Funding support for this project was provided by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2016R1A2B2006354).

1. Preparation of Media
<ol>
	<li>Prepare Yeast Extract with supplements (YES), YES without adenine (YES Low Ade), Pombe Glutamate medium (PMG12), and PMG without adenine (PMG-Ade) by mixing the components as described in Table 1. YES-Ade (Low Ade) and PMG-Ade plates have all of the same components, but the adenine is omitted from the latter.
	<ol>
		<li>Use the following salt (50x) stock: 52.5 g/L MgCl2&#183;6H2O, 2 g/L CaCl2&#183;2H2O, 50 g/L KCl, 2 g/L Na2SO4 in distilled water. Filter sterilize using a 0.22-&#181;m pore-size filter and store at 4 &#176;C.</li>
		<li>Use the following vitamin (1,000x) stock: 1 g/L pantothenate, 10 g/L nicotinic acid, 10 g/L inositol, 10 mg/L biotin in distilled water. Filter sterilize using a 0.22-&#181;m pore-size filter and store at 4 &#176;C.</li>
		<li>Use the following mineral (10,000x) stock: 5 g/L boric acid, 4 g/L MnSO4, 4 g/L ZnSO4&#183;7H2O, 2 g/L FeCl2&#183;4H2O, 0.4 g/L MoO3, 1 g/L KI, 0.4 g/L CuSO4&#183;5H2O, 10 g/L citric acid in distilled water. Filter sterilize using a 0.22-&#181;m pore-size filter and store at 4 &#176;C. 
		NOTE: YES liquid medium can be made by omitting the agar.</li>
	</ol>
	</li>
	<li>Autoclave the medium and cool it to below 60 &#176;C while stirring with a stir bar, at the rate of 200 rpm.</li>
	<li>For YES+G418 (geneticin) plates, add 1 mL/L G418 stock solution to the YES medium.
	<ol>
		<li>Use the following G418 (1,000x) stock: 100 mg/mL G418 in distilled water. Filter sterilize using a 0.22-&#181;m pore-size filter, aliquot to 1.5 mL centrifuge tubes, and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Stir for another 5 - 10 min and aliquot the media into 90-mm Petri dishes (1 L of medium aliquots to roughly 40 Petri dishes. Store the plates at 4 &#176;C.</li>
</ol>
2. Cloning of rpt4+ and its 5&#39;/3&#39; UTRs
<ol>
	<li>Perform PCR with primers p1 and p2 (Figure 1A) using fission yeast genomic DNA (gDNA) as the template in a total volume of 50 &#181;L (~1 &#181;g DNA, 20 U enzyme, 1x reaction buffer), and applying the reaction cycles described in Table 2 to amplify a 3,315-base pair (bp) fragment comprising rpt4+ and its 5&#39;/3&#39; UTRs. Purify the PCR product using a purification kit13 as directed by the manufacturer (Figure 1A).</li>
	<li>Digest the pBlueScript KS(-) vector14 and the amplified DNA fragment containing rpt4+ with its 5&#39;/3&#39; UTRs with BamH1 and Xho1 in a total volume of 20 &#181;L (~1 &#181;g DNA, 20 U enzyme, 1x digestion buffer) at 37 &#176;C in a water bath for 16 - 18 h (Figure 1B).</li>
	<li>Gel purify the digested vector (1.2% agarose gel) and DNA insert by performing TAE-based agarose gel electrophoresis (100 V, 1 h), excising the DNA bands of the desired sizes, and isolating the DNA with a gel extraction kit15.</li>
	<li>Ligate the vector and DNA insert using T4 DNA ligase by performing a 20-&#181;L ligation reaction containing 50 - 100 ng of the vector DNA, a ~3-fold molar excess of the insert DNA, 400 U T4 DNA ligase, and 1x ligation buffer. Incubate this mixture at 18 &#176;C for 16 - 18 h.</li>
	<li>Transform the ligated DNA into 100 &#181;L of E. coli DH5&#945; by applying heat shock for 70 s at 42 &#176;C. Add 1 mL of LB and incubate for 1 h at 37 &#176;C for recovery. Perform centrifugation (13,800 x&#160;g), discard the supernatant, plate all of the transformed E. coli cells on LB-Ampicillin (LA) plates, and incubate at 37 &#176;C for 16 h.</li>
	<li>Pick 4 - 8 colonies with toothpicks and grow overnight at 37 &#176;C in 3 mL LA medium.</li>
	<li>Isolate the plasmids with a plasmid purification kit16 used according to the manufacturer&#39;s protocol and perform analytical restriction enzyme digestions with 5 &#181;L of the isolated plasmid, 5 U of each restriction enzyme (BamH1 and Xho1) and 1x restriction buffer. Incubate the reaction mixture at 37 &#176;C in a water bath for 3 h. Confirm the cloning by sequencing candidate plasmids.</li>
</ol>
3. Introduction of the Silent Mutation (Xho1 Restriction Site)
<ol>
	<li>Design a pair of 33-bp primers (p3 and p4) that contain the desired mutation in the otherwise complementary sequence.</li>
	<li>Perform PCR with high-fidelity polymerase17 (Figure 1C) in a total volume of 25 &#181;L (10 ng of cloned plasmid, 2 &#181;L of 10 pmol primer mix, 2 U enzyme, 1x reaction buffer) using the cycling parameters listed in Table 3.</li>
	<li>Digest the template plasmid with DpnI in a total volume of 20 &#181;L (20 U enzyme, 1x digestion buffer) at 37 &#176;C in a water bath for 1 h.</li>
	<li>Transform, propagate, isolate, and sequence the plasmid containing the silent mutation, as described in steps 2.5 - 2.7. 
	Note: The silent mutation can also be introduced using a cloning method which allows for the joining of multiple DNA fragments in a single reaction18.</li>
</ol>
4. Random Mutagenesis of rpt4+ by Error-prone PCR
<ol>
	<li>Perform error-prone PCR, using the plasmid with the silent mutation (Xho1 site) as the template, primers p5 and p6, a total volume of 50 &#181;L (the amount of template defined in step 4.1.1, 2 &#181;L of 10 pmol primer mix, 2.5 U enzyme, 1x reaction buffer), and a special polymerase that is designed to generate a high error rate19 (Figure 1D). Perform PCR as described in Table 2.
	<ol>
		<li>Use 4 - 5 &#181;g of template plasmid to control the mutation frequency to 1 bp/kb (kilobase). 
		Note: A high-concentration (&#62;500 ng/&#181;L) of plasmid, which may be obtained using a Mini-prep kit20, is required.</li>
	</ol>
	</li>
	<li>Use gel electrophoresis to confirm a PCR product of 2670 bp (1.2% agarose gel). Gel purify this PCR product as described in step 2.5.</li>
</ol>
5. Preparation of Fusion PCR Fragments (KAN, 3&#39;UTR)
<ol>
	<li>Perform PCR using pFA6a-KANMX621 as the template, primers p7 and p8, and the conditions listed in Table 4 to obtain the marker (KAN) fragment. Gel purify the resulting 1,480-bp fragment as described in step 2.5 (Figure 1E).
	<ol>
		<li>Check if the stop codon of the gene targeted for mutation and the coding region of its adjacent gene are separated by more than 500 bp. The endogenous terminator may not be used when this region is less than 500 bp; rather, the ADH terminator should be used instead of the endogenous terminator. The protocol changes required to use the ADH terminator are presented in Table 5. 
		Note: These changes only apply when the ADH terminator, which is available in the pFA6a-3HA-KANMX6 plasmid, is used instead of the endogenous terminator.</li>
	</ol>
	</li>
	<li>Perform PCR with the cloned rpt4+-containing vector as the template and primers p9 and p10 in a total volume of 50 &#181;L (20 ng of the cloned plasmid, 2 &#181;L of 10 pmol primer mix, 2 U enzyme, 1x reaction buffer), using the conditions presented in Table 6. Gel purify the obtained 506-bp 3&#39;UTR fragment (Figure 1E).</li>
</ol>
6. Generation of the Transformation Construct by Fusion PCR (Figure 1E)
<ol>
	<li>Prepare the 3 fragments (mutagenized rpt4+, KAN, and the 3&#39;UTR) at 50 ng/&#181;L.</li>
	<li>Perform fusion PCR of the three fragments using primers p11 and p12, as described in Table 7. (The protocol for fusion PCR is a modification of the original protocol22.) Purify the major 4,398-bp PCR product.
	<ol>
		<li>Use rpt4+:KAN:3&#39;UTR fragments at a ratio of 1:3:1 for optimal results. 
		Note: The total amount of the insert DNA should not exceed 500 ng. The recommended amount of rpt4+:KAN:3&#39;UTR is 50 ng:150 ng:50 ng. The mutagenized region is approximately 1.6 kb, so the minimum number of yeast colonies that would account for all possible combinations of each base being mutated to the other three is 1,600 x 3 = 4,800 colonies. Because one transformation reaction typically yields 400 - 500 colonies, at least 10 reactions worth of fusion PCR construct is needed. This need can be met by simply increasing the reaction amount (i.e., the number of PCR tubes) by a factor of ten.</li>
	</ol>
	</li>
</ol>
7. Transformation of Fission Yeast by Electroporation (Figure 1F)
<ol>
	<li>Inoculate yeast to 10 mL of YES medium to saturation by incubating it for more than 16 h.</li>
	<li>Dilute the cells to an optical density at 600 nm (OD600) of 0.2 in 200 mL of YES medium and incubate at 30 &#176;C with shaking for 5 - 6 h, to OD600 = 0.6 - 0.8.</li>
	<li>Concentrate cells to OD600 = 30, dispense to four 50-mL conical tubes and chill on ice for 10 min.</li>
	<li>Harvest the cells by performing centrifugation at 1,050 x g for 3 min at 4 &#176;C. Place the tubes and 10 electro-cuvettes on ice. Perform the steps 7.3 - 7.8 on ice.</li>
