Name: rapid identification of chemical genetic interactions in saccharomyces cerevisiae
Uploaded: 2015-04-05T14:00:00.000+00:00
Duration: 12 min 13 s
Description: Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

Research in the CJN lab is supported by operating grants from NSERC, the Canadian Cancer Society Research Institute (CCSRI), and the Canadian Breast Cancer Foundation (BC-Yukon Branch).

NOTE: The general workflow of this procedure is outlined in Figure 2.
1. Determination of Growth-inhibitory Dose
<ol>
	<li>Preparation of yeast overnight culture 
	NOTE: The yeast-extract-peptone-dextrose growth media (YEPD) used in this protocol is a standard recipe13.
	<ol>
		<li>Streak out BY4741 (MATahis3&#916;1 leu2&#916;0 met15&#916;0 ura3&#916;0) cells on a YEPD agar plate and incubate for 48 hr at 30&#160;&#176;C&#160;or until visible colonies form.</li>
		<li>Prepare overnight cultures by inoculating 5 ml of YEPD liquid media in a sterile culture vessel with a single colony. Incubate overnight at 30&#160;&#176;C with continuous rotation or shaking. The culture will typically reach saturation by the morning (~2 &#215; 108 cells/ml).</li>
	</ol>
	</li>
	<li>Solid agar media preparation containing varying chemical doses
	<ol>
		<li>Prepare several stocks of the chemical to be tested at 100x&#160;desired final concentration in an appropriate solvent. 
		NOTE: Effective concentrations will depend on the chemical, and must be determined empirically. It is therefore advisable to initially test a broad final concentration range; i.e., from low &#181;M to high mM.</li>
		<li>For each concentration of chemical to be tested, aliquot 3 ml of molten YEPD agar to a several sterile culture tube. Place in 55 &#176;C water bath to keep from solidifying.</li>
		<li>For each concentration of chemical to be tested add 30 &#181;l of the 100x compound to molten media, vortex 2-3 sec to mix, and pipette 1 ml into each pair of duplicate wells in a 12-well plate. Be sure to include a vehicle only control. Allow plates to set overnight at room temperature. Discard any extra media.</li>
	</ol>
	</li>
	<li>Spread plating cells
	<ol>
		<li>Inoculate 10 ml of YEPD liquid media with 2 &#181;l of a saturated yeast culture, grown overnight as in Step 1.1.2.</li>
		<li>Plate 25 &#181;l of diluted yeast culture per well, spread evenly using sterile glass beads or rod, and allow the plate to dry under a flame. Incubate plate at 30&#176;C for 48 hr.</li>
		<li>Evaluate the growth of yeast (both total number and size of colonies) on the plates. Select a sub-lethal concentration of compound that does not inhibit growth by greater than 10%-15% to perform the screen.</li>
	</ol>
	</li>
</ol>
2. Systematic Chemical Genetic Screen
<ol>
	<li>Maintenance of deletion-mutant-array (DMA) and Preparation of Source Plate
	<ol>
		<li>For a detailed description on constructing deletion mutant arrays from glycerol stocks please refer to Baryshnikova et al.14. Once deletion mutants have been arrayed at a density of 384 colonies per plate, the DMA can be stored for several months at 4&#160;&#176;C. Replicate on YEPD agar containing 200 &#181;g/ml of G418 as necessary to prevent colonies from growing into each other. 
		NOTE: Multiple serial replications should be avoided to prevent loss of slow growing strains and minimize the appearance of suppressor mutants. This is particularly relevant if maintaining the collections as haploids.</li>
		<li>Perform all replica-plating steps using a microbial arraying robotic system. Alternatively, manipulate arrayed colonies using manual pinning tools. 
		NOTE: The yeast deletion collection is available as haploids and diploids from several commercial sources and is typically shipped as glycerol stocks in 96-well plates.</li>
	</ol>
	</li>
	<li>Condensing the deletion mutant array
	<ol>
		<li>Prepare 250 ml of YEPD agar media containing 200 &#181;g/ml of G418. The effective concentration of G418 may vary and each lot should be tested empirically.</li>
		<li>Prepare five YEPD agar plates by pouring 50 ml of YEPD agar containing 200 &#181;g/ml of G418 per plate. Do this one day before condensing the array and allow the plates to cool at room temperature overnight on a flat even surface. 
		NOTE: Four plates will be required for the collection at 1,536-strain density, the fifth plate is poured as an extra.</li>
		<li>Remove the 16 Petri dishes containing the mutant deletion collection at a density of 384 colonies per plate and allow to come to room temperature (~1 hr).</li>
		<li>Wipe any condensation that has formed on the lid of each plate using a delicate task wipe. Failure to do so may result in water droplets being deposited on the array and cross contamination of arrayed mutants.</li>
