我々 は、彼らの思慮深いコメントの校閲者と同様に、本稿で使用される試金の開発に彼らの支援のため Sohini Chakrabortee、サンドラ ・ ジョーンズ、ダビド ガルシア、Bhupinder Bhullar、アメリア チャン、彼女はリチャードとスーザン ・ リンドキストをありがちましょう。

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>

<ol>
	<li>Halfmann, R., Alberti, S., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions,+protein+homeostasis,+and+phenotypic+diversity.">Prions, protein homeostasis, and phenotypic diversity.</a> Trends Cell Biol. 20 (3), 125-133 (2010).</li><li>Byers, J. S., Jarosz, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pernicious+pathogens+or+expedient+elements+of+inheritance:+the+significance+of+yeast+prions.">Pernicious pathogens or expedient elements of inheritance: the significance of yeast prions.</a> PLoS Pathog. 10 (4), 1003992 (2014).</li><li>Jarosz, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-kingdom+chemical+communication+drives+a+heritable,+mutually+beneficial+prion-based+transformation+of+metabolism.">Cross-kingdom chemical communication drives a heritable, mutually beneficial prion-based transformation of metabolism.</a> Cell. 158 (5), 1083-1093 (2014).</li><li>True, H. L., Lindquist, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+prion+provides+a+mechanism+for+genetic+variation+and+phenotypic+diversity.">A yeast prion provides a mechanism for genetic variation and phenotypic diversity.</a> Nature. 407 (6803), 477-483 (2000).</li><li>Suzuki, G., Shimazu, N., Tanaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+prion,+Mod5,+promotes+acquired+drug+resistance+and+cell+survival+under+environmental+stress.">A yeast prion, Mod5, promotes acquired drug resistance and cell survival under environmental stress.</a> Science. 336 (6079), 355-359 (2012).</li><li>Hou, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAVS+forms+functional+prion-like+aggregates+to+activate+and+propagate+antiviral+innate+immune+response.">MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.</a> Cell. 146 (3), 448-461 (2011).</li><li>Cai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+polymerization+underlies+signal+transduction+in+antiviral+immune+defense+and+inflammasome+activation.">Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation.</a> Cell. 156 (6), 1207-1222 (2014).</li><li>Majumdar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+amyloid-like+oligomers+of+Drosophila+Orb2+in+the+persistence+of+memory.">Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory.</a> Cell. 148 (3), 515-529 (2012).</li><li>Khan, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloidogenic+Oligomerization+Transforms+Drosophila+Orb2+from+a+Translation+Repressor+to+an+Activator.">Amyloidogenic Oligomerization Transforms Drosophila Orb2 from a Translation Repressor to an Activator.</a> Cell. 163 (6), 1468-1483 (2015).</li><li>Fioriti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Persistence+of+Hippocampal-Based+Memory+Requires+Protein+Synthesis+Mediated+by+the+Prion-like+Protein+CPEB3.">The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3.</a> Neuron. 86 (6), 1433-1448 (2015).</li><li>Derkatch, I. L., Bradley, M. E., Hong, J. Y., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions+affect+the+appearance+of+other+prions:+the+story+of+[PIN(+)].">Prions affect the appearance of other prions: the story of [PIN(+)].</a> Cell. 106 (2), 171-182 (2001).</li><li>Alberti, S., Halfmann, R., King, O., Kapila, A., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+survey+identifies+prions+and+illuminates+sequence+features+of+prionogenic+proteins.">A systematic survey identifies prions and illuminates sequence features of prionogenic proteins.</a> Cell. 137 (1), 146-158 (2009).</li><li>Chernoff, Y. O., Lindquist, S. L., Ono, B., Inge-Vechtomov, S. G., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+chaperone+protein+Hsp104+in+propagation+of+the+yeast+prion-like+factor+[psi+].">Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+].</a> Science. 268 (5212), 880-884 (1995).</li><li>Patino, M. M., Liu, J. J., Glover, J. R., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Support+for+the+prion+hypothesis+for+inheritance+of+a+phenotypic+trait+in+yeast.">Support for the prion hypothesis for inheritance of a phenotypic trait in yeast.</a> Science. 273 (5275), 622-626 (1996).</li><li>Wickner, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[URE3]+as+an+altered+URE2+protein:+evidence+for+a+prion+analog+in+Saccharomyces+cerevisiae.">[URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae.</a> Science. 264 (5158), 566-569 (1994).</li><li>Sondheimer, N., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rnq1:+an+epigenetic+modifier+of+protein+function+in+yeast.">Rnq1: an epigenetic modifier of protein function in yeast.</a> Mol Cell. 5 (1), 163-172 (2000).</li><li>Balbirnie, M., Grothe, R., Eisenberg, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+amyloid-forming+peptide+from+the+yeast+prion+Sup35+reveals+a+dehydrated+beta-sheet+structure+for+amyloid.">An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid.</a> Proc Natl Acad Sci U S A. 98 (5), 2375-2380 (2001).</li><li>Glover, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-seeded+fibers+formed+by+Sup35,+the+protein+determinant+of+[PSI+],+a+heritable+prion-like+factor+of+S.+cerevisiae.">Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae.</a> Cell. 89 (5), 811-819 (1997).</li><li>King, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-inducing+domain+2-114+of+yeast+Sup35+protein+transforms+in+vitro+into+amyloid-like+filaments.">Prion-inducing domain 2-114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments.</a> Proc Natl Acad Sci U S A. 94 (13), 6618-6622 (1997).</li><li>Shorter, J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp104+catalyzes+formation+and+elimination+of+self-replicating+Sup35+prion+conformers.">Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers.</a> Science. 304 (5678), 1793-1797 (2004).</li><li>Ferreira, P. C., Ness, F., Edwards, S. R., Cox, B. S., Tuite, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elimination+of+the+yeast+[PSI+]+prion+by+guanidine+hydrochloride+is+the+result+of+Hsp104+inactivation.">The elimination of the yeast [PSI+] prion by guanidine hydrochloride is the result of Hsp104 inactivation.</a> Mol Microbiol. 40 (6), 1357-1369 (2001).</li><li>Caudron, F., Barral, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+super-assembly+of+Whi3+encodes+memory+of+deceptive+encounters+by+single+cells+during+yeast+courtship.">A super-assembly of Whi3 encodes memory of deceptive encounters by single cells during yeast courtship.</a> Cell. 155 (6), 1244-1257 (2013).</li><li>Wickner, R. B., Edskes, H. K., Shewmaker, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+find+a+prion:+[URE3],+[PSI+]+and+[beta].">How to find a prion: [URE3], [PSI+] and [beta].</a> Methods. 39 (1), 3-8 (2006).</li><li>Chernoff, Y. O., Derkach, I. L., Inge-Vechtomov, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicopy+SUP35+gene+induces+de-novo+appearance+of+psi-like+factors+in+the+yeast+Saccharomyces+cerevisiae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heritable+switch+in+carbon+source+utilization+driven+by+an+unusual+yeast+prion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+product+of+the+het-s+heterokaryon+incompatibility+gene+of+the+fungus+Podospora+anserina+behaves+as+a+prion+analog.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+pathways+and+phenotypes+by+systematic+gene+overexpression.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+Disordered+Proteins+Drive+Emergence+and+Inheritance+of+Biological+Traits.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaching+a+complete+repository+of+sequence-verified+protein-encoding+clones+for+Saccharomyces+cerevisiae.">Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae.</a> Genome Res. 17 (4), 536-543 (2007).</li><li>Swain, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+time+derivatives+including+cell+growth+rates+using+Gaussian+processes.">Inferring time derivatives including cell growth rates using Gaussian processes.</a> Nat Commun. 7, 13766 (2016).</li><li>Jarosz, D. F., Lancaster, A. K., Brown, J. C., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evolutionarily+conserved+prion-like+element+converts+wild+fungi+from+metabolic+specialists+to+generalists.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+in+the+budding+yeast+Saccharomyces+cerevisiae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+high+efficiency+transformation+of+intact+yeast+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppressors+of+the+temperature+sensitivity+of+DNA+polymerase+alpha+mutations+in+Saccharomyces+cerevisiae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+proteome+turnover+analyses+of+the+Yeasts+S.+cerevisiae+and+S.+pombe.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLAAC:+a+web+and+command-line+application+to+identify+proteins+with+prion-like+amino+acid+composition.">PLAAC: a web and command-line application to identify proteins with prion-like amino acid composition.</a> Bioinformatics. 30 (17), 2501-2502 (2014).</li><li>Halfmann, R., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+amyloid+aggregation+by+Semi-Denaturing+Detergent-Agarose+Gel+Electrophoresis.">Screening for amyloid aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis.</a> J Vis Exp. (17), (2008).</li><li>Rogoza, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Mendelian+determinant+[ISP+]+in+yeast+is+a+nuclear-residing+prion+form+of+the+global+transcriptional+regulator+Sfp1.">Non-Mendelian determinant [ISP+] in yeast is a nuclear-residing prion form of the global transcriptional regulator Sfp1.</a> Proc Natl Acad Sci U S A. 107 (23), 10573-10577 (2010).</li><li>Shorter, J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions+as+adaptive+conduits+of+memory+and+inheritance.">Prions as adaptive conduits of memory and inheritance.</a> Nat Rev Genet. 6 (6), 435-450 (2005).</li><li>Tanaka, M., Chien, P., Naber, N., Cooke, R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+variations+in+an+infectious+protein+determine+prion+strain+differences.">Conformational variations in an infectious protein determine prion strain differences.</a> Nature. 428 (6980), 323-328 (2004).</li><li>Tanaka, M., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+protein+transformation+protocol+for+introducing+prions+into+yeast.">An efficient protein transformation protocol for introducing prions into yeast.</a> Methods Enzymol. 412, 185-200 (2006).</li><li>Roberts, B. T., Wickner, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+activity:+a+prion+that+propagates+by+covalent+autoactivation.">Heritable activity: a prion that propagates by covalent autoactivation.</a> Genes Dev. 17 (17), 2083-2087 (2003).</li><li>Ozbudak, E. M., Thattai, M., Lim, H. N., Shraiman, B. I., Van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistability+in+the+lactose+utilization+network+of+Escherichia+coli.">Multistability in the lactose utilization network of Escherichia coli.</a> Nature. 427 (6976), 737-740 (2004).</li></ol>