	<li>Discard the supernatant and add 15 mL of 1.2 M sorbitol. Gently shake the tubes to resuspend the cells.</li>
	<li>Centrifuge the cells at 1050 x g for 3 min at 4 &#176;C.</li>
	<li>Repeat steps 7.4 and 7.5. Discard the supernatant. Add 500 &#181;L of 1.2 M sorbitol to each tube, resuspend the cells, and collect all of the cells in one 15-ml conical tube.</li>
	<li>Add 1.2 M sorbitol up to 2.4 mL (OD600 = 10 per 0.2 mL). Keep the tube on ice.</li>
	<li>Add 200 &#181;L of sorbitol-suspended cells (make sure they are fully suspended) to an EP tube containing the fusion PCR construct, mix well, and transfer the sample to an electro-cuvette.</li>
	<li>Electroporate the cells with the following options: Fungi, ShS (2.00kV, 1 pulse).</li>
	<li>Add 600 &#181;L of 1.2 M sorbitol to each electro-cuvette for a total volume of 800 &#181;L, and then spread the cells on four YES plates (200 &#181;L/plate). Ten electro-cuvettes will dispense to 40 YES plates.</li>
	<li>Incubate the plates at 30 &#176;C for 24 h.</li>
	<li>Perform replica plating to YES+G418 and incubate the plates at 30 &#176;C for 3 days.</li>
</ol>
8. Selection of Heterochromatin-destabilizing Mutants and Checking for False Positives
<ol>
	<li>For each YES+G418 plate, perform replica plating to YES-Ade (Low Ade) and PMG-Ade (No Ade) plates. Incubate the replica plates for 1 - 2 days at 30 &#176;C, until some of the colonies on the YES-Ade plates show a red coloration (Figure 1G).</li>
	<li>Compare YES-Ade and PMG-Ade plates and select cells that show pink or white on the YES-Ade plate and also grow on the PMG-Ade plate. Do not select colonies without growth on PMG-Ade, as they are false positives (e.g., reflecting that the KAN cassette has been integrated at a non-target site elsewhere in the genome).</li>
	<li>Pick approximately 1 x 105 cells from each colony and incubate in 10 &#181;L of SPZ solution containing 2.5 mg/mL zymolyase 100 T at 37 &#176;C for at least 30 min. Use 1 &#181;L of this solution to perform as the starting template for colony PCR (Figure 1H).
	<ol>
		<li>Make SPZ (50 mL) by mixing 30 mL of 2 M sorbitol, 4.05 mL of 1 M Na2HPO4, 0.95 mL NaH2PO4 and 15 mL of distilled water (final pH, 7.5). Samples (5 mL) can be supplemented with zymolyase and stored in 500-&#181;L aliquots at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Perform gel electrophoresis (1.2% agarose gel, 180V, 20 min) to visualize the results of +the colony PCR. Select reactions that exhibit PCR bands of the proper size and digest 5 &#181;L of each reaction product with XhoI (4 U enzyme, 1x digestion buffer, 37 &#176;C water bath, 1 h) to screen out false positives (Figure 1H).</li>
	<li>Perform gel electrophoresis to visualize the XhoI digests (1.2% Agarose gel, 180 V, 20 min). Mark the colonies whose PCR products are cut by XhoI, and patch them to YES+G418 plates (Figure 1I).</li>
	<li>Isolate gDNA from the fission yeast cells and perform sequencing of the PCR products23. Compare the obtained sequence with the wild-type sequence to identify mutations (Figure 1I).</li>
	<li>Directly re-introduce the mutation(s) to wild-type cells by transforming them with fusion constructs amplified by PCR from mutant cells23. Use spotting to confirm the phenotype in the newly made mutant cells (Figure 1J).
	<ol>
		<li>Fusion transformation construct can be easily amplified by PCR with primers p1 and p10 using the genomic DNA of mutants as the template in a total volume of 50 &#181;L (~1 &#181;g DNA, 20 U enzyme, 1x reaction buffer), and applying the reaction cycles described in Table 2. Transformation of the construct can be done by following the steps 7.1 - 7.13.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Random mutagenesis via error-prone PCR is a powerful tool for generating a diverse pool of mutants in a given region. This technique is especially useful for studies that seek to assess the function of a protein under a specific circumstance. For example, we herein used error-prone PCR to assess the function of the 19S proteasome subunit, Rpt4, in heterochromatin maintenance. By varying the region targeted by the error-prone PCR and adjusting the screening conditions, we were able to mutate cells at the genomic region of...

