Name: microarray analysis for saccharomyces cerevisiae
Uploaded: 2011-04-07T13:00:00
Duration: 13 min 17 s
Description: Watch this Scientific Journal Video about Microarray Analysis for Saccharomyces cerevisiae at JoVE.com

University of Vermont, Vermont Genetics Network. This publication was made possible by the Vermont Genetics Network, through Grant Number P20 RR16462 from the BRIN Program and Grant Number P20 RR16462 from the INBRE Program of theNational Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.
We would like to thank all our participating outreach institutions for their collaboration and input in refining this module Castleton State College, Green Mountain College, Johnson State College, Lyndon State College, Marlboro College, Middlebury College, Norwich University and Saint Michael's College.

<ol>
	<li>Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc Natl Acad Sci U S A. 87, 1663-1667 (1990).</li><li>Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., Botstein, D., Brown, P. O. <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> Mol Biol Cell. 11, 4241-4257 (2000).</li><li>D'Autr&#233;aux, B., Toledano, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+as+signalling+molecules:+mechanisms+that+generate+specificity+in+ROS+homeostasis.">ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis.</a> Nat Rev Mol Cell Biol. 8, 813-824 (2007).</li><li>Shenton, D., Smirnova, J. B., Selley, J. N., Carroll, K., Hubbard, S. J., Pavitt, G. D., Ashe, M. P., Grant, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+translational+responses+to+oxidative+stress+impact+upon+multiple+levels+of+protein+synthesis.">Global translational responses to oxidative stress impact upon multiple levels of protein synthesis.</a> J Biol Chem. 281, 29011-29021 (2006).</li><li>Godon, C., Lagniel, G., Lee, J., Buhler, J. M., Kieffer, S., Perrot, M., Boucherie, H., Toledano, M. B., Labarre, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+H2O2+stimulon+in+Saccharomyces+cerevisiae.">The H2O2 stimulon in Saccharomyces cerevisiae.</a> J Biol Chem. 273, 22480-229 (1998).</li><li>Vermont Genetics Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microarray Outreach. , (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Affymetrix GeneChip Expression Analysis: Technical Manual. , (2004).</li><li>Huang, D. W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+Bioinformatics+Resources.">Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources.</a> Nature Protoc. 4, 44-57 (2009).</li><li>Dennis, G., Sherman, B. T., Hosack, D. A., Yang, J., Gao, W., Lane, H. C., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAVID:+Database+for+Annotation,+Visualization,+and+Integrated+Discovery.">DAVID: Database for Annotation, Visualization, and Integrated Discovery.</a> Genome Biol. 4, P3-P3 (2003).</li></ol>

The production of this video-article was sponsored by EMD-Millipore.

