我们感谢他们协助制定检测用于本文，以及审阅者提出深思熟虑的意见 Sohini Chakrabortee、 桑德拉 · 琼斯、 大卫 · 加西亚、 Bhupinder Bhullar、 阿米莉亚昌、 理查德 · 她和苏珊林德奎斯特。

1. Initial Overexpression
<ol>
	<li>Transform the yeast cells (in this case, BY4741 MAT a haploids) previously grown in YPD liquid (10 g of yeast extract, 20 g of peptone, and 20 g of glucose per 1 L) with the desired candidate constructs from the FLEXGene ORFeome library (yeast ORFs under the control of a galactose-inducible promoter in the URA3-marked centromericplasmid backbone, pBY01129).</li>
	<li>Use autoclaved toothpicks to pick four separate colonies from these transformations to serve as biological replicates. Grow them for 48-72 h in 150 &#181;L of SRaffinose-URA (0.74 g of CSM-URA, 6.7 g of yeast nitrogen base without amino acids, and 20 g of raffinose per 1 L) in 96-well plates. Inoculate colonies from the first ORF in well A1 of all plates, colonies from the second ORF in A2 of all plates, etc. 
	NOTE: All growth steps are conducted at 30 &#176;C and at atmospheric levels of CO2 (407.05 ppm), unless stated otherwise.</li>
	<li>Confirm that the cultures are saturated (cells visible by eye at the bottom of each well) and use a liquid handling robot to inoculate a 1:4 array of 1 &#181;L from each 96-well plate well into 4 separate wells of a 384-well plate filled with 45 &#181;L of SGal-URA (0.74 g of CSM-URA, 6.7 g of yeast nitrogen base without amino acids, and 20 g of galactose per 1 L) per well. 
	NOTE: This creates a composite plate in which 4 biological replicates of each ORF are arrayed in a square pattern.</li>
	<li>At the same time, prepare separate 384-well plates containing 45 &#181;L of SGal-URA with the stressors of interest (e.g., manganese chloride at 20 mM) per well. Inoculate this plate in the same manner as in step 1.3. 
	NOTE: To achieve the greatest dynamic range for phenotypic detection, a 2-fold dilution series around the LD50 concentration of a given stressor is recommended. This can be done beforehand to determine the optimum concentration at which to observe both enhanced and reduced growth.</li>
	<li>As a final parallel control, inoculate a third 384-well plate in the same manner, with the same stressor included in medium that will not induce plasmid expression. Ensure that the plate contains 45 &#181;L of SD-URA (0.74 g of CSM-URA, 6.7 g of yeast nitrogen base without amino acids, and 20 g of glucose per 1 L) per well and is inoculated as in steps 1.3 and 1.4.</li>
	<li>Immediately place plates with cells on a microplate stacker and set the protocol for a 72-h continuous loop, measuring the OD600 with a microplate reader equipped with stackers at room temperature and atmospheric CO2 (407.05 ppm). 
	Note: The frequency of the growth measurement will depend on the number of plates (ultimately determined by the number of genes and conditions the investigator wants to probe) used in the run. The more plates used, the longer it will take the plate reader to complete a loop and thus the longer time between measurements (measurement time per plate is ~45 s, depending on the instrumentation employed).</li>
	<li>After the growth measurements, use the same liquid handling robot to transfer 1 &#181;L per well of the SGal-URA-induced cultures (i.e., those that experienced protein overexpression) to new 384-well plates containing 45 &#181;L of SD-URA per well (medium that does not permit protein expression of the plasmid).
	<ol>
		<li>In parallel, perform analogous inoculations of a second set of 384-well plates containing 45 &#181;L of SD-URA per well from the cultures that were grown in SD-URA in the presence of the stressors (i.e., those from step 1.5).</li>
		<li>Grow the plates for 48 h at 30 &#176;C to saturation in a humidified chamber (i.e., a sealable plastic bin with a damp paper towel inside).</li>
	</ol>
	</li>
	<li>Take the plates from the previous step and reinoculate 1 &#181;L per well in 384-well plates containing 45 &#181;L of SD-URA. Then, perform a separate reinoculation of 1 &#181;L per well from the same source plate into a separate 384-well plate containing 45 &#181;L of SD-URA with the stressor. 
	NOTE: This will determine if the sensitivity/resistance phenotypes to a stressor persist after the protein overexpression has ceased.</li>
	<li>Immediately place plates with cells on a microplate stacker and set the protocol for a 48-h continuous loop measuring the OD600 with a microplate reader at room temperature and atmospheric CO2 (407.05 ppm).</li>
	<li>Export the time versus OD600 measurements as an XY table using the plate reader software. Group columns of the OD600 for each biological replicate together and calculate the mean. Create an XY plot of time versus OD600 to generate growth curves.