Forward genetics is a classical method in which researchers seek naturally occurring mutants that display a particular phenotype, and perform genetic analyses. In reverse genetics, mutations are introduced into a gene of interest and the phenotype is examined. In the latter case, random mutagenesis of a target gene is often used to generate a pool of mutants that are subsequently selected for desired phenotypes, such as temperature sensitivity or altered enzymatic activity. Various methods may be used to achieve random mutagenesis, including error-prone PCR1; UV irradiation2; chemical mutagens, such as ethyl methanesulfonate (EMS) or nitrous acid3; the use of temporary mutator strains, such as those over-expressing mutD5 4; and DNA shuffling5.
Here, we describe a reverse-genetics strategy in which we utilize error-prone PCR to generate mutant pools for a target gene in the fission yeast. As one might guess from its name, this method generates mutations by deliberately introducing errors during PCR. Unlike other mutagenesis methods, error-prone PCR allows the user to define the region to be mutagenized. This makes it particularly useful in efforts to study the function of a protein/domain of interest.
To demonstrate this random mutagenesis procedure, we herein use rpt4+, which encodes the 19S proteasome subunit, as an example. Rpt4 has been shown to have proteolysis-independent functions in organisms other than fission yeast6,7,8,9, and a defect in proteolysis could cause indirect effects by altering proteins levels. We, therefore, screened for mutants that triggered proteolysis-independent changes, with the goal of investigating the function of the proteasome on heterochromatin.
Error-prone PCR can be applied to any gene region by tuning the location at which the primers bind. Mutants that exhibit the desired phenotypic change can be identified with appropriate reporters. Here, we utilized an ade6+ reporter inserted at the centromere 1 outer repeat (otr) region10. Constitutive heterochromatin is formed at this region11, so the ade6+ reporter is silenced in the wild-type condition; this is indicated by red colonies10. A mutation that destabilizes the constitutive heterochromatin at the centromere will lead to the expression of the ade6+ reporter, which is visualized as white colonies.