Applications and significance.
The Vermont Genetics Network outreach program, at the University of Vermont, conducts undergraduate outreach to eight partner baccalaureate colleges throughout the state. The goal of the VGN Outreach Core is to expose undergraduates in the state of Vermont to state-of-the-art scientific technology &amp; resources using hands-on experiences. The microarray module described was developed in 2003 and subsequently upgraded. It has been offered as a mini-course or integrated into existing laboratory curriculum at the participating institutions.
This project, between research university cores and undergraduate colleges, promotes collaborations in education and research. Microarray outreach throughout the state has enhanced curriculum and created research and networking opportunities for core facilities, faculty and students.
As undergraduate faculty adopt the program they are encouraged to design unique experiments provided there are appropriate Affymetrix GeneChips and annotation available. All information for this module including the laboratory manual, references and PowerPoint presentations are available online (Vermont Genetics Network, 2008).
Critical steps:
Spheroplasting: It is important to observe successful spheroplasting under the microscope. The use of SDS is highly beneficial as it causes the yeast swelling resulting in spheroids. It is important to observe that the budding cells form spheroids as well. If partial spheroplasting is observed, an extended treatment with lyticase is recommended.
Pellet cleaning: During the precipitation reaction and subsequent ethanol washing step, it is very easy to lose the cDNA pellet. Extreme care and meticulous attention to the pellet is imperative. 
Applying the water to the center of the membrane: During the RNA elution step from the membrane, it is important to "squirt" the 30 &mu;l of water directly to the center of the silica membrane. If this is not accomplished, the column can be centrifuged and the resulting elutant can be re-applied to the silica membrane again. This can be repeated as many times as necessary.
Modifications and Product Substitutions:
<ol>
 <li>Alternative RNA clean-up columns can be substituted. Examples are the USB prep-ease and the Invitrogen Pure Link RNA kit, and the Omega RNA clean-up kit to name a few.</li>
 <li>Schizosaccharomyces pombe is an optional yeast. The Affymetrix GeneChip contains both genomes on the same chip</li>
 <li>Various treatments and times may be used. This may include drug treatments, temperature treatments, anti-oxidants, etc. Treatment times may also be adjusted.</li>
 <li>DNase treatment may not be required because the methods described employ the use of an Oligo (dT) primer.</li>
 <li>It is not required to perform a double elution of the RNA off the column with the same 30 &mu;l aliquot. Once is almost always adequate. This is suggested to insure that a complete recovery is obtained. Additionally, the water used to elute the RNA from the column can be pre-heated to 65&deg;C to further enhance the ability to recover RNA from the silica column.</li>
 <li>Many methods are available to quantify RNA. The Eppendorf and Nanodrop have only been selected for their ease of use and tested accuracy.</li>
 <li>Agarose gel electrophoresis many be performed using standard laboratory equipment. The E-Gel is not required but is desirable because it is RNase free and does not require gel solidification waiting time.</li>
 <li>Ordering information for products has been supplied. However, many general reagents can be ordered from multiple vendors.</li>
 <li>One Cycle cDNA reagents can be purchased separately if that is more cost effective.</li>
 <li>There are other target prep methods but we feel this one offers more teaching opportunities for the student.</li>
</ol>