	<ol>
		<li>Compare the growth rates of cultures that had been subjected to a past overexpression (i.e., those originally induced in galactose-containing medium before reinoculation in SD-URA) versus cultures that had not (i.e., those propagated in glucose), as described previously30.</li>
	</ol>
	</li>
	<li>For cultures that show significant growth differences in response to a given stressor, dependent upon ancestral protein overexpression, take 1 &#181;L from each biological replicate, dilute it in 10 mL of water, and then plate 50 &#181;L on plates containing 5-FOA (0.74 g of CSM-URA, 1 g of 5-FOA, 50 mg of uracil, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per liter). Grow at 30 &#176;C for 3 days.
	<ol>
		<li>If this results in too many or too few colonies per plate, adjust the dilution factor accordingly. 
		Note: 5-FOA is converted into a toxic intermediate by the uracil biosynthesis pathway. This will cause a loss of the URA3-marked expression plasmid transformed in the first step.</li>
	</ol>
	</li>
	<li>Pick 8-32 single colonies with autoclaved toothpicks and pin them to 96-well plates containing 150 &#181;L of SD-CSM. Grow to saturation for 48-72 h in a humidified chamber at 30 &#176;C. Use these cultures to inoculate two new sets of 96-well plates containing 150 &#181;L of SD-CSM, both with and without the stressors from step 1.11.</li>
	<li>Place the plates on a microplate stacker and set the protocol to measure the OD600 on a 48-h continuous loop.</li>
	<li>Analyze the data as in step 1.10 and confirm that the significant growth differences seen in step 1.10 are maintained after plasmid loss. 
	NOTE: These cells harbor stable phenotypic states that might be indicative of protein-based inheritance.</li>
	<li>Test the cells that maintained the induced phenotypes for classic hallmarks of protein-based inheritance (see below).</li>
</ol>
2. Tests for Prion-like Inheritance
<ol>
	<li>Chaperone-mediated &#34;curing&#34;
	<ol>
		<li>Test Hsp104 chaperone dependence.
		<ol>
			<li>Pick a colony of a yeast strain harboring a stable phenotypic state, streak to single colonies on a YPD plate (10 g of yeast extract, 20 g of peptone, 20 g of glucose, and 20 g of agar per 1 L) containing 3 mM GdnHCl and grow at 30 &#176;C for 3 days. In parallel, streak out a colony from a na&#239;ve strain to single colonies on YPD+ 3 mM GdnHCl as a control in the same manner.</li>
			<li>Repeat 2 more times on fresh YPD plates containing 3 mM GdnHCl.</li>
			<li>Because GdnHCl exposure can increase the frequency at which petite cells arise, pick multiple colonies that have undergone 3 passages on GdnHCl and check for functional mitochondrial respiration by examining their ability to grow into visible colonies on YP-Glycerol plates (10 g of yeast extract, 20 g of peptone, 20 mL of glycerol, and 20 g of agar per 1 L) after 7 days.</li>
			<li>Pick multiple colonies from the YP-Glycerol plates and test for the maintenance of stable phenotypic states using growth in the presence of the stressor compared to non-&#34;cured&#34; and na&#239;ve (i.e., isogenic cells that do not harbor the phenotypic state, such as BY4741 MATa haploids) controls in a similar manner as described in step 1.13.</li>
		</ol>
		</li>
		<li>Test Hsp70 chaperone dependence.
		<ol>
			<li>&#34;Cure&#34; via a plasmid.
			<ol>
				<li>Transform both na&#239;ve cells and cells harboring stable phenotypic states with URA3-marked plasmids expressing a dominant negative allele of Hsp70 (K69M)25,31 from a strong constitutive promoter (GPD).</li>
				<li>Pick single transformants for each, streak to single colonies on an SD-URA plate (0.74 g of CSM-URA, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per 1 L), and grow for 3 days at 30 &#176;C and atmospheric CO2.</li>
				<li>Repeat this passaging 2 more times on SD-URA plates.</li>
				<li>Pick multiple colonies and streak to single colonies on 5-FOA plates to eliminate the plasmid.</li>
				<li>Pick multiple single colonies for each isolate and test for retention (or loss) of the stable phenotypic states in a similar manner as described in step 1.13.</li>
			</ol>
			</li>
			<li>&#34;Cure&#34; via genetic crossing.
			<ol>
				<li>Cross BY4741 MATa haploid strains harboring stable phenotypic states and na&#239;ve strains of the opposite mating type (in this case, BY4742 MAT&#945;) harboring genetic deletions in two of the four yeast Hsp70 paralogs (ssa1&#916; ssa2&#916;). 
				NOTE: This strain is deficient in Hsp70 chaperone function.</li>
				<li>Select for the growth of diploids by streaking to single colonies on SD-LYS-MET (0.74 g of CSM-LYS-MET, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per 1 L) double-dropout medium.</li>
