Research in the author&#8217;s laboratory is supported by the Texas Higher Education Coordinating Board&#8217;s Norman Hackerman Advanced Research Program and start-up funds from Baylor University.

1. Growth of Yeast Cells
<ol>
	<li>Select the appropriate yeast strains to be utilized for the mRNA decay rate measurements. To inhibit transcription using the temperature sensitive allele of RNA polymerase II, use yeast strains harboring the rpb1-1 mutation 1. Obtain this yeast strain from a laboratory that already has one, or generate it in the laboratory using standard techniques if a specific genetic background is required9.</li>
	<li>Using sterile technique, prepare yeast media using standard procedures 10. If no selection is required, prepare rich media such as YPD. Alternatively, prepare selective media if the yeast cells are transformed with plasmids and selection is required. Autoclave the media.</li>
	<li>Grow the yeast cells at 28 &#176;C, which is the permissive temperature for this yeast strain. Do this in two steps:
	<ol>
		<li>For the first O/N, grow the yeast cells in 5 ml of growth media to saturation.</li>
		<li>Set up the second O/N at the end of the second day. For the second O/N, inoculate different amounts of yeast cells from the first O/N into 100 to 150 ml of growth media (ranging from 100 &#181;l to 1 ml, 2 ml or more, depending on when the yeast cells need to be harvested the following day). Do this step to ensure that one of the cultures is at the correct OD600 the following day.</li>
	</ol>
	</li>
</ol>
2. Harvest the Yeast Cells
<ol>
	<li>Prepare to harvest the yeast cells . The timing for this step is critical; label all the required tubes and gather all required equipment and materials in one place. Preheat a water bath to 39 &#176;C and preheat two 15 ml test tubes of the growth media at 28 &#176;C and at 60 &#176;C. 
	NOTE: The 15 ml growth media volume can be varied depending on the number of time points the cells are to be harvested.</li>
	<li>Harvest the yeast cells when they reach an OD600 of 0.4 to 0.7. Transfer the cells to 4 - 6 sterile 50 ml screw cap bottles. Centrifuge at 7,500 x g in a high speed centrifuge for 5 min at RT.</li>
	<li>Discard the supernatant and resuspend the pellets in the 15 ml growth media that was equilibrating at 28 &#176;C. Pool the yeast cells in one sterile 250 ml flask and equilibrate them for ~5 min at the 28 &#176;C growth temperature.</li>
	<li>After the yeast cells have equilibrated at the growth temperature, immediately add the 15 ml of the growth media equilibrated at 60 &#176;C to raise the temperature to 39 &#176;C (the nonpermissive temperature) and immediately place the flask in the 39 &#176;C water bath. Make sure that the culture medium remains in the 39 &#176;C water bath throughout the remaining cell harvesting steps to ensure that transcription is turned off.</li>
	<li>Harvest the cells at different times after placing the flask in the 39 &#176;C water bath. For the first time point, harvest the cells immediately after placing the flask at 39 &#176;C. Harvest a 3 ml aliquot and distribute the 3 ml into two, 1.5 ml microcentrifuge tubes. Pellet the cells for 10 sec in a mini centrifuge or picofuge with rapid deceleration and pour out the supernatant.</li>
	<li>Immediately freeze the pellet in a dry ice/ethanol bath or in liquid nitrogen. Designate the first time point the cells are harvested at as the zero time point.</li>
	<li>Experimentally determine the time points the cells are harvested at thereafter by a pilot experiment, depending on the mRNAs of interest. Normally, harvest yeast cells at the following time points: 0, 3, 6, 9, 12, 18, 25 and 35 min (Figure 2B). If the mRNA half-life cannot be determined using the above mentioned time points, adjust these time points. For example, for mRNAs with anticipated short half-lives, use shorter times points, (i.e., 0, 1, 2, 3, 4, 6, 9, 12 and 18 min) and for mRNAs with anticipated long half-lives, extend the time points.</li>
	<li>After the cells are harvested, store them in a -80 &#176;C freezer until RNA extraction is carried out.</li>
</ol>
3. Extract RNA from the Yeast Cells
<ol>
	<li>For the RNA extraction portion of the protocol, use RNase free techniques to prevent degradation of the RNA by RNases. Prepare RNase free solutions, glassware and plastic ware to be used in the extraction of the RNA.</li>
	<li>Extract the RNA from the yeast cells according to standard protocols. Typically, use the hot phenol method 12. Alternatively, use kits that can be used to extract RNA from yeast cells.</li>
	<li>Determine the quantity and purity of the RNA, normally by measuring the absorbance at A260 and A280 of 2 &#181;l of RNA using a Nanodrop. Determine the concentration of the RNA from the A260. Based on the concentration, dilute the RNA to 1 &#181;g/&#181;l using DEPC treated water. 
	NOTE: The ratio of the A260/A280 provides information on the purity of the RNA. An RNA sample is pure if the A260/A280 ratio is 2.0 &#177; 0.1.</li>