		<li>Using a microbial array pinning robot, condense the DMA from a density of 384 colonies per plate (16 Petri dishes) to 1,536 colonies per plate (4 Petri dishes). Perform this so that the 1st colony from plate 1 pins to row 1 column 1 of the condensed array, the 1st colony of plate 2 to row 1 column 2, the 1st colony of plate 3 to row 2 and column 1, and the 1st colony of plate 4 to row 2 column 2.</li>
		<li>Incubate the consolidated array at 30&#176;C overnight.</li>
		<li>Examine the array to ensure uniform transfer of colonies from source plates. There will be several blank positions, purposely incorporated into the array, which serve as a guide to ensure the DMA has been condensed correctly (Figure 4A). 
		NOTE: These will be the source plates for replica plating onto media containing the test compound. A single source plate can be used for multiple pinnings. Also many robots will have an offsetting feature, which will ensure similar numbers of yeast are being transferred each time.</li>
	</ol>
	</li>
	<li>Replica plate deletion mutant array
	<ol>
		<li>Prepare 700 ml of control (vehicle only) and 700 ml of experimental media (chemical-containing). This is enough for performing the assay in triplicate (12 plates per condition, plus two extras).</li>
		<li>Pour 50 ml of YEPD agar containing test chemical or control per plate. Allow the plates to cool at room temperature overnight on a flat even surface.</li>
		<li>Using the robot to replica plate, inoculate the DMA onto three sets of plates containing chemicals at the experimentally determined concentration, as well as three sets of plates containing the vehicle control. Incubate plates at room temperature for 24-48 hr.</li>
	</ol>
	</li>
</ol>
3. Imaging Plates and Data Analysis
<ol>
	<li>Remove plates from incubator and allow them to come to room temperature (~1 hr) prior to imaging. This will prevent condensation from forming while imaging the plates.</li>
	<li>Image plates at 24 and 48 hr by removing the lid and placing face down on a flat bed scanner. Capture images at a resolution of at least 300 dpi. Alternatively, use a digital camera to capture images. If doing so, place plates face up on a dark background and remove the lids to image. 
	NOTE: The file format and positioning of plates will depend on the quantification program used.</li>
	<li>Perform quantification and comparison of array colony sizes using one of several open source programs, including Balony 15, SGAtools 16, and ScreenMill 17.</li>
</ol>
4. Validation of Screen
NOTE: At this stage several yeast deletions mutants will score as hypersensitive to the chemical of interest. These chemical-genetic interactions must next be validated in two ways. First the identity of sensitive strains should be confirmed by PCR. Second, chemical sensitivity must be independently scored. Outlined below is a brief protocol for performing spotting assays to validate strain hypersensitivity, a technique common in yeast biology.
<ol>
	<li>Streak out desired (hypersensitive) mutants from the yeast deletion collection onto YEPD agar containing 200 &#181;g/ml of G418. Incubate at 30&#160;&#176;C until colonies form. Verify the genotype of the strain by diagnostic PCR with primers according to the manufacturer&#8217;s protocol.</li>
	<li>Inoculate 5 ml of the YEPD liquid for each strain and incubate overnight at 30&#160;&#176;C with rotation or shaking.</li>
	<li>Measure OD600 and dilute each strain to OD600&#160;= 1 in sterile dH2O. These are now the normalized yeast cultures.</li>
	<li>Dispense 100 &#181;l of normalized wild-type yeast (BY4741) culture to well A1 of a 96-well plate. Dispense 100 &#181;l of up to seven additional strains to wells B1-H1.</li>
	<li>Use a multichannel pipettor to dispense 90 &#181;l of sterile dH2O&#160;to wells A2-H2 through A6-H6. Finally, prepare a series of 1:10 dilutions of normalized yeast cultures. Start by serially pipetting 10 &#181;l of column 1 to column 2 with a multichannel pipettor, and continue across the 96-well plate until six dilutions are made (i.e., 100 to 10-5).</li>
	<li>Transfer 2-5 &#181;l of diluted yeast cultures in a grid using a multichannel pipettor onto YEPD solid media containing chemical and vehicle control. Incubate plates at the appropriate temperature for 24-48 hr.</li>
	<li>Inspect plates and compare the sensitivity of select mutants to chemical (relative to BY4741 control). Image plates at 24 and 48 hr as in step 3.2. 
	NOTE: While the identification of several hypersensitive strains with related gene ontologies or functions lends confidence to the validity of the screen, transformation of a hypersensitive deletion strain with a plasmid containing a wild-type copy of the gene is required to formally establish that a gene deletion of interest (and not hidden second site mutations) causes drug sensitivity.</li>
</ol>

