We thank Sue Lindquist, Aaron Gitler, and Martin Duennwald for kindly sharing reagents. Our studies were supported by: an American Heart Association Post-Doctoral Fellowship (M.E.J); NIH Director's New Innovator Award DP2OD002177, NIH grants R21NS067354, R21HD074510, and R01GM099836, a Muscular Dystrophy Association Research Award (MDA277268), Packard Center for ALS Research at Johns Hopkins University, Target ALS, and an Ellison Medical Foundation New Scholar in Aging Award (J.S.).

1. Herstellung von Bibliotheken <ol><li> Um Bibliotheken von Hsp104 mit domänenspezifischen fehleranfällige PCR konstruieren zunächst verstärken die Domäne von Interesse mit einem fehleranfälligen DNA-Polymerase 20. </li><li> Reinige den PCR-Produkt durch Gel-Extraktion. </li><li> Führen Megaprimer-Extensionsschritt unter Verwendung einer Standardortsgerichtete Mutagenese-Protokoll: Kombinieren 50 ng Templat-Plasmid, 250 ng Megaprimer, 200 uM dNTPs und High-Fidelity-DNA-Polymerase in PCR-Puffer, und mit Wasser auf 50 &amp; mgr; l Gesamtvolumen mit PCR-Wasser 20 . Führen Sie einen Standard-PCR-Programm. HINWEIS: Spezifische Primer verwendet wird, basierend auf dem bestimmten Bereich des Gens, das zu amplifizierende variieren. <ol><li> Nach der PCR verdauen Eltern Template DNA mit 1 &amp; mgr; l DpnI-Restriktionsenzym 2 Stunden bei 37 ° C. </li></ol></li><li> Verwandeln Sie die Bibliothek durch Elektroporation oder andere Mittel in ultrakompetente E. coli und reinigen es durch Miniprep. HINWEIS: Bibliothek Generationvariiert im wesentlichen von den Zielen einer gegebenen Experiment basiert. Hsp 104 ist ein sehr großes Protein, so dass es unpraktisch ist, das gesamte Gen, weshalb wir nutzen domänenspezifischen fehleranfällige PCR zufällig ist. Darüber hinaus bleibt die Struktur des Hsp104 schlecht verstanden, dass das Design von gerichteten Bibliotheken anspruchs 21. Bibliotheken können mit gerichteten oder zufälligen Ansätze zur Mutagenese konstruiert werden, mit der einzigen Einschränkung, dass die Templat-Plasmid-Rückgrat ist das URA3-Gen für die 5-FOA Gegen Selektion auf Dextrose-Medium erlauben. </li></ol> 2. Transformation des Hsp104 Bibliothek <ol><li> Integrieren Sie die krankheitsassoziierten Substrat in W303aΔ hsp104 Hefe mit einem Standard-Lithium-Acetat / PEG Transformationsprotokoll 22. Wählen einzelne Kolonien zu screenen, und sie für Toxizität, einen Stamm mit einer hohen Toxizität der Krankheit assoziiert Substrat 23 zu isolieren. HINWEIS: Verwenden Sie eine integrierte Belastung und isolate einer Einzelkolonie auf Gleich Expression in allen Zellen zu gewährleisten. Klonen Sie die Krankheit Substrat in ein Plasmid ermöglicht die Integration des Gens unter einem anderen als Uracil-Marker (wir verwenden Histidin) und dem Hsp104 Bibliothek in das pAG416GAL Plasmid (Uracil-Marker) 24. Für 5-FOA Gegenselektion zu ermöglichen, sicherzustellen, dass die Bibliothek aus einem Plasmid mit der Uracil-Marker exprimiert. Andere Hefe-Stämme können ebenfalls verwendet werden. Wir haben eine ähnliche Toxizität Unterdrückung Phänotyp mit W303a und BY4741 Hefestämme in beiden WT und Δ hsp104 Hintergründe (MEJ und JS, unveröffentlichte Beobachtungen) festgestellt. </li><li> Transformieren Sie das Hsp104 Bibliothek in diesen Stamm mit dem gleichen Lithiumacetat / PEG Transformationsprotokoll 22. Scale up die Transformation entsprechend dem Sequenzraum der Bibliothek zu erhalten und um die vorhergesagte Größe der Bibliothek zu erhalten. </li><li> Platte der Transformationsansatz auf noninducing, Selektivplatten (zB SD-His-Ura) mit genug Plattensicherzustellen Wachstum einer großen Anzahl von Kolonien. Verwenden Sie große Petrischalen (150 mm), die Anzahl der Platten erforderlich minimieren. Gewinnen Anten unter Verwendung von Platten ermöglicht Transformationseffizienz zu bewerten. </li><li> Trans Hsp104 WT und Vektor Negativkontrollen