				<li>Pick single diploid colonies and grow for 24 h at 30 &#176;C in 8 mL of pre-sporulation medium (0.8% yeast extract, 0.3% peptone, 10% dextrose, and 100 mg/L adenine sulfate).</li>
				<li>Spin down the cultures for 3 min at 3,000 xg, aspirate the pre-sporulation (pre-SPO) medium, and wash the pellets once with sterile water.</li>
				<li>Re-suspend the pellets in 2 mL of sporulation medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose, and 0.01% amino acid add-back mix (2 g of histidine, 10 g of leucine, 2 g of lysine, and 2 g of uracil)). Grow at 25&#176;C for 5 days.</li>
				<li>Move the cultures to 30 &#176;C and incubate for an additional 48 h.</li>
				<li>Assess the efficiency of sporulation by looking for the presence of tetrads using a light microscope32.</li>
				<li>Take 40 &#181;L of culture, spin down for 1 min at 3,000 xg in a 1.5-mL tube, and re-suspend in an equal volume of zymolyase enzyme mix (1 M sorbitol, 0.1 M EDTA, and 10 mg/mL zymolyase 100T)</li>
				<li>Incubate at 25 &#176;C for 5 min.</li>
				<li>Apply 10 &#181;L of digested culture to a very dry, very level YPD agar plate and allow the culture to slowly spread down the plate in a straight line.</li>
				<li>Allow the plates to dry for at least 30 min in a laminar flow hood.</li>
				<li>Using a dissection microscope, separate the spores into tetrads and array them on the plate.</li>
				<li>Allow the individual haploid spores to grow into colonies and test for their ability to grow on SD-HIS-LEU (0.67 g of CSM-HIS-LEU, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per 1L) plates, indicating that they harbor both genetic deletions (ssa1&#916; ssa2&#916;).</li>
				<li>Test each for the maintenance of the phenotypic state in the Hsp70-deficient background in a similar manner as described in step 1.13. 
				NOTE: Crossing these spores back to a wildtype strain with restored Hsp70 function should not restore the prion phenotype.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Testing for non-Mendelian inheritance.
	<ol>
		<li>Cross BY4741 MATa haploid strains harboring stable phenotypic states and isogenic na&#239;ve controls to an isogenic na&#239;ve strain of the opposite mating type (in this case, BY4742 MAT&#945;).</li>
		<li>Select for the growth of diploids by streaking to single colonies on SD-LYS-MET double-dropout medium.</li>
		<li>Pick single diploid colonies and grow for 24 h at 30 &#176;C in 8 mL of pre-sporulation medium.</li>
		<li>Repeat steps 2.2.2.3-2.2.2.12, as described above.</li>
		<li>Allow the individual haploid spores to grow into colonies and test each for maintenance of the phenotypic state through meiosis using the growth assay, as in step 1.13. 
		Note: Another defining feature of prions is that they can be heritably eliminated through the removal of the original casual protein. Thus, analogously crossing a prion strain with a strain harboring a genetic deletion of the original induced protein will abrogate prion phenotypes in the spores that inherit the deletion. Also note that is possible for the prion to be maintained in a &#34;cryptic&#34; state in such a mutant if the deleted gene is required to manifest the prion phenotype but not to propagate the prion itself. To distinguish between these two possibilities, cross the spores back to a na&#239;ve wildtype strain and test whether the prion phenotype re-emerges in a diploid genetic background, where the function of the gene has been restored.</li>
	</ol>
	</li>
	<li>Cytoduction.
	<ol>
		<li>Generate the initial BY4742 recipient strain.
		<ol>
			<li>Through transformation, introduce a defective KAR allele (kar1-15) that prevents nuclear fusion during mating23.</li>
			<li>Make this strain &#34;petite&#34; (i.e., incompetent for mitochondrial respiration) by inoculating a single colony in YPD broth with 0.25% ethidium bromide.</li>
			<li>Grow the culture at 30 &#176;C until late exponential/stationary phase (OD600 ~1).</li>
			<li>Dilute 1:1,000 in fresh YPD with 0.25% ethidium bromide and repeat twice.</li>
			<li>Once the culture reaches late exponential/early stationary phase (OD600 0.8-1.2), plate to single colonies by diluting 1:10,000 in sterile water and plating 50 &#181;L on a YPD plate. After growing for 3 days at 30 &#176;C, pick multiple colonies and test each for respiration incompetence by examining their ability to grow into visible colonies on YP-Glycerol plates after 7 days.</li>
		</ol>
		</li>
		<li>Perform initial cytoduction into BY4742.
		<ol>
			<li>Mix cells of the donor BY4741 strain harboring a stable phenotypic state with cells of the na&#239;ve BY4742 kar1-15 recipient strain on the surface of a YPD agar plate.</li>