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The authors declare they have no competing financial interests.

Outlined here is an approach for generating chemical-genetic interaction profiles. The method is simple: by comparing colony sizes of each strain in comprehensive gene deletion collections in the presence and absence of a chemical, all genes needed to tolerate a chemical insult are identified. Annotation enrichment analysis of the resulting list of sensitive strains can then be used to provide insight into a chemicals mode of action. While this protocol has been optimized for budding yeast, it can also be adapted for use...

The genetic tools and resources available in the model organism Saccharomyces cerevisiae have enabled large-scale functional genomics studies that collectively provide new insight into how genes function as networks to fulfill the requirements of biological systems. The cornerstone of these tools was the collaborative creation of a complete set of non-essential gene deletions of all open reading frames in yeast1,2. A striking observation was that only ~20% of yeast genes are required for viability when grown as haploids under standard laboratory conditions. This highlights&#160;the ability of a cell to buffer against genomic perturbations through the utilization of alternative biological pathways. Genetic mutants that are viable individually, but lethal in combination, signal connected or convergent parallel biological pathways and form genetic interaction networks that describe biological function. With the development of conditional&#160;temperature-sensitive and hypomorphic alleles of essential genes the technology has not been limited to the study of non-essential genes3,4. This concept has been applied at a genomic scale producing an unbiased genetic interaction map illustrating how genes involved in similar cellular processes cluster together5.
Chemical perturbations of genetic networks mimic gene deletions (Figure 1)6. Querying growth-inhibitory compounds against a high-density array of deletion strains for hypersensitivity identifies a chemical-genetic interaction profile, i.e. a list of genes that is required to tolerate chemical stress. Like genetic interactions, large-scale screens of chemical libraries have shown that compounds with a similar mode of action cluster together7. Therefore, by establishing the chemical-genetic interaction profile of a compound the mode of action may be inferred by comparing it with large-scale synthetic genetic and chemical genetic interaction datasets8,9.
Large-scale chemical-genetic screens, where scores of compounds are interrogated, have been performed by barcode competition assays. In this approach, the pooled collection of deletion strains is grown en masse for several generations in a small volume of media containing a chemical. Since each deletion mutant harbors a unique genetic barcode, the viability/growth of individual mutants within the pool of deletion strains is tracked by microarray or high-throughput sequencing10.
Inferring fitness by monitoring colony size of physically arrayed mutants grown on solid agar containing a bioactive compound is also an effective method to identify chemical-genetic interactions11,12. This approach provides a cost-effective alternative to competition-based screening and is well suited for assaying small libraries of chemicals. Outlined here is a simple methodology for producing a list of chemical-genetic interactions in S. cerevisiae that does not rely on molecular biology manipulations or infrastructure. It requires only a yeast deletion collection, a robotic or manual pinning apparatus, and freely available image analysis software.

<table><tbody><tr><td>Yeast Extract</td><td>BioBasic</td><td>G0961</td><td>For YEPD liquid/solid media add to 1% final concentration (w/v)</td></tr><tr><td>Tyrptone Powder</td><td>BD Biosciences</td><td>211820</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v)</td></tr><tr><td>Dextrose</td><td>Anachemia</td><td>31096-380</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v) &amp;#8212; do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td></tr><tr><td>Agar A</td><td>Bio Basic</td><td>FB0010</td><td>For YEPD solid media add to 2% final concentration (w/v)</td></tr><tr><td>G418</td><td>A.G. Scientific Inc.</td><td>G-1033</td><td>Prepare 1,000x stock at 200&amp;nbsp;mg/ml in dH&lt;sub&gt;2&lt;/sub&gt;O and filter sterilize.&amp;nbsp;</td></tr><tr><td>12-well plate</td><td>Greiner Bio One</td><td>655180</td><td></td></tr><tr><td>5 ml culture tubes</td><td>Evergreen Scientific</td><td>222-2376-080</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>ROTOR HDA</td><td>Singer Instruments&amp;nbsp;</td><td>ROT-001</td><td>high-throughput microbial array pinning robot</td></tr><tr><td>PLUSPLATE&amp;copy; Petri Dish</td><td>Singer Instruments&amp;nbsp;</td><td>PLU-001</td><td>Box of 200 dishes</td></tr><tr><td>384 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-384</td><td>Box of 1,000 pads</td></tr><tr><td>1536 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-1536</td><td>Box of 1,000 pads</td></tr><tr><td colspan="4">Alternative Pinning Tools:</td></tr><tr><td>Fully Automated Robtic Systems</td><td>S&amp;amp;P robotics</td><td>http://www.sprobotics.com</td><td>Several automated colony handling robitic and imagining systems available.</td></tr><tr><td>Manual Pinning Tools</td><td>V&amp;amp;P Scientific</td><td>http://www.vp-scientific.com</td><td>Handheld replication tools and accessories.&amp;nbsp;</td></tr></tbody></table>

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.

As a validation of this approach we performed a representative chemical genetic interaction screen of the chemotherapeutic agent 5-fluorouracil (5-FU) following the above outlined protocol. 5-FU is known to disrupt thymidylate synthase as well as DNA and RNA metabolism18. The chemical genetic interactions of 5-FU are well studied and have been investigated by yeast barcode microarray techniques using both heterozygous and homozygous deletion collections8,19. Here we show that similar results can be ...

Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

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.

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. ...

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Biology

10.3K Views.  University of Victoria. 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.

Video: Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

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

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

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

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

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

Genetics

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Biochemistry

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 ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

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 ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Summary

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Chapters in this video

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

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Microarray Analysis for Saccharomyces cerevisiae

Competitive Genomic Screens of Barcoded Yeast Libraries

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

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe