Name: high-throughput screening for protein-based inheritance in s. cerevisiae
Uploaded: 2017-08-08T14:00:00.000+00:00
Duration: 8 min 12 s
Description: Watch this Scientific Journal Video about High-throughput Screening for Protein-based Inheritance in S. cerevisiae at JoVE.com

Ringraziamo Sohini Chakrabortee, Sandra Jones, David Garcia, Bhupinder Bhullar, Amelia Chang, Richard She e Susan Lindquist per la loro assistenza nello sviluppo i dosaggi utilizzati in questa carta, come pure gli utenti per i loro commenti riflessivo.

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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I prioni lievito primi sono stati identificati dai loro fenotipi insoliti e perplessi modelli di ereditarietà. Le caratteristiche di questi prioni furono poi utilizzate per costruire algoritmi e strumenti computazionali alla schermata per proteine prioniche aggiuntive. Il metodo descritto qui, al contrario, è sperimentale e si basa sulla sovraespressione transitoria per creare un duraturo cambiamento-a stabile stato codificato nella conformazione della proteina. Tuttavia, se l'efficienza di "semina" Assemblea del prion da sovraespressione per qualsiasi data proteina è molto bassa, che la proteina continuamente emergerà come un falso negativo nelle schermate di sovraespressione di questo tipo. Una tale modifica per risolvere il problema sarebbe utilizzare un plasmide di 2 micron per la sovraespressione della proteina in futuro esperimenti. Infine, ogni prionica indotto ha relativo proprio insieme unico dei fenotipi di crescita e sarà non essere evidente in ogni condizione analizzati. Così, il numero di condizioni diverse e dosi testate limita il numero di colpi.
Cosa importante, non tutti i tipi di ereditarietà su base proteica saranno ugualmente recuperati utilizzando questo metodo. Ovviamente ci mancherà continuamente proteine che non possono essere sovraespresso in modo efficiente o senza tossicità. Elementi mitotically instabili, ad esempio "mnemons," non avrebbero mai propagare a figlie seguire la sovraespressione iniziale22. Al contrario, altri tipi di Interruttori bistabili longevo teoricamente potrebbero essere indotto tramite sovraespressione transiente42,43. Tuttavia, questi Stati sono in genere non dipendente da macchinari omeostatico della proteina o trasmissibile tramite proteine "seminato". Inoltre, i prioni si basano su altri chaperoni (di fuori di Hsp70 e Hsp104) o bracci aggiuntivi della rete omeostasi della proteina per propagazione fallirebbe i dosaggi di chaperone-dipendenza descritti qui. Infine, una proteine di abbondanza bassa che anche formano amiloide potrebbero avere tariffe inferiori al limite di rilevazione di infettività nel programma di installazione di trasformazione proteina.
Questo protocollo descrive una tecnica per indurre stati epigenetici basati su proteine stabili tramite sovraespressione della proteina, come pure più ulteriormente a valle procedura per convalidare se ciascuno indotta stato epigenetico è un prione bona fide . L'esempio presentato in questa carta, Psp1, è un esempio di una proteina che visualizza una polarizzazione dell'amminoacido "del prion-like" e potrebbe teoricamente essere recuperato utilizzando precedentemente sviluppato algoritmi bioinformatici. Tuttavia, l'incapacità di Psp1 per formare amiloide e la sua dipendenza insolito chaperone (Hsp104) sarebbe rapidamente squalificato da ulteriori analisi e quindi eliminato dal conto del prion. Tuttavia, le tecniche di screening presentate in questa carta sono indipendente da questi presupposti e invece concentrano sui sottostanti modelli di ereditarietà e la sufficienza della proteina da solo per trasmettere i fenotipi corrispondenti. Infatti, la stragrande maggioranza della base di proteine eredità recuperata con questo metodo era privo di pregiudizi sequenza N/Q-ricco.
Questo metodo è stato utilizzato per sondare il lievito intero ORFeome per la sua capacità di suscitare ereditarietà a base di proteine in modo imparziale, utilizzando solo un piccolo numero di fattori di stress (cloruro di cadmio di 25 mM, 1 mM di cloruro di cobalto, solfato di rame di 2 mM, 1 mM diammide, fluconazole 0,2 mM, 50mm hydroxyurea, cloruro di manganese 20 mM, il paraquat 0,75 mM, 50mm radicicol, 80 J/m2 irradiazione UV e solfato di zinco 10 mM). Tuttavia, questo approccio potrebbe essere facilmente modificato per reti genetico schermo o specifiche risposte cellulari in modo più mirato. Per esempio, proteine funzionalmente correlate o tutte le proteine regolato una discreta rete di segnalazione potrebbe essere indotta tramite sovraespressione transitoria e proiettata con fattori di stress legate alla loro funzione biologica. Al contrario, un più ampio insieme di proteine potrebbe proiettato con un set più completo di fattori di stress per indagare se specifiche risposte cellulari si sono evoluti naturalmente per harbor interruttori da prioni. Infine, anche se abbiamo condotto questi studi in lievito, molti aspetti degli esperimenti (ad esempio, l'espressione della proteina transitoria, chaperon "curare", ecc.) potrebbe essere generalizzata ad altri sistemi di modello in futuro. Ad esempio, coltura di tessuti dei mammiferi è favorevole alla sovraespressione tramite sistemi basati su plasmide e fuochi di fluorescenza potrebbero essere anche essere utilizzato come una lettura per ereditabile auto-assemblaggio, come descritto in precedenza28. Inoltre, sequenze codificanti proteine da altri organismi potrebbero essere espresso in lievito e testati per la loro capacità di suscitare da prioni-come eredità utilizzando i metodi descritti qui.