</ol>
4. Northern Blot Analysis
NOTE: Use northern blots to quantify mRNA levels and obtain information on the size of the transcripts. In addition, use northern blots to detect mRNAs that produce multiple isoforms of the same mRNA.
<ol>
	<li>Prepare a 1.0% agarose-formaldehyde gel. Run equal amounts of the sample RNA on the agarose formaldehyde gel by electrophoresis according to standard protocols 12. Run a RNA ladder alongside the RNA samples and use it to determine the sizes of the RNA that are detected on the northern blot. Before transferring the RNA separated on the agarose formaldehyde gel to a membrane, cut the lane with the RNA ladder from the gel and visualize using ethidium bromide. 
	NOTE: Alternatively, the whole gel can be stained with ethidium bromide before transfer of the RNA.</li>
	<li>After the RNA samples have migrated to an appropriate distance on the gel, transfer the RNA to a membrane using RNase free techniques. Use one of several protocols available to transfer RNA to membranes 12-13. To transfer the RNA to a membrane, cut the membrane to approximately the same size as the gel. Transfer the RNA according to standard protocols 12.</li>
	<li>UV cross-link the RNA to the membrane using a UV crosslinker following the manufacturer&#8217;s instructions. Alternatively, bake the membrane in an oven set to 80 &#176;C for 1 hr. Store the membrane at -20 &#176;C in a plastic bag indefinitely until it is hybridized to specific probes. 
	NOTE: The dry membrane can also be stored at RT between filter papers.</li>
</ol>
5. Hybridize Probes Complementary to the RNA of Interest to the Membrane
NOTE: One way to detect mRNA on the membrane is to hybridize 32P labeled DNA probes. CAUTION: Researchers working with 32P need to use protective procedures to prevent contamination. Follow institutional guidelines on use of radioactive material.
<ol>
	<li>Prepare the DNA probe by labeling 25 - 50 ng of the DNA to be hybridized to the membrane according to standard protocols 12. Generate the DNA fragment by PCR or plasmid digestion. Determine the specific activity of the DNA probe by counting 1 &#181;l of the radiolabelled DNA probe after the unincorporated nucleotides are removed 12.</li>
	<li>Preheat prehybridization/hybridization buffer at 42 &#176;C. Use ~10 ml of the prehybridization/hybridization buffer per 100 cm2 of the membrane 12.</li>
	<li>Hybridize 1 - 5 x 106 cpm of radiolabeled DNA probe per ml of hybridization buffer to the membrane O/N in a hybridization oven to detect the mRNA of interest 12. Make sure that during the hybridization the hybridization oven rotation is in a direction that causes the entire membrane to be exposed to the hybridization solution, to ensure proper hybridization.</li>
	<li>Wash the membrane two times at RT for 15 min with 50 ml of 2x SSPE and one time at 65 &#176;C with 50 ml of 2x SSPE/2% SDS for 15 min 12. Wrap the membrane in plastic wrap to ensure that it does not dry out or contaminate the Phosphor screen.</li>
	<li>Using a Geiger counter, estimate the intensity of the radioactivity on the membrane. 
	NOTE: This estimate ensures that the membrane is exposed to the Phosphor screen for the appropriate amount of time. Use longer exposure times for membranes with low amounts of radioactivity.</li>
	<li>For northern blots, use a loading control to confirm that equal amounts of RNA are loaded on each spot. To do this, strip the membrane and reprobe it with an RNA that is not affected by the process being examined. Strip the membrane by placing it in a glass tray containing 200 ml of boiling stripping solution (0.1% SDS/0.01 X SSC) for 2 min. Pour out the stripping solution and repeat the procedure five times 12. 
	NOTE: An example of an RNA used as a loading control is SCR1. SCR1 is an RNA polymerase III transcript that is stable and abundant. SCR1 RNA abundance should not change significantly during short periods of RNA polymerase II inhibition.</li>
</ol>
6. Quantify the RNA that is Bound to the Membrane
NOTE: To quantify the amount of radioactivity on membrane, expose the membrane to a Phosphor screen. After the appropriate exposure time, scan the Phosphor screen using a phosphorimager.
<ol>
	<li>Scan the Phosphor screen using the instructions provided by the manufacturer of the phosphorimager.</li>
	<li>Quantify the mRNA at each time point. Quantify mRNA levels using ImageQuant software or related software. Normalize the mRNA at each time point to the loading control. If SCR1 was used as the loading control, normalize the half-life northerns to SCR1.</li>
	<li>The half-life of the mRNA is calculated by dividing the amount of mRNA remaining at each time point by the amount of mRNA present at time point 0 (the initial time point). Graph the percent mRNA remaining versus time on a semilogarithmic plot (Figure 2C). Calculate mRNA half-lives by the following equation: 
	t1/2 = -0.693/k (negative 0.693)</li>
	<li>k is the slope of the best fit line and t1/2 is the mRNA half-life.</li>
</ol>

