiş

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

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

Yaşlı Hücre akıbet tarafından Maya Kronolojik Ömrü miktarının

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Fırıncı Maya saccharomyces cerevisiae Mitokondriyal Genom İfade Ürünlerinin Etiketlenmesi ve Görselleştirilmesi

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Biyokimya

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Fisyon Maya Kantitatif Canlı Hücre Floresan mikroskopi Analizi

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Tomurcuklanma Maya Floresan Time-lapse Filmler Kazanılması ve GREFTLERİ kullanarak Tek hücreli Dynamics analiz

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Tütün Geçici İfade Sisteminin Kullanılması bir MAPK sinyal yolu Mekansal Reorganizasyon Görüntüleme

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Fisyon Maya Cytokinetic Olayların zamanmekansal Analizi

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...