Sistemi biologici spesso esperienza transitorie fluttuazioni in abbondanza di proteine. Se questi hanno un impatto duraturo nel plasmare il fenotipo di un organismo o delle generazioni future rimane poco chiaro. I più noti casi di questa biologia coinvolgono una rara classe di proteine, i prioni, che guidano l'emergere dei tratti ereditari senza alcuna modifica del genoma. Invece, queste particelle di fectious proteinaceous e neltrasmettono fenotipi tramite auto-perpetuante cambiamenti di conformazione della proteina1,2. Questo tipo di ereditarietà è stato scoperto come la causa dei modelli di ereditarietà insoliti di una devastante malattia neurodegenerative. Tuttavia, gli studi negli organismi che variano dai funghi ai mammiferi3,4,5,6,7,8,9,10 da allora hanno rivelato che il prione-come gli elementi possono conferire valore adattativo. Ciò nonostante, i prioni sono stati visti come un affascinante ma rara stranezza biologica.
Questa saggezza prevalente si svolge in parte perché la caratterizzazione di base proteica eredità lungo è stata limitata da un piccolo insieme di esempi. I recenti sforzi di screening sistematico hanno ampliato significativamente questa immagine individuando diversi nuovi bona fide prioni11 e quasi due dozzine di proteina domini12 con la capacità di conversione conformazionali del prione-come combustibile. Tuttavia, poiché questi approcci sono generalmente concentrati su pregiudizi di sequenza dell'amminoacido forte, i prioni che sono stati scoperti condividono le proprietà biochimiche del fondatore lievito prioni [PSI+]13,14, [URE3]15e [RNQ+]11,16. Questi includono: 1) modulare domini che sono ricchi di tratti lungo polimerici di asparagina (N) e glutammina (Q), 2) assembly in un amiloide [PRION+] conformazione17,18,19e 3) completano dipendenza da disaggregase funzione di Hsp104 per la propagazione di fedeli da madre a figlia13,20,21. Infatti, molti bona fide prioni, tra cui [GAR+], [Het-s] e anche l'originale del prion (PrPSc), sarebbe saltato sotto tali criteri rigorosi. Forse ancora più importante, sarebbero in grado di catturare qualsiasi nuovi meccanismi di ereditarietà su base proteica22. Quindi, l'ampiezza biologica vera di tali fenomeni può essere molto più comune in natura di quanto si pensasse.
Per studiare questa domanda, una strategia di alto-rendimento, tutto il proteoma è stato impiegato. Un segno distintivo di tutti i prioni, tra cui PrPSc, [GAR+], e [Het-s], è che la sovraespressione transitoria delle proteine causale aumenta fortemente il tasso del prione acquisizione15,23,24,25,26. Abbiamo approfittato di questa funzionalità per sistematicamente chiedere, su tutto il lievito ORFeome, se stati stabili basati su proteine, epigenetici potrebbero essere iniziati inducendo transitoriamente la sovraespressione delle singole proteine. È ben noto che la sovraespressione della proteina può alterare fenotipi27. Tuttavia, proteine prioniche sono insoliti perché loro sovrapproduzione temporanea produce un cambiamento nel fenotipo che è ereditabile per molte centinaia di generazioni dopo la sovraespressione iniziale. Abbiamo precedentemente preso vantaggio di questa funzione, così come i modelli di ereditarietà insoliti di elementi genetici basati su proteine, per identificare decine di proteine che sono in grado di heritably necessità di cablaggio fenotipiche paesaggi senza alterare il genoma28. Anche se alcuni identificati proteine precedentemente erano conosciuti come i prioni, la maggior parte erano non, sottolineando la potenza di questo approccio per scoprire nuove forme di ereditarietà a base di proteine.

