Name: rapid identification of chemical genetic interactions in saccharomyces cerevisiae
Uploaded: 2015-04-05T14:00:00
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 unt...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+strategy+enabling+high-resolution+functional+analysis+of+the+yeast+genome.">A comprehensive strategy enabling high-resolution functional analysis of the yeast genome.</a> Nat Methods. 5, 711-718 (2008).</li><li>Li, Z., 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=Systematic+exploration+of+essential+yeast+gene+function+with+temperature-sensitive+mutants.">Systematic exploration of essential yeast gene function with temperature-sensitive mutants.</a> Nat Biotechnol. 29, 361-367 (2011).</li><li>Costanzo, M., 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=The+genetic+landscape+of+a+cell.">The genetic landscape of a cell.</a> Science. 327, 425-431 (2010).</li><li>Parsons, A. B., 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=Integration+of+chemical-genetic+and+genetic+interaction+data+links+bioactive+compounds+to+cellular+target+pathways.">Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways.</a> Nat Biotechnol. 22, 62-69 (2004).</li><li>Parsons, A. B., 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=Exploring+the+mode-of-action+of+bioactive+compounds+by+chemical-genetic+profiling+in+yeast.">Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast.</a> Cell. 126, 611-625 (2006).</li><li>Hillenmeyer, M. 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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 border="0" cellpadding="0" cellspacing="0" width="987">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Name of Material/ Equipment&#160;</td>
			<td style="width:161px;">Company&#160;</td>
			<td style="width:156px;">Catalog Number&#160;</td>
			<td style="width:457px;">Comments/Description&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Yeast Extract</td>
			<td style="width:161px;">BioBasic</td>
			<td>G0961</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 1% final concentration (w/v)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Tyrptone Powder</td>
			<td style="width:161px;">BD Biosciences</td>
			<td>211820</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Dextrose</td>
			<td style="width:161px;">Anachemia</td>
			<td style="width:156px;">31096-380</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v) - Do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Agar A</td>
			<td style="width:161px;">Bio Basic</td>
			<td>FB0010</td>
			<td style="width:457px;">For YEPD solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:213px;">G418</td>
			<td style="width:161px;">A.G. Scientific Inc.</td>
			<td style="width:156px;">G-1033</td>
			<td style="width:457px;">Prepare 1000x stock at 200mg/ml in dH2O and filter sterilize.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">12 well plate</td>
			<td>Greiner Bio One</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">5ml culture tubes</td>
			<td>Evergreen Scientific</td>
			<td>222-2376-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">10cm Petri Dish</td>
			<td style="width:161px;">VWR</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROTOR HDA</td>
			<td>Singer Instruments&#160;</td>
			<td>ROT-001</td>
			<td>high-throughput microbial array pinning robot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PLUSPLATE&#169; Petri Dish</td>
			<td>Singer Instruments&#160;</td>
			<td>PLU-001</td>
			<td>Box of 200 dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-384</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1536 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-1536</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alternative Pinning Tools:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fully Automated Robtic Systems</td>
			<td>S&#38;P robotics</td>
			<td>http://www.sprobotics.com</td>
			<td>Several automated colony handling robitic and imagining systems available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manual Pinning Tools</td>
			<td>V&#38;P Scientific</td>
			<td>http://www.vp-scientific.com</td>
			<td>Handheld replication tools and accessories.&#160;</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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