<table><tbody><tr><td>&lt;strong&gt;1. Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270-10KG</td><td></td></tr><tr><td>Yeast extract</td><td>BD Biosciences</td><td>212720</td><td></td></tr><tr><td>L-Leucine</td><td>JUNSEI</td><td>87070-0310</td><td></td></tr><tr><td>Adenine sulfate</td><td>ACR</td><td>16363-0250</td><td></td></tr><tr><td>Uracil&amp;nbsp;</td><td>Sigma-Aldrich</td><td>U0750</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma-Aldrich</td><td>H8125</td><td></td></tr><tr><td>KH2PO4</td><td>Sigma-Aldrich</td><td>1.05108.0050</td><td></td></tr><tr><td>NaCl</td><td>JUNSEI</td><td>19015-0350</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>63140</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>12095</td><td></td></tr><tr><td>Potassium phthalate</td><td>Sigma-Aldrich</td><td>P6758-500g</td><td></td></tr><tr><td>Inositol</td><td>Sigma-Aldrich</td><td>I7508-50G</td><td></td></tr><tr><td>Biotin</td><td>Sigma-Aldrich</td><td>B4501-100MG</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B6768-1KG</td><td></td></tr><tr><td>MnSO4</td><td>Sigma-Aldrich</td><td>M7634-500G</td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>JUNSEI</td><td>83060-031</td><td></td></tr><tr><td>FeCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>KANTO</td><td>CB8943686</td><td></td></tr><tr><td>Sodium molybdate dihydrate</td><td>YAKURI</td><td>31621</td><td></td></tr><tr><td>KI</td><td>JUNSEI</td><td>80090-0301</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>YAKURI</td><td>09605</td><td></td></tr><tr><td>D-myo-inositol</td><td>MP biomedicals</td><td>102052</td><td></td></tr><tr><td>Pantothenate</td><td>YAKURI</td><td>26003</td><td></td></tr><tr><td>Nicotinic Acid</td><td>Sigma-Aldrich</td><td>N4126-500G</td><td></td></tr><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A4418-100G</td><td></td></tr><tr><td>Agarose</td><td>Biobasic</td><td>D0012</td><td></td></tr><tr><td>G418, geneticin</td><td>LPS</td><td>G41805</td><td></td></tr><tr><td>&lt;strong&gt;2. Enzyme reactions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PfuUltra II Fusion HS DNA Polymerase</td><td>Aglient</td><td>600380</td><td>For site-directed mutagenesis</td></tr><tr><td>GeneMorphII Random mutagenesis Kit</td><td>Aglient</td><td>200500</td><td>For error-prone PCR</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Fisher</td><td>F-530L</td><td>For fusion PCR</td></tr><tr><td>Ex Taq&amp;nbsp;DNA Polymerase</td><td>Takara</td><td>RR001b</td><td>For general PCR</td></tr><tr><td>BamH1</td><td>New England BioLabs</td><td>R0136S</td><td></td></tr><tr><td>Xho1</td><td>New England BioLabs</td><td>R0146S</td><td></td></tr><tr><td>Dpn1</td><td>New England BioLabs</td><td>R0176S</td><td></td></tr><tr><td>&lt;strong&gt;3. Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Velveteen square, black</td><td>VWR</td><td>89033-116</td><td>For replica</td></tr><tr><td>Replica-plating tool</td><td>VWR</td><td>25395-380</td><td>For replica</td></tr><tr><td>MicroPulser Electroporator</td><td>Biorad</td><td>1652100&amp;nbsp;</td><td>For fission yeast transformation</td></tr><tr><td>Electroporation Cuvettes, 0.2 cm gap</td><td>Biorad</td><td>1652086&amp;nbsp;</td><td>For fission yeast transformation</td></tr><tr><td>Thermal Cycler</td><td>Bioer</td><td>BYQ6078</td><td>For fusion PCR ramp reaction&amp;nbsp;</td></tr></tbody></table>

gene-targeted random mutagenesis to select heterochromatin-destabilizing proteasome mutants in fission yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

The acquired Rpt4 mutants by following the procedures described in Figure 1 can be analyzed by assessing the colors of the colonies. The colors of the colonies are spotted onto the relevant plates in decreasing cell number in Figure 2. The ade6+ reporter inserted at the heterochromatin region is silenced in wild-type and shows red colonies in YES-Ade plate. Once the heterochromatin is destabilized and the ade6+ ...

Watch this Scientific Journal Video about Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast at JoVE.com

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

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Research

JoVE Journal

Genetics

10.6K Views.  Korea Advanced Institute of Science and Technology. Dit artikel beschrijft een gedetailleerde methodologie voor willekeurige mutagenese van een target-gen in kernsplijting gist. Als voorbeeld, wij de doelstelling van rpt4 +, die codeert een subeenheid van de 19S proteasoom, en het scherm voor mutaties die heterochromatin te destabiliseren.

Video: Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

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 Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Biology

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

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.

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

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