<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

La codifica di informazioni biologiche che sono accessibile alle generazioni future è generalmente ottenuta tramite modifiche alla sequenza di DNA. Longeva eredità codificati in proteina conformazione (anziché sequenza) lungamente è stato osservato come lo spostamento di paradigma, ma rara. Esempi di tali elementi epigenetici meglio caratterizzati sono i prioni, che possiedono un comportamento auto-assemblante che può guidare la manifestazione ereditabile di nuovi fenotipi. Molti prioni archetipici visualizzare un notevole pregiudizio sequenza N/Q-ricco e assemblare un ovile dell'amiloide. Queste caratteristiche insolite hanno informato la maggior parte degli sforzi di screening per identificare nuove proteine prioniche. Tuttavia, almeno tre prioni noti (tra cui il prione fondatore, PrPSc) non porto queste caratteristiche biochimiche. Abbiamo pertanto sviluppato un metodo alternativo per sondare l'ambito dell'eredità a base di proteine basata su una proprietà di azione di massa: la sovraespressione transiente di proteine prioniche aumenta la frequenza in cui acquisiscono una conformazione di auto-applicazione di modelli. Questo articolo descrive un metodo per analizzare la capacità del lievito ORFeome a suscitare l'ereditarietà a base di proteine. Utilizzando questa strategia, abbiamo trovato in precedenza che &gt; 1% di proteine del lievito potrebbe alimentare l'emergere dei tratti biologici che erano longevi, stabile e ha presentato più di una mutazione genetica. Questo approccio può essere impiegato in elevato throughput attraverso ORFeomes intero o come un paradigma di screening mirato per reti genetiche specifiche o stimoli ambientali. Come schermi genetici avanti definiscono numerose vie di segnalazione e di sviluppo, queste tecniche forniscono una metodologia per indagare l'influenza dell'eredità di base di proteine nei processi biologici.