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	<li>Nonet, M., Scafe, C., Sexton, J., Young, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+RNA+polymerase+conditional+mutant+that+rapidly+ceases+mRNA+synthesis.">Eukaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.</a> Mol. Cell. Biol. 7, 1602-1616 (1987).</li><li>Santiago, T. C., Purvis, I. J., Bethany, A. J., Brown, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+mRNA+stability+and+length+in+Saccharomyces+cerevisiae.">The relationship between mRNA stability and length in Saccharomyces cerevisiae.</a> Nucleic Acids Res. 14 (21), 8347-8360 (1986).</li><li>Jimenez, A., Tipper, D. J., Davies, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+of+thiolutin,+an+inhibitor+of+macromolecular+synthesis+in+Saccharomyces+cerevisiae.">Mode of action of thiolutin, an inhibitor of macromolecular synthesis in Saccharomyces cerevisiae.</a> Antimicrob Agents Chemother. 3 (6), 729-738 .</li><li>Herrick, D., Parker, R., Jacobson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Comparison+of+stable+and+unstable+mRNAs+in+Saccharomyces+cerevisiae.">Identification and Comparison of stable and unstable mRNAs in Saccharomyces cerevisiae.</a> Mol Cell Biol. 10 (5), 2269-2284 .</li><li>Grigull, J., Mnaimneh, S., Pootoolal, J., Robinson, M. D., Hughes, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+Analysis+of+mRNA+Stability+Using+Transcription+Inhibitors+and+Microarrays+Reveals+Postranscriptional+Control+of+Ribosome+Biogenesis+Factors.">Genome-wide Analysis of mRNA Stability Using Transcription Inhibitors and Microarrays Reveals Postranscriptional Control of Ribosome Biogenesis Factors.</a> Mol Cell Biol. 24 (12), 5534-5547 (2004).</li><li>Tani, H., Akimitsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+technology+for+determining+RNA+stability+in+mammalian+cells.">Genome-wide technology for determining RNA stability in mammalian cells.</a> RNA Biology. 9 (10), 1233-1238 (2012).</li><li>Pelechano, V., Perez-Ortin, J. 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The authors declare that they have no competing financial interest.