著者が明らかに何もありません。

最初の酵母プリオンは、彼らの異常な表現型と継承の複雑なパターンによって識別されました。これらのプリオンの特性は、アルゴリズムと計算ツール追加プリオン蛋白質のため画面を構築するそれから使用されました。ここで説明する方法は対照的は実験的一時的な過剰発現タンパク質の立体構造の状態でエンコードされた永続的な変更は安定を作成するに依存しています。ただし、任意?...

生物学的システムはよくタンパク質豊富で過渡変動を経験します。これらは有機体のまたは将来の世代の表現型を形作る上で永続的な影響をあるかどうかは不明します。この生物の最も有名なインスタンスを含むタンパク質、プリオンは、ゲノム修正なしの遺伝性の特性の出現をドライブの珍しいクラス。代わりに、これらのプロの teinaceous とのfectious 粒子タンパク質構造1,2の自己永続的変更による表現型を送信します。この型を継承は、壊滅的な神経変性疾患の異常な継承のパターンの原因として発見されました。しかし、菌類から哺乳類3,4,5,6,7,8,9,10まで及ぶ有機体の研究は以来プリオンのような要素が適応値を与えることができること明らかに。それにもかかわらず、プリオンは、魅惑的で珍しい生物風変わりなしかしとして表示されています。
この相場の知恵は、継承の蛋白質ベースの特性は長い例の小さなセットによって制限されているので一部で開催されます。最近の組織的スクリーニングの努力は大幅燃料プリオンのような構造変換する能力を持ついくつかの新しいbona fideプリオン11およびほぼ 2 つのダース蛋白質ドメイン12を識別することによってこの画像を拡大しています。ただし、これらのアプローチは、一般的に強力なアミノ酸シーケンス バイアスを当てている、ので、発見されているプリオンは創立酵母プリオン [PSI+]13,14、[URE3]15[RNQ+]11,16の生化学的性質を共有します。これらが含まれます: 1) モジュラー ドメイン アスパラギン (N) とグルタミン (Q)、2)、アミロイド [プリオン+] 構造17,18,に19アセンブリの長い高分子の伸張に富んでいる、3) 完了 disaggregase 母から娘13,20,21に忠実な伝播のための Hsp104 関数に依存します。確かに、多くbona fideプリオンを含む [ガー+] [ヘット s]、元プリオン (PrPSc) にも、このような厳しい条件下では見逃されること。おそらくもっと重要なは、彼らことはできません蛋白質ベースの継承22の任意の新規メカニズムをキャプチャします。したがって、このような現象の真の生物学的幅は、はるか以前の仮定より自然で一般的な可能性があります。
この問題を調べるためには、高スループット、プロテオーム的戦略が採用されました。PrPSc[ガー+] を含む全てのプリオンの特徴 [ヘット-s] は、原因タンパク質の一過性発現が強くプリオン買収15,23,24,25,26の率を増加することとします。一過性個々 のタンパク質の発現を誘導することによって安定した蛋白質ベース、エピジェネティック状態を開始できる場合、体系的に全体にわたって酵母の ORFeome、お願い、この機能の利用をしました。その蛋白の過剰発現は、表現型27を変更できますをよく知られています。しかし、彼らの一時的な過剰世代初期の過剰発現後の数百の遺伝性は表現型の変化を生成するため、プリオン蛋白質は通常ありません。以前、ホリールード ゲノム28を変えないで表現型の風景を再配線することができる蛋白質の多数を識別するために、タンパク質ベース遺伝的要素の異常な継承のパターンだけでなく、この機能を利用しました。いくつかの特定蛋白質以前として知られていたプリオン、ほとんどなかった、タンパク質ベースの継承の新たな形態を明らかにするこのアプローチの力を強調します。

<table width="958">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="width:356px;height:21px;">Guanidine hydrochloride</td>
			<td style="width:299px;">Sigma</td>
			<td style="width:125px;">Cat#G3272-25G</td>
			<td style="width:179px;">Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manganese chloride</td>
			<td>Sigma</td>
			<td>Cat#M8054-100G</td>
			<td>Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethidium bromide</td>
			<td>Sigma</td>
			<td>E1510</td>
			<td>Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Fluoroorotic Acid</td>
			<td>Sigma</td>
			<td>Cat#F5013-50MG</td>
			<td>Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BY4741 MATa (his3&#916;1 leu2&#916;0 LYS2 met15&#916;0 ura3&#916;0)</td>
			<td>Winston et al., 1995; Brachmann et al., 1998</td>
			<td>N/A</td>
			<td>Yeast strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BY4741 MAT&#945; (his3&#916;1 leu2&#916;0 lys2&#916;0 MET15 ura3&#916;0)</td>
			<td>Winston et al., 1995; Brachmann et al., 1998</td>
			<td>N/A</td>
			<td>Yeast strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hsp70 (K69M)&#160;</td>
			<td>Jarosz et al., 2014b</td>
			<td>N/A</td>
			<td>Plasmid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FLEXGene library</td>