			<li>Grow for 24 h at 30 &#176;C and atmospheric CO2 (407.05 ppm) and transfer to methionine dropout medium containing glycerol (SGly-MET: 0.74 g of CSM-MET, 6.7 g of yeast nitrogen base without amino acids, 20 mL of glycerol, and 20 g of agar per 1 L). 
			NOTE: This selects for both BY4742 nuclear markers, along with allowing the restoration of functional mitochondria via cytoplasmic exchange.</li>
			<li>After 3-5 days, pick multiple single colonies and perform another round of selection on SD-MET (0.74 g of CSM-MET, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per 1 L).</li>
			<li>In parallel, confirm that the colonies are not diploids by passaging on SD-LYS-MET medium.</li>
		</ol>
		</li>
		<li>Perform reverse cytoductions.
		<ol>
			<li>Mix the new donor strains (this time, the successful BY4742 kar1-15 cytoductants) with petite na&#239;ve BY4741 cells (generated as above) on YPD agar.</li>
			<li>Repeat cytoductions, as described previously (steps 2.3.2.1-2.3.2.4), except selecting for BY4741 recipient nuclear markers on glycerol medium lacking lysine (SGly-LYS: 0.74 g of CSM-LYS, 6.7 g of yeast nitrogen base without amino acids, 20 mL of glycerol, and 20 g of agar per 1 L).</li>
			<li>Pick multiple cytoductants and test for the maintenance of stable phenotypic states using the growth assay, as before in step 1.13.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Protein transformation.
	<ol>
		<li>Prepare the lysate.
		<ol>
			<li>Grow 50 mL of cells harboring stable phenotypic states in YPD for 18 h at 30 &#176;C.</li>
			<li>Pellet the cultures at 3,000 x g for 4 min and then wash twice: once in autoclaved H2O and then in 1 M sorbitol.</li>
			<li>Re-suspend the cells in 200 mL of SCE buffer (1 M sorbitol, 10 mM EDTA, 10 mM DTT, 100 mM sodium citrate, and 1 mini-EDTA-free protease inhibitor tablet per 50 mL, pH 5.8) with 50 U/mL of yeast lytic enzyme 100T.</li>
			<li>Incubate the mix at 35 &#176;C for 30 min.</li>
			<li>Sonicate the cells at 20 kHz and 20% intensity for 10 s on ice with a sonic dismembrator.</li>
			<li>Remove the cell debris by centrifugation at 10,000 x g for 15 min at 4 &#176;C.</li>
			<li>Move the supernatants to a clean tube and add RNase I and biotinylated DNase at a 3-fold excess (as determined by units of activity) and incubate at 37 &#176;C for 1 h.</li>
			<li>Remove the DNase I by adding excess streptavidin-agarose beads, incubating for 5 min, and pelleting by centrifugation at 10,000 x g for 1.5 min.</li>
			<li>Transfer the supernatant to a fresh tube.</li>
		</ol>
		</li>
		<li>Prepare the recipient spheroplast.
		<ol>
			<li>Grow 5 mL of na&#239;ve recipient cells in YPD to mid-exponential phase (OD600 ranging from 0.5-1).</li>
			<li>Harvest by centrifugation (3,000 x g for 4 min) and wash 4 times: twice in H2O and twice in 1 M sorbitol.</li>
			<li>Re-suspend the cells in 1 M sorbitol containing 200 U/mL yeast lytic enzyme and incubate for 15 min at 35 &#176;C to digest the cell walls.</li>
			<li>Harvest the resulting spheroplasts with gentle centrifugation at 600 x g for 5 min and wash with 1 mL of 1 M sorbitol and then again with 1 mL of STC buffer (1 M sorbitol, 10 mM Tris pH 7.5, and 10 mM CaCl2).</li>
			<li>Resuspend the washed spheroplasts in 50 &#181;L of STC buffer. 
			NOTE: Use scissors to cut off the last ~0.7 cm of a pipette tip; this will create a wider mouth and prevent the lysis of the spheroplasts.</li>
		</ol>
		</li>
		<li>Transform the na&#239;ve cells with protein.
		<ol>
			<li>Transform 50 &#181;L of spheroplast aliquots with 50 &#181;L of donor lysate, 20 &#181;L of salmon sperm DNA (2 mg/mL), and 5 &#181;L of a URA3-marked selection plasmid (e.g., pAG426-GFP)28,33.</li>
			<li>Incubate the mix at 25 &#176;C for 30 min.</li>
			<li>Centrifuge for 5 min at 600 x g to collect the spheroplasts and re-suspend in 150 &#181;L of SOS-buffer (1 M sorbitol, 7 mM CaCl2, 0.25% yeast extract, and 0.5% peptone).</li>
			<li>Recover for 30 min at 30 &#176;C.</li>
			<li>Plate the entire culture on SD-URA plates.</li>
			<li>Quickly overlay the plated culture with warm (~45 &#176;C) SD-URA containing 0.8% agar. 
			Note: This will prevent the spheroplasts from bursting</li>
			<li>Incubate the transformation plates for 2-3 days at 30 &#176;C.</li>
			<li>Using a toothpick, pick successfully growing cells from the overlaid plate and re-streak to single colonies on SD-URA plates.</li>
			<li>Pick dozens of colonies for each and streak on 5-FOA plates to eliminate URA3-marked carrier plasmid.</li>
