Name: determination of s-phase duration using 5-ethynyl-2'-deoxyuridine incorporation in saccharomyces cerevisiae
Uploaded: 2022-10-21T13:30:00
Description: Watch this Scientific Journal Video about Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae at JoVE.com

The authors wish to acknowledge Agence Nationale de la Recherche (ANR) and Association pour la Recherche sur le Cancer (ARC) for the PhD fellowships to J.d.D.B.T. and the Agence Nationale pour la Recherche (ANR) for financial support (grant ANR-18-CE12-0018-01). Cytometry and microscopy were performed at the Montpellier MRI BioCampus imaging facility.

1. S. cerevisiae cell culture
NOTE: The yeast strains used are listed in Table 1
NOTE: The S-phase duration can be monitored in different ways. Depending on the question to be addressed, the cells can be grown asynchronously or synchronously following G1 arrest.
<ol>
	<li>From asynchronously growing S. cerevisiae cells 
	NOTE: This method allows for the determination of the percentage of cells in ...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORC-dependent+and+origin-specific+initiation+of+DNA+replication+at+defined+foci+in+isolated+yeast+nuclei.">ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei.</a> Genes &amp; Development. 11 (12), 1504-1518 (1997).</li><li>Lengronne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+S+phase+progression+globally+and+locally+using+BrdU+incorporation+in+TK++yeast+strains.">Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains.</a> Nucleic Acids Research. 29 (7), 1433-1442 (2001).</li><li>Magiera, M. M., Gueydon, E., Schwob, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+and+spindle+checkpoints+cooperate+during+S+phase+to+delay+mitosis+and+preserve+genome+integrity.">DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity.</a> The Journal of Cell Biology. 204 (2), 165-175 (2014).</li><li>Talarek, N., Petit, J., Gueydon, E., Schwob, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EdU+incorporation+for+FACS+and+microscopy+analysis+of+DNA+replication+in+budding+yeast.">EdU incorporation for FACS and microscopy analysis of DNA replication in budding yeast.</a> Methods in Molecular Biology. 1300, 105-112 (2015).</li><li>Ivanova, 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=Budding+yeast+complete+DNA+synthesis+after+chromosome+segregation+begins.">Budding yeast complete DNA synthesis after chromosome segregation begins.</a> Nature Communications. 11 (1), 2267 (2020).</li></ol>

Yeast is a prime model organism for cell cycle studies, yet the characterization of its S phase has long been hampered by its inability to incorporate exogenous nucleosides, such as BrdU, which are used as tracers of DNA replication. Equipping yeast with a high expression of herpes simplex thymidine kinase (TK) and the addition of a human nucleoside transporter (hENT) has largely solved this problem15,16. EdU is more versatile than BrdU as its detection with smal...

Genome stability through mitotic division is ensured by the transmission of a complete and equal set of chromosomes to the two produced cell progenies. This relies on the accurate completion of a series of events occurring in a given time in each phase of the cell cycle. In G1, the replication origins are licensed upon the recruitment of several licensing factors, including Cdc61. In the S phase, whole-genome duplication is initiated from multiple active replication origins and performed by replication machineries that gather in microscopically visible foci named replication factories2. In the M phase, duplicated sister chromatids are attached and bioriented on the mitotic spindle to allow their segregation to the opposite poles of the mitotic cell3. The regulation, proper completion, and duration of each phase are key to ensure genome stability. Indeed, premature exit from any of these phases leads to genome instability. For instance, a shorter G1 induced by deletion of the budding yeast CDK inhibitor Sic1 or by the overexpression of G1 cyclins will alter the subsequent S phase4,5,6. Consequently, these deregulations, associated or not with replication stress, result in chromosome breaks, rearrangements, and mis-segregation4,5,6. Therefore, monitoring the duration of the S phase and, more broadly, the duration of the other phases of the cell cycle may be crucial to identify the defects occurring in different mutants and in different stressful conditions.
A traditional method for measuring cell cycle phase duration includes simple DNA content flow cytometry (Figure 1A) and relies on a fitting algorithm (available in most cytometry software) used to separate the population into G1, S, and G2 + M phase fractions from the 1C and 2C peaks. The fractions are then multiplied by the population doubling time7. However, this method gives only estimated values, requires a homogeneous cell size distribution within a given fraction, and is not applicable to synchronized cultures. To study the S-phase duration in mammalian cells, several thymidine analogs have been developed and widely used, including EdU. Their uptake from the extracellular medium and phosphorylation by thymidine kinase (hereafter referred to as TK) make them available for DNA polymerases to incorporate them at sites of DNA synthesis (replication, recombination, repair). To bypass the absence of the TK gene in Saccharomyces cerevisiae cells, yeast strains have been engineered to allow stable and constitutive expression of the herpes simplex virus TK8 and the human equilibrative nucleoside transporter (hENT1)9. Once incorporated into DNA, EdU is detected via the selective Click reaction, which chemically couples its alkyne moiety to azide-modified fluorochromes10.
This paper provides two optimized comprehensive protocols to pulse-label asynchronous and synchronous TK-hENT1 engineered cells with EdU in order to precisely visualize and measure DNA replication duration and dynamics, as well as the duration of the other phases of the cell cycle, with high spatial and temporal resolution at both the single-cell and population levels by microscopy and flow cytometry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#945;-factor&#160;</td>
			<td>Genescript</td>
			<td>RP01002</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Euromedex</td>
			<td>04-100--812-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper sulfate</td>
			<td>Sigma</td>
			<td>C1297</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-sulfo-Cyanine5 azide (Cy5 azide)</td>
			<td>Interchim</td>
			<td>FP-JV6320</td>
			<td>Alternative to Alexa647-Azide</td>
		</tr>
		<tr>
			<td>Dy-530 azide</td>
			<td>Dyomics</td>
			<td>&#160;530-10</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU (5-ethynyl-2&#8217;-deoxyuridine)</td>
			<td>Carbosynth</td>
			<td>NE08701</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Carlo Erba reagents</td>
			<td>P013A10D16</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>L- ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Euromedex</td>
			<td>EU0090</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase</td>
			<td>SIGMA</td>
			<td>R5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sytox Green</td>
			<td>Invitrogen</td>
			<td>S-7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>OLS</td>
			<td>CASY</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Agilent</td>
			<td>NovoSampler Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Infors</td>
			<td>444-4230</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Shaking water bath</td>
			<td>Julabo</td>
			<td>SW22</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics</td>
			<td>Vibra cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field microscopy</td>
			<td>Leica</td>
			<td>THUNDER Imager</td>
			<td>or equivalent</td>
		</tr>
	</tbody>
</table>