			<td>Hu et al., 2007</td>
			<td>N/A</td>
			<td>Plasmid library</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose (glucose)</td>
			<td>Fisher Scientific</td>
			<td>D16-3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Raffinose</td>
			<td>Sigma</td>
			<td>R0250-25G</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galactose</td>
			<td>Fisher Scientific</td>
			<td>BP656-500</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CSM</td>
			<td>Sunrise Science</td>
			<td>1001-100</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CSM-URA</td>
			<td>Sunrise Science</td>
			<td>1004-100</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CSM-LYS</td>
			<td>Sunrise Science</td>
			<td>1032-100</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CSM-MET</td>
			<td>Sunrise Science</td>
			<td>1019-100</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CSM-LYS-MET</td>
			<td>Sunrise Science</td>
			<td>1035-100</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peptone</td>
			<td>Research Products International</td>
			<td>P20240-5000</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bacto-peptone</td>
			<td>BD</td>
			<td align="right">211677</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol</td>
			<td>EMD Millipore</td>
			<td>GX0185-2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">yeast nitrogen base w/o amino acids</td>
			<td>BD</td>
			<td align="right">291920</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">agar</td>
			<td>IBI Scientific</td>
			<td>IB49172</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenine sulfate</td>
			<td>Sigma</td>
			<td>A3159-25G</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium acetate</td>
			<td>Sigma</td>
			<td>P1190-500G</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td>Sigma</td>
			<td>U0750-100G</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histidine</td>
			<td>Sigma</td>
			<td>H8000-100G</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leucine</td>
			<td>Sigma</td>
			<td>L8000-25G</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysine</td>
			<td>Sigma</td>
			<td>L5501-25G</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase I&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0601</td>
			<td>Enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">biotinylated DNase</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1906</td>
			<td>Enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">zymolyase 100T (yeast lytic enzyme)</td>
			<td>Sunrise Science</td>
			<td>N0766555</td>
			<td>Enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy H1</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate stacker</td>
			<td>BioTek</td>
			<td>BioStack3</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate filler</td>
			<td>BiotTek</td>
			<td>EL406</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