			<li>Pick colonies that grow on the 5-FOA plates and test for the transfer of stable phenotypic states using the growth assay, as before (step 1.13).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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<li>Chernoff, Y. O., Derkach, I. L., Inge-Vechtomov, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multicopy+SUP35+gene+induces+de-novo+appearance+of+psi-like+factors+in+the+yeast+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Curr+Genet%22%5BJournal%5D)">Multicopy SUP35 gene induces de-novo appearance of psi-like factors in the yeast Saccharomyces cerevisiae.</a> Curr Genet. 24 (3), 268-270 (1993).</li>
<li>Brown, J. C., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+heritable+switch+in+carbon+source+utilization+driven+by+an+unusual+yeast+prion%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">A heritable switch in carbon source utilization driven by an unusual yeast prion.</a> Genes Dev. 23 (19), 2320-2332 (2009).</li>
<li>Coustou, V., Deleu, C., Saupe, S., Begueret, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+protein+product+of+the+het-s+heterokaryon+incompatibility+gene+of+the+fungus+Podospora+anserina+behaves+as+a+prion+analog%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog.</a> Proc Natl Acad Sci U S A. 94 (18), 9773-9778 (1997).</li>
<li>Sopko, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+pathways+and+phenotypes+by+systematic+gene+overexpression%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Mapping pathways and phenotypes by systematic gene overexpression.</a> Mol Cell. 21 (3), 319-330 (2006).</li>
<li>Chakrabortee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrinsically+Disordered+Proteins+Drive+Emergence+and+Inheritance+of+Biological+Traits%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Intrinsically Disordered Proteins Drive Emergence and Inheritance of Biological Traits.</a> Cell. 167 (2), 369-381 (2016).</li>
<li>Hu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Approaching+a+complete+repository+of+sequence-verified+protein-encoding+clones+for+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae.</a> Genome Res. 17 (4), 536-543 (2007).</li>
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<li>Jarosz, D. F., Lancaster, A. K., Brown, J. C., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+evolutionarily+conserved+prion-like+element+converts+wild+fungi+from+metabolic+specialists+to+generalists%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">An evolutionarily conserved prion-like element converts wild fungi from metabolic specialists to generalists.</a> Cell. 158 (5), 1072-1082 (2014).</li>
<li>Neiman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sporulation+in+the+budding+yeast+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Sporulation in the budding yeast Saccharomyces cerevisiae.</a> Genetics. 189 (3), 737-765 (2011).</li>
<li>Gietz, D., St Jean, A., Woods, R. A., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+method+for+high+efficiency+transformation+of+intact+yeast+cells%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Improved method for high efficiency transformation of intact yeast cells.</a> Nucleic Acids Res. 20 (6), 1425(1992).</li>
<li>Formosa, T., Nittis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppressors+of+the+temperature+sensitivity+of+DNA+polymerase+alpha+mutations+in+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Mol+Gen+Genet%22%5BJournal%5D)">Suppressors of the temperature sensitivity of DNA polymerase alpha mutations in Saccharomyces cerevisiae.</a> Mol Gen Genet. 257 (4), 461-468 (1998).</li>
<li>Christiano, R., Nagaraj, N., Frohlich, F., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+proteome+turnover+analyses+of+the+Yeasts+S.+cerevisiae+and+S.+pombe%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Global proteome turnover analyses of the Yeasts S. cerevisiae and S. pombe.</a> Cell Rep. 9 (5), 1959-1965 (2014).</li>
<li>Lancaster, A. K., Nutter-Upham, A., Lindquist, S., King, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PLAAC%3A+a+web+and+command-line+application+to+identify+proteins+with+prion-like+amino+acid+composition%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">PLAAC: a web and command-line application to identify proteins with prion-like amino acid composition.</a> Bioinformatics. 30 (17), 2501-2502 (2014).</li>
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<li>Shorter, J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prions+as+adaptive+conduits+of+memory+and+inheritance%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Prions as adaptive conduits of memory and inheritance.</a> Nat Rev Genet. 6 (6), 435-450 (2005).</li>
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</ol>