parallel. </li></ol> 3. Screening zur Unterdrückung von proteotoxicity <ol><li> Verwendung Raffinose ergänzt Dropout Medien (zB sraff-His-Ura) Waschen Sie die Kolonien von den Platten. Verwenden Sie eine serologische Pipette und sterilen Holz Applikatoren, die Kolonien von den Platten lösen. Übertragen Sie die Flüssigkeit wäscht auf eine 50 ml konischen Röhrchen und vortexen, um alle Klumpen von Zellen zu trennen. Verdünnen, um eine leicht trübe Kultur, OD 600 ~ 0,025. HINWEIS: Hier Raffinose dient dazu, die Zellen Glucose-vermittelte Unterdrückung der Induktion zu entlasten, damit die Zellen Grundierung für Galaktose-vermittelte Induktion. </li><li> Wachsen die verdünnte Kultur über Nacht in Raffinose Dropout Medien mit 3 Schütteln0 ºC. Wachsen die Hsp104 WT und Vektorkontrollen parallel. </li><li> Am folgenden Morgen, Platte eine Reihe von Konzentrationen auf einzelne Galactose (zB SGal-His-Ura) Platten (1 ul bis 2 ml in 400 ul Gesamtvolumen konzentriert Kultur). Plattieren eines breiten Konzentrationsbereich wird sichergestellt, dass eine Platte wird einzelne Kolonien haben. Die tatsächliche erforderliche Konzentration hängt von der Toxizität des Krankheits Substrat von Interesse. </li><li> Normalisierung des Vektors und Hsp104 WT Kulturen der OD 600 der Bibliothek und der Platte gleichen Volumina, um das Wachstum der Bibliothek relativ zu den Kontrollen zu vergleichen. </li><li> Um die Auswahl Stringenz zu bewerten, Plate Die Bibliothek auf Glukose (Repression) Medien. Inkubation erfolgt für 2-3 Tage bei 30 ° C, bis Kolonien erscheinen (Abbildung 2). Bewerten Sie die resultierenden Kolonien und im Vergleich zu den Kontrollen. HINWEIS: Oft große und kleine Kolonien erhalten werden, aber keine Korrelation zwischen Koloniegröße und handelnduktivität beobachtet. Bildschirm diese potenziellen Hits mit einem 5-FOA Gegenselektionstechnik (Schritt 4), um die Sequenzierung übertragenen Fehlalarme zu minimieren. </li></ol> 4. 5-FOA Gegenselektion und Spek, Fehlalarme zu eliminieren <ol><li> Streak aus Einzelkolonien in zweifacher Ausfertigung auf Doppel- und Einzel Dropout Medien (zB SD-His-Ura und SD-His-Platten) und über Nacht wachsen bei 30 ºC. Wiederholen Sie mit Vektor- und Hsp104 WT Kontrollen. Plattieren auf SD-His vor 5-FOA erhöht die Effizienz der 5-FOA Schritt durch die Erhöhung der Wahrscheinlichkeit, dass die Zellen das Hsp104-Plasmid verloren haben, wenn sie auf 5-FOA plattiert sind. </li><li> Um Platten vor dem Austrocknen zu verhindern, wickeln Sie das SD-His-Ura Platten in Parafilm und bei 4 ºC während der Durchführung der 5-FOA-Bildschirm. Diese Platten werden schließlich für die Sequenzierung verwendet werden. </li><li> Streak die Kolonien von der SD-His-Platten, um einzelne Kolonien auf 5-FOA-Platten (YPD media + 1g / L 5-FOA) und Inkubation für 1-2 Tage bei 30 &amp;# 186; C, bis einzelne Kolonien erscheinen. Dabei wird 5-FOA in einen toxischen Stoff (5-Fluordeoxyuridin) in Zellen, die das URA3-Gen enthalten, umgewandelt. Somit werden alle Zellen noch beherbergen die Hsp104 Plasmid sterben. </li><li> Streak die Einzelkolonien aus den 5-FOA-Platten in doppelter Ausführung auf SD-Ura und SD-His-Platten. Test 3 Kolonien für jede getroffen, um die Wahrscheinlichkeit, dass mindestens einer Kolonie, für jeden Treffer wird das Hsp104-Plasmid verloren haben, zu erhöhen. Inkubation für 1-2 Tage bei 30 ° C. Kolonien, die Hsp104 Plasmid wird auf SD-His-Platten, aber nicht SD-Ura Platten wachsen verloren haben. </li><li> Wachsen die Stämme, die Hsp104 Plasmide für Spek Assays verloren haben. Wachsen die Stämme in Raffinose Dropout Mediensättigung (zB sraff-His) in 96 Deepwell 2 ml Platten über Nacht bei 30 ºC unter Schütteln. Achten Sie darauf, um die 5-FOA behandelten Kontrollstämme gehören. </li><li> Bereiten Platten für Spotting-Assay. Mit einer Mehrkanalpipette, aliquoten 200 ul Raffinose Dropout Medien (zB