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 a protein of unknown function that can suppress mutants in the replication machinery (e.g., in pol alpha34). Overexpression of the PSP1 ORF modulated cellular phenotypes in a variety of stressors. Strikingly, one phenotype (resistance to manganese chloride) was maintained for hundreds of generations in cells long after overexpression had ceased (i.e., progeny who had never experienced Psp1 induction directly, but whose ancestors had) (Figure 1A). The half-life of Psp1 is ~5 h35, making it extremely unlikely that the resistance phenotype is due to the propagation of long-lived, stable Psp1 protein from mothers to their daughters over this timescale (as the original protein would be degraded within a handful of generations).
Because the MnCl2 resistance phenotype arose at a high frequency and in multiple independent transient overexpression experiments, it is extremely unlikely that this phenotype was due to de novo mutation. A possible explanation for the heritable nature of this phenotype is the induction of a self-templating element (i.e., a prion). Indeed, the Psp1 amino acid content harbors some small stretches of asparagine and glutamine, reminiscent of canonical prions such as [PSI+], which are driven by proteins that contain long stretches of these amino acids. Predictive algorithms36 score the N-terminus of Psp1 as moderately &#34;prion-like&#34; (Figure 1B, Rank 173 in the yeast proteome). However, unlike canonical prion proteins, Psp1 protein from cells harboring the stable phenotypic state did not form amyloid fibers, as judged by semi-denaturing agarose gel electrophoresis28,37. To determine whether Psp1 formed a bona fide prion, we tested whether the pattern of inheritance of the Psp1-induced phenotypic state was consistent with hallmarks of prion biology: reliance on protein homeostasis machinery for faithful propagation and non-Mendelian inheritance in meiosis. Finally, a &#34;gold standard&#34; test of transmission was performed using only assembled protein.
Unlike mutations or other forms of epigenetic inheritance, prions are uniquely reliant on the function of molecular chaperones and other arms of the protein homeostasis network that regulate protein folding in the cell. Most canonical prions require the Hsp104 disaggregase to act on the amyloid fibers that they adopt. This process generates heritable &#34;seeds&#34; that transmit the prion conformation from mothers to daughters13. One notable exception to this requirement is the [ISP+] prion, which is curable via Hsp104 inhibition but does not require its disaggregase activity for propagation38. Furthermore, we and others have recently reported many examples of prions that are Hsp104-independent and are instead regulated by other molecular chaperones (primarily Hsp70 and, in rare cases, Hsp90)25,28. We first tested whether the inheritance of the Psp1-induced MnCl2 resistance depended on Hsp104. Cells harboring this state were passaged 3 times on rich medium (YPD) supplemented with a low dose of guanidine hydrochloride (an Hsp104 inhibitor). We then returned the cells to standard rich medium to restore full Hsp104 function. This regimen did not affect the heritability of the Psp1-induced MnCl2 resistance, establishing that the cellular state was Hsp104-independent (Figure 2).
Next, a crossing approach was employed to test whether the Psp1-induced phenotypic state depended on Hsp70. The MnCl2 resistant cells were mated to an Hsp70-deficient strain, with genetic deletions in two of the four yeast Hsp70 paralogs (ssa1&#916; ssa2&#916;). Diploids were selected and then sporulated to generate haploid spores. We tested for the maintenance of the Psp1-dependent state and found that the Psp1-induced MnCl2 resistance phenotype had been completely eliminated in spores harboring the ssa1&#916; ssa2&#916; double deletion (Figure 2). Thus, the Psp1-induced phenotypic state requires the activity of Hsp70 to pass from one generation to the next.
Another defining characteristic of prions is a non-Mendelian pattern of inheritance. Traits encoded by mutations in DNA segregate 2:2 in meiotic crosses: half of the progeny inherit one parental allele and other half inherit the other parental allele. The inheritance of prions, in contrast, does not depend upon changes in DNA, but rather transmissible changes in protein conformation. Thus, they are inherited by most, if not all, meiotic progeny in genetic crosses (3:1 or 4:0 inheritance)39. We tested the inheritance pattern of the Psp1-induced MnCl2 resistance by crossing strains harboring the state to na&#239;ve controls of the opposite mating type. The resulting diploids were sporulated and tested for the presence of the Psp1 MnCl2 resistance phenotype within spores derived from the same tetrads. All spores from 2 separate tetrads in which one parent harbored the Psp1-dependent epigenetic state inherited the corresponding MnCl2 resistance phenotype (Figure 2). In contrast, no cells from na&#239;ve control crosses displayed the phenotype. These data establish that the Psp1-induced phenotypic state is not encoded by a DNA-based mutation. However, it is important to note that while the Psp1 state is not encoded in DNA, the state does require the continual presence of the PSP1 gene. Crossing the Psp1-dependent phenotypic state into a genetic background lacking the PSP1 gene (psp1&#916;) heritably eliminated the MnCl2 resistance phenotype (Figure 2). Thus, overexpression of Psp1 does not simply orchestrate the formation of a highly stable feedback loop that functions autonomously of Psp1 once initiated.
The very definition of protein-based inheritance is the transmission of stable traits to progeny using only assembled protein as the heritable material. To carry out such &#34;protein transformations,&#34; a method that is agnostic with respect to protein stoichiometry, modification, or interaction was used40,41. Separate cultures of cells harboring the Psp1-induced phenotypic state and na&#239;ve controls were grown to mid-exponential phase, and their cell walls were lysed to generate donor spheroplasts. These spheroplasts were then sonicated and spun down to pellet the resulting cell debris. Finally, the remaining lysates were subjected to over-digestion with both RNase and DNase to eradicate nucleic acid (to aid steps later in the protocol, the biotinylated DNase enzyme was subsequently removed via affinity purification).
These nucleic acid-depleted lysates were used to transform na&#239;ve cells. To aid in the uptake of heritable protein &#34;seeds,&#34; the cell walls of the na&#239;ve recipients were enzymatically digested prior to transformation. A URA3-marked carrier plasmid with the protein donor lysates was also included to enable the selection of cells that were competent for the uptake of foreign material. The transformants were plated on medium lacking uracil, resistant colonies were selected, and the URA3-marked carrier plasmid was removed by growth on 5-FOA. The resistance of the resulting colonies to MnCl2 was then examined. Strikingly, over half of the transformants tested harbored heritable MnCl2 resistance (Figure 3). In contrast, no cells transformed with na&#239;ve lysates displayed any such phenotype. Importantly, these efficiencies were gleaned from cellular lysates expressing endogenous levels of Psp1 protein (~340 molecules per cell). This is remarkably more efficient than the transformation of other yeast prions, such as [PSI+]. These results establish that Psp1 is capable of forming a protein-based heritable element-a &#34;prion&#34;-henceforth known as [PSP1+]. Furthermore, the protein transformation technique described here is sufficient for screening for non-amyloid, Hsp104-independent, protein-based inheritance in a high-throughput manner.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56069/56069fig1.jpg" /> 
Figure 1. Transient overexpression of PSP1 drives heritable MnCl2 resistance. (A) Growth of uninduced ([psp1-]) and Psp1-induced ([PSP1+]) strains in SD-CSM with 10 mM MnCl2. The error bars represent the standard error of the mean from three biological replicates. (B) Left: The Psp1 primary amino acid sequence scores as highly prion-like in prion-predicting algorithms (PLAAC). Right: Representative PLAAC analysis for most inducing proteins recovered in our screen predicted virtually no significant prion-like sequence features. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56069/56069fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56069/56069fig2.jpg" /> 
Figure 2. Psp1 chaperone dependence and pattern of inheritance. The growth of strains in SD-CSM with 10 mM MnCl2, normalized to a corresponding na&#239;ve [psp1-] control. The inhibition of Hsp104 (GdnHCl &#34;cured&#34;) does not impair Psp1-dependent MnCl2 resistance, whereas the removal of the Hsp70 chaperone (ssa1&#916; ssa2&#916;) and the PSP1 gene (psp1&#916;) heritably eliminates this phenotype. All spores from single tetrads of crosses between strains harboring the Psp1-dependent phenotypic state and na&#239;ve strains display MnCl2 resistance, indicating non-Mendelian (4:0) inheritance of the phenotype (n=2 tetrads). The error bars represent the standard error of the mean from three biological replicates. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56069/56069fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56069/56069fig3.jpg" /> 
Figure 3. Transmission of Psp1-dependent phenotypic state via heritable protein &#34;seeds.&#34; Rates of infectivity for protein transformation comparing mock ([psp1-] lysates) and [PSP1+]- harboring lysates as the transmissible material. Infectivity is calculated as the percent of transmission divided by the amount of seeded protein used. Over 53% of na&#239;ve cells transformed with seeded [PSP1+] received the corresponding MnCl2 resistance phenotype, whereas no cells transformed with [psp1-] displayed MnCl2 resistance (p=7.4 x 10-4 by Student&#39;s t-test). The infectivity rate of the previously characterized yeast prion [PSI+] is displayed via the dotted horizontal line40. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56069/56069fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Questo protocollo descrive una metodologia di alto-rendimento allo schermo funzionalmente per base di proteine di ereditarietà in S. cerevisiae.