作者没有透露。

他们不寻常的表型和令人费解的继承模式，确定了第一酵母朊病毒。这些朊病毒特征被用于生成算法和计算工具，屏幕为额外的朊病毒蛋白。在这里描述的方法相反，是实验和依赖瞬态的过度表达，创造一个持久变化一稳定状态编码的蛋白质构象。然而，如果"播种"朊病毒大会由为任何给定的蛋白质表达的效率是很低，这种蛋白质会不断出现作为过度屏幕这种类型的假阴性。一个此类修改要更正?...

生物系统经常经历的瞬态波动蛋白质丰富。这些是否有持久的影响，在塑造的有机体或未来几代人的表型仍不清楚。这个生物学的著名实例涉及一类罕见的蛋白质，朊病毒，开车遗传性基因组原封不动的出现。相反，这些临的 teinaceous 和在发感染粒子传输通过自我延续的变化对蛋白质构象1，2的表型。这种类型的继承被发现不寻常的继承模式的破坏性的神经退行性疾病的原因。然而，在生物体从真菌到哺乳动物3，4，，56，7，8，，910研究以来发现朊病毒样元素可以赋予自适应值。尽管如此，朊病毒已被视为美丽的向往但罕见生物古怪。
这个普遍的智慧是部分举行因为表征蛋白质基础继承长期以来一直受一小套的示例。最近系统筛查努力扩大这张照片明显确定几个新的善意取得制度朊病毒11和几乎两打蛋白域12与燃料朊病毒样构象转化的能力。然而，因为这些方法一般集中在强氨基酸序列偏见，朊病毒被发现分享创始酵母朊病毒 [PSI+]13，14，[URE3]15和 [RNQ+]11，16的生化性质。这些措施包括: 1） 模块化域，富含长高分子绵延的天冬酰胺 (N) 和谷氨酰胺 (Q) 2） 组装成淀粉样 [朊病毒+] 构象17，，1819，和 3） 完成 disaggregase 忠实传播从母亲到女儿13，，2021Hsp104 函数依赖。事实上，许多善意朊病毒，包括 [噶尔+]，[Het-s]，和甚至原始朊蛋白 (PrPSc)，将错过下这样严格的标准。也许更重要的是，他们将无法捕获任何新的机制的蛋白质基础继承22。因此，真正的生物学宽度，这种现象可能是在自然比以前想象中更为常见。
为了调查这一问题，采用高通量蛋白质组全战略。所有的朊病毒，包括 PrPSc，[噶尔+]，标志和 [Het-s]，是瞬变的因果蛋白过度表达强烈增加的朊病毒采集15，23，，2425，26率。我们利用此功能，以便系统地问，跨整个酵母 ORFeome，如果稳定的蛋白质基础、 表观遗传状态可以由瞬态诱发个别蛋白质的表达。众所周知，蛋白过度表达可以改变表型27。然而朊病毒蛋白是不寻常的因为他们临时生产过剩产生许多数百代后的初始的过度表达是可遗传的表型变化。我们以前采取了利用此功能，以及基于蛋白质的遗传元件，以查明的蛋白质能够重新 heritably 接线表型景观而不改变基因组28几十件不寻常的继承模式。虽然有一些蛋白质被以前称为朊病毒，大多数没有，彰显了这种方法来发现新的基于蛋白质的继承形式。