determination of s-phase duration using 5-ethynyl-2'-deoxyuridine incorporation in saccharomyces cerevisiae

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome segregation. As DNA synthesis defects underlie chromosome rearrangements, monitoring DNA replication has become essential to understand the basis of genome instability. Saccharomyces cerevisiae is a classical model to study cell cycle regulation, but key DNA replication parameters, such as the fraction of cells in the S phase or the S-phase duration, are still difficult to determine. This protocol uses short and non-toxic pulses of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, in engineered TK-hENT1 yeast cells, followed by its detection by Click reaction to allow the visualization and quantification of DNA replication with high spatial and temporal resolution at both the single-cell and population levels by microscopy and flow cytometry. This method may identify previously overlooked defects in the S phase and cell cycle progression of yeast mutants, thereby allowing the characterization of new players essential for ensuring genome stability.

To determine the S-phase duration and, more broadly, the duration of G1 and G2 + M (protocol step 1.1), S. cerevisiae W303 wild-type cells (WT, Table 1) were grown asynchronously in SC medium for 7 h. Every hour, the cell concentration was monitored to determine the doubling time (Figure 2B). In these growth conditions, the calculated doubling time was 120 min &#177; 13 min at 25 &#730;C (Table 2). When the cells were in the ex...

Watch this Scientific Journal Video about Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae at JoVE.com

Determination of S-Phase Duration Using 5-Ethynyl-2&#039;-deoxyuridine Incorporation in Saccharomyces cerevisiae

We describe two complementary protocols to accurately determine S-phase duration in S. cerevisiae using EdU, a thymidine analog, which is incorporated in vivo and detected using Click chemistry by microscopy and flow cytometry. It allows for the easy characterization of the duration of DNA replication and overlooked replication defects in mutants.

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome ...