High throughput Screening per eredità a base di proteine in S. cerevisiae

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

Universidad San Sebastian

Research

JoVE Journal

Biology

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

6.2K Views.  Stanford University School of Medicine. Questo protocollo descrive una metodologia di alto-rendimento allo schermo funzionalmente per base di proteine di ereditarietà in S. cerevisiae.

Video: 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.

Sovraespressione di accoppiamento-based Library Screening in lievito

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

Ricerca

Genetica

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.

Una schermata di soppressore Deep-sequenziamento-assistita, spontanea nel lievito a fissione 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 ...

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.

Uno schermo soppressore per la caratterizzazione dei collegamenti genetici che regolano la durata cronologica 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 ...

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.

Un high-throughput metodo per studiare la morfologia globale organello in S. cerevisiae

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

Biologia

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.

High-throughput iperespressione schermo lievito plasmidi

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.

Schermi competitivo genomica delle Biblioteche di lievito con codice a barre

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.

Phenotipizzazione del ceppo di lievito ad alta velocità effettiva con sequenziamento dell'RNA basato su droplet

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

Bioingegneria

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.

Probing ad alta densità di microarrays della proteina funzionali per rilevare le interazioni proteina-proteina

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.

Rapida identificazione dei chimici genetici Interactions in<em&gt; Saccharomyces cerevisiae</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.

Diretto Evolution Metodo<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Biblioteca Creazione e screening

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

High throughput Screening per eredità a base di proteine in S. cerevisiae

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