将来の世代にアクセス可能な生物学的情報のエンコードは、DNA シーケンスの変更を介して達成される一般に。(シーケンスではなく) 蛋白質立体構造でエンコードされた長寿命の継承は、パラダイム シフトしますが、珍しいとして表示されている長い。などのエピジェネティックな要素の最高の特徴の例は、プリオンは、新しい表現型の遺伝的症状をドライブすることができます自己組織化挙動を所有しています。多くの典型的なプリオンは印象的な豊富な N/Q シーケンス バイアスを表示し、アミロイドの折りにアセンブルします。これらの珍しい特徴は、新しいプリオン蛋白質を識別するほとんどのスクリーニングの努力を知らせています。ただし、(創設のプリオン、PrPScを含む)、少なくとも 3 つの知られているプリオンは、これらの生化学的な特徴を港ないです。したがってプローブ質量作用のプロパティに基づいて蛋白質ベースの継承の範囲に代替法を開発した: プリオン蛋白質の一時的な過剰発現する彼らは自己テンプレート構造を取得頻度が増加します。本稿では、蛋白質ベースの継承を引き出す酵母 ORFeome の能力を分析するためのメソッドについて説明します。この戦略を使用して、我々 は以前ことを発見した &gt; イースト蛋白質の 1% は長寿命、安定した遺伝の突然変異よりも頻繁に起こったされ生物学的形質の出現を燃料でした。全体 ORFeomes 全体または特定の遺伝子ネットワークまたは環境刺激の標的スクリーニング パラダイムとして、高スループットでこの方法を採用できます。同様に前方遺伝的画面多数発達とシグナル伝達経路を定義する、これらの技術は生物学的過程での蛋白質ベースの継承の影響を調査するための方法論を提供します。

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 ...

Watch this Scientific Journal Video about High-throughput Screening for Protein-based Inheritance in S. cerevisiae at JoVE.com

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

このプロトコルでは、出芽酵母でタンパク質に基づく継承のため画面の機能的に高スループット方法論について説明します。

出芽酵母におけるタンパク質ベース継承の高速スクリーニング

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

high-throughput-screening-for-protein-based-inheritance-s

Research

JoVE Journal

Biology

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

6.2K ビュー.  Stanford University School of Medicine. このプロトコルでは、出芽酵母でタンパク質に基づく継承のため画面の機能的に高スループット方法論について説明します。

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

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

S. cerevisiaeの接着と寒天の侵略のためのアッセイ

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

生物学

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.

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

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

Identification of protein complexes with quantitative proteomics in S. cerevisiae

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

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

S. cerevisiaeの定量的プロテオミクスとタンパク質の複合体の同定

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

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

蛍光とハイスループットスクリーニングとバイオセンシング C.エレガンス菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

における真菌エンドグルカナーゼ活性のハイスループットスクリーニング大腸菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

のフローサイトメトリーベースの精製<em&gt; S.セレビ</em&gt;受精卵

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向き整流カリウムチャネルの小分子モジュレーターのためのハイスループットスクリーニング

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

自家製デュアルグロールシフェラーゼアッセイを用いて、ハイスループット機能スクリーニング

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

で産生された組換えジスルフィド豊富な毒タンパク質に印加される高スループット定量的発現スクリーニングおよび精製<em&gt; E.大腸菌の</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...