This protocol allows a precise determination of S-phase duration in synchronized budding yeast cells. It is a quick, easy, and reproducible method. In addition, it is sensitive enough to detect mild synthesis defects undetected by classical protocols.This method will be useful for many researchers working on cell cycle regulation in budding yeast. Demonstrating this protocol will be Nicolas Talarek, a senior researcher, and Juan De Dios Barba Tena, a PhD student in the lab. Inoculate Saccharomyces cerevisiae cells in 10 milliliters of SC medium at a low cell concentration for an overnight culture at 30 degree Celsius with orbital agitation at 130 rotations per minute.The following day, dilute the cells in 20 milliliters of fresh SC medium at a final concentration of two to three times 10 to the six cells per milliliter. At 40 microliters of one milligram per milliliter alpha factor diluted in water. Then culture the cells at 30 degree Celsius, with orbital agitation at 130 rotations per minute for one hour.Repeat this step once. Visualize the cells under a light microscope to monitor G1 arrest. Proceed if more than 90%of the cells display a shmoo, and the others are rounded unbutted cells.Centrifuge the samples for three minutes at 1, 500 G.Then discard the supernatant with the vacuum pipette and resuspend the cells in 20 milliliters of SC medium. Repeat this step once. Collect one milliliter of cells twice every five minutes and proceed to EDU labeling.Add 400 microliters of the alpha factor 30 minutes after the release. Transfer one milliliter of the cell culture to a two milliliter microfuge tube containing one microliter of 10 millimolar EDU, mixed well by inversion. To discriminate the EDU positive cells from the EDU negative cells on a Pi EDU bivariant facts, transfer another one milliliter of cell culture to a two milliliter microfuge tube containing one microliter of DMSO.Incubate for three to five minutes at 30 degree Celsius under agitation in a shaking water bath. Stop the reaction by adding 100 microliters of 100%ethanol. Pellet the cells for two minutes at 10, 000 G in a microfuge.Remove the supernatant using a vacuum pipette. Resuspend the cell pellet in 500 microliters of 70%ethanol, and mix well by vortexing. Leave the samples at room temperature for at least one hour at 20 tilts per minute on a variable speed rocker to permeablizie the cells.Pellet the cells for two minutes at 10, 000 G in a microcentrifuge and discard the supernatant with a vacuum pipette. Wash the cells twice with 500 microliters of 10%ethanol and PBS. Pellet the cells for two minutes at 10, 000 G in a microfuge and discard the supernatant with a vacuum pipette.Resuspend the pellet in 200 microliters of PBS containing RNase A and Proteinase K.Incubate from one to two hours at 50 degree Celsius with occasional shaking, or overnight at 37 degree Celsius. Pellet the cells for two minutes at 10, 000 G in a micro centrifuge and discard the supernatant with a vacuum pipette. Wash the cells with 500 microliters of PBS.Then pellet the cells and discard the supernatant as demonstrated earlier. Resuspend the cell pellet in 200 microliters of PBS with 1%BSA. Incubate for 30 minutes at room temperature.Then pellet the cells, discard the supernatant as demonstrated earlier, and resuspend the pellet in 300 microliters of PBS containing 1%BSA. Distribute the cells into two, 1.5 milliliter microcentrifuge tubes. The first one should consist of 200 microliters of cell culture for the Click reaction, and the second tube should contain 100 microliters of cell culture for the sytox green staining.Then, pellet the cells and discard the supernatant as demonstrated earlier. For sytox green staining, resuspend the cell pellet in 100 microliters of PBS. Then depending on the cell concentration, transfer 10 to 30 microliters to a flow cytometer tube containing 300 microliters of sytox green mixture.Sonicate the samples twice for two seconds at an amplitude of 40 to 50%Leave in the dark until processing the samples on a flow cytometer. For the Click reaction, resuspend the cell pellet with 40 microliters of freshly prepared Azide dye buffer. Incubate at room temperature in the dark for 60 minutes.Pellet the cells and discard the supernatant as demonstrated earlier. Then wash the cells three times with 300 microliters of 10%ethanol in PBS. Next resuspend the cells in 100 microliters of 50 micrograms per milliliter propidium iodide in PBS and leave for 10 minutes in the dark.Depending on the cell concentration, transfer 10 to 30 microliters of the cell suspension to a flow cytometer tube containing 300 microliters of 50 millimolar Tris HCl, pH 7.5. Sonicate twice for two seconds at an amplitude of 40 to 50. Leave the samples in the dark until processing them on a cytometer.Read the sytox green samples using an excitation blue laser at 488 nanometers, and a 530 by 30 band pass filter. Read the Bavaria Pi EDU samples on a dot plot using an excitation blue laser at 488 nanometers and a 615 by 20 band pass filter for the Pi X-axis, and a red excitation laser at 640 nanometers, a 660 by 20 band pass filter for the Y-axis. Three populations of cells were observed in the bivariant EDU Pi cytometer analysis.At 25 degree Celsius, approximately 27%of the cell population was in the G1 phase, 29%was in the S phase, and about 44%was in G2 plus M phase. In synchronized cells, the duration of the S phase may be about 25 minutes based on the time when the DNA content changed from one C to two C on the sytox green flow cytometer profile. EDU pulse labeling accurately determined the S phase duration.15 minutes after the release, a fraction of EDU positive cells was detected, indicating the start of the S phase. The progression through the S phase was seen by the cell cloud moving upward and rightward in the Bavaria Pi EDU graph. Finally, at 35 minutes from the release, a fraction of cells was EDU negative, but with twice the amount of DNA indicating that the S phase ended and the cells were in the G2 plus M phase.Despite the high synchrony observed, some cells finished the S phase 60 minutes after the release and the S phase was complete for the whole population approximately 65 minutes after the release. It is crucial to have a perfect synchronization and to prevent cells entering the second S phase. Cells can be imaged with a microscope where nuclear DNA replication foresight can be monitored and quantified.This technique will help to identify overlook mutants that have mild S phase defects, as well as cell cycle duration defects.

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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 ...

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 ...

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 ...

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 ...

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.

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.

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

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.