Inhibition of mRNA synthesis and monitoring mRNA turnover in the absence of new synthesis is a method that is frequently used to measure mRNA decay rates. In S. cerevisiae, measurement of mRNA decay rates by inhibiting transcription using the temperature sensitive allele of RNA polymerase II is one of the most frequently used methods. This method specifically inhibits RNA polymerase II. The most critical steps for determination of mRNA decay rates using this technique are: 1) Prior to harvesting the yeast cells,...

The transcription and decay of specific mRNA are crucial determinants of gene expression. The rate of synthesis and decay of specific mRNAs determines the steady-state level of that particular mRNA. The steady state levels of mRNAs govern the abundance of mRNAs and determine how much of each mRNA is available for protein synthesis. Measurements of mRNA half-lives are used extensively to determine the decay rate of mRNAs. Specific mRNAs decay at different rates that are related to features of the mRNA, the function of the protein encoded by the mRNA and the environmental conditions. Depending on the technique utilized to determine mRNA decay rates, decay rate measurements can be determined either globally or for individual transcripts. In the yeast S. cerevisiae, the techniques that are most commonly used to measure global mRNA decay rates include utilizing a yeast strain harboring the temperature sensitive allele of RNA polymerase II and chemical transcriptional inhibitors such as thiolutin and 1,10-phenanthroline 1-5. These methods can also be utilized to measure individual mRNA decay rates 4. Other methods can also be utilized to measure mRNA decay rates. These methods include approach to steady state labeling or utilization of mRNA molecules that are expressed from a regulated promoter that is expressed only in select conditions. Each of these techniques has certain advantages and limitations. The technique described here utilizes the temperature sensitive allele of RNA polymerase II. This method uses S. cerevisiae as the model, but can been modified and utilized in other systems using specific transcriptional inhibition techniques 6.
mRNA half-life measurements using the temperature sensitive allele of RNA polymerase II are extensively used for both genome-wide and individual measurements of mRNA decay 4-5. This technique requires the use of a specific yeast strain that harbors a temperature sensitive allele of RNA polymerase II, rpb1-11. The rationale for this technique is that exposure of the temperature sensitive yeast strain to the nonpermissive temperature inhibits mRNA synthesis. Subsequently, decay of the preexisting mRNA is monitored at different time points after transcription has been inhibited. The disappearance of the preexisting mRNA is monitored by extracting RNA from the yeast cells at different time points after transcription has been turned off. The time points at which the yeast cells are harvested are predetermined by a pilot experiment, and depend on the transcripts and system being investigated. Quicker time points are used for short-lived transcripts, while longer time points are used for longer lived transcripts. Afterwards, the decay of the mRNA is monitored by either northern blot analysis, quantitative PCR or RNAseq.
Measurement of mRNA half-lives using a temperature sensitive allele of RNA polymerase II has its advantages. First, this technique is easy and straight forward. Second, once the yeast strain is acquired or generated in the laboratory the mRNA half-life measurements can be determined in different growth conditions; enabling determination of environmental influence on mRNA decay. Third, mRNA decay rates can be monitored genome-wide. Use of other transcription inhibition techniques also has advantages and limitations. For example, use of an inducible promoter requires subcloning to generate an mRNA that is under the control of the regulated promoter. Thiolutin is not readily available and is expensive when available. In addition, thiolutin&#8217;s mode of action is not completely understood and it has been reported to affect other cellular processes including inhibiting mRNA decay 7. Alternatively, 1,10-phenanthroline, is more readily available. Furthermore, all of the techniques used to inhibit transcription can perturb cellular function and can affect different mRNAs in distinct ways. An investigator needs to determine the most appropriate method to use in their experimental conditions to attain the most reliable results. To determine which method is most suitable for their application, a researcher needs to identify the transcripts and function of the proteins encoded by the transcripts being investigated. The most reliable mRNA decay rate measurements are those that are determined using multiple techniques and show the same decay rate. No single technique is always the best, and the most appropriate technique depends on the specific situation.
Numerous studies in S. cerevisiae have measured mRNA decay rates in various conditions and genetic backgrounds. The conditions that mRNA decay rates are measured in depend on the specific experiment being investigated. Measuring mRNA decay rates in different cellular environments determines whether the conditions being examined preferentially affect the decay rates of specific mRNAs. The decay rates of mRNAs can also vary depending on the yeast strain being used. For example, mRNA decay rates can be determined in wild-type yeast cells and yeast cells with a nonfunctional nonsense-mediated mRNA degradation (NMD) pathway. This mRNA degradation pathway is found in all eukaryotic organisms that have been examined so far and it triggers the degradation of mRNAs that prematurely terminate translation 8. NMD was initially identified as a pathway that degrades mRNAs with premature termination codons or nonsense codons, but is now recognized as a pathway that also regulates the expression of non-nonsense containing natural mRNAs. mRNAs that are targets of the pathway are rapidly degraded in yeast cells with a functional NMD pathway and stabilized in yeast cells with a nonfunctional NMD pathway. Thus, the half-lives of mRNAs that are direct targets of this pathway are shorter in wild-type yeast cells compared to yeast cells with a nonfunctional NMD pathway.