<table border="1">
	<tbody>
		<tr>
			<td>General Laboratory Equipment</td><td>Company</td><td>Product number</td></tr>
		<tr>
			<td>Spetrophotometer</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Thermocyler</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Microcentrifuge</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Vortex</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Stir Plates (2)</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>P-10s pipettors</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>P-20's pipettors:</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>P-200's pipettors:</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>P-1000's pipettors:</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Camera and transilluminator</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td colspan="3">&nbsp;</td></tr>
		<tr>
			<td>Supplies - recommended</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>E-Gel Apparatus</td><td>Invitrogen</td><td>G6000-08</td></tr>
		<tr>
			<td>E-Gels</td><td>Invitrogen</td><td>G6018-02</td></tr>
		<tr>
			<td>Axygen 1.7 ml. Clear</td><td>Krackler</td><td>383-MCT175C</td></tr>
		<tr>
			<td>Axygen 0.5 ml clear tubes</td><td>Krackler</td><td>383-MCT060C</td></tr>
		<tr>
			<td>Axygen 2ml capture tubes</td><td>Krackler</td><td>383-MCT200NC</td></tr>
		<tr>
			<td>boxes of P-10 ART tips:</td><td>CLP</td><td>BT10XL</td></tr>
		<tr>
			<td>Boxes of P-100 ART tips:</td><td>CLP</td><td>BT200</td></tr>
		<tr>
			<td>Boxes of P-20 ART Tips:</td><td>CLP</td><td>BT20</td></tr>
		<tr>
			<td>Boxes of P-1000 ART Tips:</td><td>CLP</td><td>BT1000</td></tr>
		<tr>
			<td>Yeast Chips</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td colspan="3">&nbsp;</td></tr>
		<tr>
			<td>General Laboratory Supplies</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>25 ml sterile disposable pipets-</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>5ml sterile disposable pipets-</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Microfuge tube racks</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>KimWipes:</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Lab Coats</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Stir Bars</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Culture flasks</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Innoculating loops</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Sharpies</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Gloves</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Autoclave tape</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Stirrer bars</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td colspan="3">&nbsp;</td></tr>
		<tr>
			<td>Reagents</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>30% Hydrogen Peroxide</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-386790?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>386790</td></tr>
		<tr>
			<td>HBSS without Ca, Mg, or dye</td><td>Sigma-Aldrich</td><td>H6648-100ml</td></tr>
		<tr>
			<td>Qiagen Rneasy Mini Kit:</td><td>Qiagen</td><td>74104</td></tr>
		<tr>
			<td>Qiagen DNase Kit:</td><td>Qiagen</td><td>79254</td></tr>
		<tr>
			<td>Rnase Remover</td><td>Denville Scientific</td><td>D1180</td></tr>
		<tr>
			<td>Harleco Alcohol 100%</td><td><a href="http://www.emdchemicals.com/harleco-alcohol/EMD_CHEM-65347/p_uuid?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>65347</td></tr>
		<tr>
			<td>ENZO IVT Kit</td><td>ENZO</td><td>ENZ-42655-10</td></tr>
		<tr>
			<td>Lyticase: one bottle</td><td>VWR</td><td>IC19012310</td></tr>
		<tr>
			<td>Water, Cell Culture grade</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-4.86505?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>4.86505</td></tr>
		<tr>
			<td>Yeast culture:</td><td>ATCC</td><td>18824</td></tr>
		<tr>
			<td>YPD broth powder</td><td><a href="http://www.emdchemicals.com/products/EMD_CHEM-4.85040?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>4.8504</td></tr>
		<tr>
			<td>D(+) Glucose, Anhydrous</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-346351?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>346351</td></tr>
		<tr>
			<td>Sorbitol</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-56755?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>56755</td></tr>
		<tr>
			<td>EDTA</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-4005?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>4005</td></tr>
		<tr>
			<td>SDS solution, 20%</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-7990-OP?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>7990-OP</td></tr>
		<tr>
			<td>Pellet Paint</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-69049?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>69049</td></tr>
		<tr>
			<td>PCI RNase DNase free (7 aliquots):</td><td>Fisher</td><td>AC32711-500</td></tr>
		<tr>
			<td>7.5M NH4OAc</td><td>Fisher</td><td>ICN1987 5980</td></tr>
		<tr>
			<td>Fragmentation Buffer (200 mM Tris-Acetate, pH 8.1, 500 mM KOAc, 150 mM MgOA)</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Trizma Base</td><td>Fiaher</td><td>50-899-90034</td></tr>
		<tr>
			<td>KOAc</td><td>Fisher</td><td>NC9757725</td></tr>
		<tr>
			<td>MgOA</td><td>Fisher</td><td>NC9681042</td></tr>
		<tr>
			<td>Loading Dye</td><td>Invitrogen</td><td>10482055</td></tr>
		<tr>
			<td>PCR Markers</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-69278-3?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>69278-3</td></tr>
		<tr>
			<td>Phase Lock Gels</td><td>Fisher</td><td>FP2302820</td></tr>
		<tr>
			<td>One-Cycle cDNA Kit</td><td>Invitrogen</td><td>A10752-030</td></tr>
		<tr>
			<td>Includes;</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>E. coli DNA Pol</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>dNTP's</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>T4 DNA Pol.</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>T-7 oligod(T)</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>0.5ml EDTA pH 8.0</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>First Strand Buffer</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>E. Coli RNaseH</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Second Strand Buffer</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>E. Coli DNA ligase</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>SuperScript II</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>DTT</td><td>&nbsp;</td><td>&nbsp;</td></tr>
	</tbody>
</table>

microarray analysis for saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Watch this Scientific Journal Video about Microarray Analysis for Saccharomyces cerevisiae at JoVE.com

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

microarray-analysis-for-saccharomyces-cerevisiae

Research

JoVE Journal

Biology

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

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.

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

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).