<table><tbody><tr><td>Guanidine hydrochloride</td><td>Sigma</td><td>Cat#G3272-25G</td><td>Chemical</td></tr><tr><td>Manganese chloride</td><td>Sigma</td><td>Cat#M8054-100G</td><td>Chemical</td></tr><tr><td>Ethidium bromide</td><td>Sigma</td><td>E1510</td><td>Chemical</td></tr><tr><td>5-Fluoroorotic Acid</td><td>Sigma</td><td>Cat#F5013-50MG</td><td>Chemical</td></tr><tr><td>BY4741 &lt;em&gt;MATa (his3&amp;Delta;1 leu2&amp;Delta;0 LYS2 met15&amp;Delta;0 ura3&amp;Delta;0)&lt;/em&gt;</td><td>Winston et al., 1995; Brachmann et al., 1998</td><td>N/A</td><td>Yeast strain</td></tr><tr><td>BY4741 &lt;em&gt;MAT&amp;alpha; (his3&amp;Delta;1 leu2&amp;Delta;0 lys2&amp;Delta;0 MET15 ura3&amp;Delta;0)&lt;/em&gt;</td><td>Winston et al., 1995; Brachmann et al., 1998</td><td>N/A</td><td>Yeast strain</td></tr><tr><td>Hsp70 (K69M)&amp;nbsp;</td><td>Jarosz et al., 2014b</td><td>N/A</td><td>Plasmid</td></tr><tr><td>FLEXGene library</td><td>Hu et al., 2007</td><td>N/A</td><td>Plasmid library</td></tr><tr><td>Dextrose (glucose)</td><td>Fisher Scientific</td><td>D16-3</td><td>Media component</td></tr><tr><td>Raffinose</td><td>Sigma</td><td>R0250-25G</td><td>Media component</td></tr><tr><td>Galactose</td><td>Fisher Scientific</td><td>BP656-500</td><td>Media component</td></tr><tr><td>CSM</td><td>Sunrise Science</td><td>1001-100</td><td>Media component</td></tr><tr><td>CSM-URA</td><td>Sunrise Science</td><td>1004-100</td><td>Media component</td></tr><tr><td>CSM-LYS</td><td>Sunrise Science</td><td>1032-100</td><td>Media component</td></tr><tr><td>CSM-MET</td><td>Sunrise Science</td><td>1019-100</td><td>Media component</td></tr><tr><td>CSM-LYS-MET</td><td>Sunrise Science</td><td>1035-100</td><td>Media component</td></tr><tr><td>yeast extract</td><td>Fisher Scientific</td><td>BP1422-2</td><td>Media component</td></tr><tr><td>peptone</td><td>Research Products International</td><td>P20240-5000</td><td>Media component</td></tr><tr><td>bacto-peptone</td><td>BD</td><td>211677</td><td>Media component</td></tr><tr><td>glycerol</td><td>EMD Millipore</td><td>GX0185-2</td><td>Media component</td></tr><tr><td>yeast nitrogen base w/o amino acids</td><td>BD</td><td>291920</td><td>Media component</td></tr><tr><td>agar</td><td>IBI Scientific</td><td>IB49172</td><td>Media component</td></tr><tr><td>Adenine sulfate</td><td>Sigma</td><td>A3159-25G</td><td>Media component</td></tr><tr><td>Potassium acetate</td><td>Sigma</td><td>P1190-500G</td><td>Media component</td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td>Media component</td></tr><tr><td>Histidine</td><td>Sigma</td><td>H8000-100G</td><td>Media component</td></tr><tr><td>Leucine</td><td>Sigma</td><td>L8000-25G</td><td>Media component</td></tr><tr><td>Lysine</td><td>Sigma</td><td>L5501-25G</td><td>Media component</td></tr><tr><td>RNase I&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>EN0601</td><td>Enzyme</td></tr><tr><td>biotinylated DNase</td><td>Thermo Fisher Scientific</td><td>AM1906</td><td>Enzyme</td></tr><tr><td>zymolyase 100T (yeast lytic enzyme)</td><td>Sunrise Science</td><td>N0766555</td><td>Enzyme</td></tr><tr><td>Microplate reader</td><td>BioTek</td><td>Synergy H1</td><td>Equipment</td></tr><tr><td>Microplate stacker</td><td>BioTek</td><td>BioStack3</td><td>Equipment</td></tr><tr><td>Plate filler</td><td>BiotTek</td><td>EL406</td><td>Equipment</td></tr><tr><td>Liquid handling robot</td><td>Beckman Coulter</td><td>Biomek FX</td><td>Equipment</td></tr></tbody></table>