<table border="0" cellpadding="0" cellspacing="0" width="1077">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:272px;">Name of Material/Equipment</td>
			<td style="width:181px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:479px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Speed Centrifuge</td>
			<td>Eppendorf</td>
			<td>22628169</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mini Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>05-090-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Genescreen Plus membrane</td>
			<td>PerkinElmer</td>
			<td>50-905-0169</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hybridization Oven</td>
			<td>Fisher Scientific</td>
			<td>95-0030-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nanodrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-8000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphor screen</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9564-78</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Typhoon phosphorimager</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29004080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV cross-linker</td>
			<td>GE Healthcare Life Sciences</td>
			<td>80-6222-31</td>
			<td>Alternatively the membrane can be baked in an oven set to 80&#176; for 1 hour</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:272px;">NorthernMax prehybridization/hybridization buffer&#160;</td>
			<td>Life Technologies</td>
			<td>AM8677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">Yeast strains harboring the rpb1-1 mutation</td>
			<td></td>
			<td>Yeast strains can be obtained from a laboratory or created with a specific background</td>
		</tr>
	</tbody>
</table>

measurement of mrna decay rates in saccharomyces cerevisiae using rpb1-1 strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

The ability of this protocol to accurately measure mRNA decay rates depends on inhibition of transcription, the harvesting of yeast cells at the appropriate time points, and utilization of RNase free techniques while extracting RNA and northern blotting. Probing for two control mRNAs known to be unstable and stable, respectively, provides confidence that the experiment worked. For example, this can be accomplished by probing with a probe that detects both the CYH2 pre-mRNA and mRNA. Figure 2B sh...

Watch this Scientific Journal Video about Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains at JoVE.com

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

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12.5K Aufrufe.  Baylor University.  Die Steady-State-Level spezifischer mRNAs wird durch die Geschwindigkeit der Synthese und der Zerfall der mRNA bestimmt. Genomweite mRNA Abbauraten oder die Zerfallsraten spezifischer mRNAs kann durch Bestimmung mRNA Halbwertszeiten gemessen werden. Dieses Protokoll konzentriert sich auf die Messung der mRNA-Zerfallsraten in Saccharomyces cerevisiae. 

Serie: Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

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 ...

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.

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

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 ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

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. ...

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).