high-throughput screening for protein-based inheritance in s. cerevisiae

对后代是容易接近的生物信息的编码通常被通过 DNA 序列的变化。长寿遗传编码的蛋白质构象 （而不是序列） 长期以来被视为范式转移，但很少。这种表观遗传的元素的最佳的特征的示例包括朊病毒，拥有可以推动新的表型的遗传表现的自组装行为。许多原型朊病毒显示醒目的富含 Q 的 N 序列偏见和组装成淀粉样蛋白的折叠。这些不寻常的特征已通知筛选确定新的朊病毒蛋白的大多数努力。然而，至少三个已知的朊病毒 （包括创始的朊病毒，PrPSc） 不要怀有这些生化特性。因此，我们开发了一种替代的方法，探讨基于蛋白质的继承，基于属性的大规模行动的范围: 瞬态的朊病毒蛋白过度表达增加，他们获得自我模板构象的频率。本文介绍了一种分析酵母 ORFeome 能够引出基于蛋白质的继承方法。使用此策略，我们以前发现 &gt; 1%的酵母蛋白质能燃料出现的生物性状，寿命长，性能稳定，并比遗传突变更频繁地出现。这种方法可以从事高吞吐量，横跨整个 ORFeomes 或作为特定遗传网络或环境刺激有针对性的筛选模式。正如前的遗传屏幕定义许多发展和信号转导途径，这些技术提供一种方法来探讨基于蛋白质的继承在生物过程中的影响。

Protein overexpression is known to dramatically alter cellular phenotypes27. Indeed, with an initial screening approach, hundreds of new phenotypes were reproducibly recovered from the overexpression of clones from the yeast ORFeome using just ten stressors. However, the assays described above allow for the assessment of whether cells retain any long-term stable phenotypes following this overexpression. One protein capable of encoding such a state is Psp1. Psp1 is ...

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High-throughput Screening for Protein-based Inheritance in S. cerevisiae

此协议说明一种高通量方法来功能屏幕在酿酒酵母中的蛋白质基础继承。

基于蛋白质的继承在酿酒酵母中的高通量筛选

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

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High-throughput Screening for Protein-based Inheritance in S. cerevisiae

6.3K 次观看.  Stanford University School of Medicine. 此协议说明一种高通量方法来功能屏幕在酿酒酵母中的蛋白质基础继承。

视频: High-throughput Screening for Protein-based Inheritance in S. cerevisiae

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.

基于交配的酵母超表达文库筛选

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

科研

遗传学

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.

一种在裂变酵母中的深度固序辅助自发抑制屏

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 ...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

糖精中遗传链接调控时间寿命的抑制 器屏幕

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

生物学

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

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.

条形码酵母图书馆竞争力的基因屏幕

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

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

生物工程

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

定向进化法<em&gt;酿酒酵母</em&gt;:突变体库的创建和筛选

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

基于蛋白质的继承在酿酒酵母中的高通量筛选

摘要

探索更多视频

基于交配的酵母超表达文库筛选

一种在裂变酵母中的深度固序辅助自发抑制屏

糖精中遗传链接调控时间寿命的抑制器屏幕

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

高通量酵母质粒过表达屏幕

条形码酵母图书馆竞争力的基因屏幕

高通量酵母菌株，带基于滴滴的RNA测序

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

化学遗传相互作用的快速鉴定

定向进化法

基于蛋白质的继承在酿酒酵母中的高通量筛选

摘要

探索更多视频

基于交配的酵母超表达文库筛选

一种在裂变酵母中的深度固序辅助自发抑制屏

糖精中遗传链接调控时间寿命的抑制 器屏幕

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

高通量酵母质粒过表达屏幕

条形码酵母图书馆竞争力的基因屏幕

高通量酵母菌株，带基于滴滴的RNA测序

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

化学遗传相互作用的快速鉴定

定向进化法

糖精中遗传链接调控时间寿命的抑制器屏幕