sraff-His) proVertiefung einer 96-Well-Platte, Reservierung Spalten 1 und 7 für die gepflegte Kulturen. Aliquots von 250 &amp; mgr; l von jeder der 16 gesättigten Kulturen in den Spalten 1 und 7 der Platte. </li><li> Seriell verdünnten Kulturen 5-fach in jeder Spalte der Platte, gründliches Mischen mit einer Mehrkanalpipette. Entfernen 50 ul der verdünnten Kultur aus den Spalten 6 und 12, um das Endvolumen jeder Vertiefung 200 ul gewährleisten. Dies ist unerlässlich, um auch Schmierblutungen zu gewährleisten. </li><li> Mit einem 96-Loch-Replikator-Werkzeug (Frogger), vor Ort die Kulturen in zweifacher Ausfertigung auf SD-His und SGal-His-Platten. Die Inkubation bei 30 ° C für 2-3 Tage. </li><li> Wählen Sie für die Sequenzierung Stämme, die Toxizität auf den SGal-His-Platten ähnlich dem der Kontrollen zeigen. Verwerfen als Fehlalarme Stämme, die nicht so giftig wie die Kontrollen sind. 5-FOA ist auch häufig auf eine eigene giftig, so verwerfen Stämme, die einen Wachstumsdefekt auf SD-His-Platten oder dass eine wesentlich größere Toxizität aufweisen als die Krankheit Substrat allein ( <strong&gt; 3). </li></ol> 5. Sequenzierung des Hsp104 Varianten durch Kolonie-PCR <ol><li> Scrape ~ 10 ul Hefe aus den SD-His-Ura-Platten in 3 ul 20 mM NaOH in PCR Streifen Rohren und Lyse der Zellen durch Einfrieren und Auftauen. Legen Sie die Streifen Röhrchen in einem -80 ° C Gefrierschrank für 10 min oder in flüssigem Stickstoff, dann bei 99 ° C inkubieren in einem Thermocycler für 10 min. Verdünnen Sie die Stämme bis 100 &amp; mgr; l mit PCR-Wasser. </li><li> Bereiten PCR-Reaktionen: 5 ul PCR-Matrize, 0,5 ul 100 uM jeder Primer, 0,5 ul 10 mM dNTPs, 0,25 ul DNA-Polymerase in PCR-Puffer, und stellen bis zu 25 &amp; mgr; l Gesamtvolumen mit PCR-Wasser. Design-Primer, die randomisierten Bereich plus etwa 100 bp an beiden Enden zu verstärken. Minimierung der Größe des amplifizierten Region, um die Wahrscheinlichkeit einer erfolgreichen Amplifikation erhöhen. Führen Sie einen Standard-PCR-Programm. </li><li> Analyse Proben durch Agarosegelelektrophorese zur Amplifikation eines PCR-Produkt der geeigneten siz bestätigene. Wiederholen PCR, wenn keine vorhanden ist. HINWEIS: PCR Ausfall ist in der Regel durch zu viel Hefe in Schritt 5.1 verwendet. </li><li> Vorbereitung der Proben für die DNA-Sequenzierung. Entfernen PCR-Primer entweder durch PCR Aufreinigung oder unter Verwendung eines PCR-Produkt Bereinigung Reagens wie Exonuklease I und Shrimp Alkaline Phosphatase-Enzym (ExoSAP-IT), um einzelsträngige DNA abzubauen. Dieses Reagenz enzymatisch abbaut unincorporated Primern und dNTPs, so dass für einen höheren Durchsatz. </li><li> Sequenz, die die PCR-Produkte. Achten Sie darauf, gründlich zu analysieren Sequenzierung Chromatogramme für Mutationen. Mutationen können als Mischung von zwei Nukleotiden an einer bestimmten Stelle (4) beobachtet werden, wie Hefe häufig beherbergen mehreren Plasmiden. </li></ol>

<ol>
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The authors have nothing to disclose.

Hier präsentieren wir unseren Ansatz zur Isolierung potenziert Hsp104-Varianten, die die Toxizität von krankheitsassoziierten Substraten unter Verwendung von Hefe proteinopathy Modelle zu unterdrücken. Mit diesem Ansatz können große Bibliotheken von Varianten im Hochdurchsatz untersucht werden, wobei die einzige Einschränkung ist die Anzahl der Varianten, die das 5-FOA sekundären Sieb passieren. Durch Durchführen dieser Schritte in 96-Well-Format, routinemäßig auf die wir bis zu 200 Zugriffe zu einem Zeitpunkt...

Hefe proteinopathy Modelle wurden für Proteinfehlfaltung Störungen, einschließlich amyotropher Lateralsklerose (ALS) und Parkinson-Krankheit (PD) 1-3 entwickelt. Expression der Proteine ​​TDP-43 und FUS, die in ALS-Patienten misfold, sind toxisch und mislocalize cytoplasmatischen Aggregaten in Hefe 1,2 bilden. Ebenso Expression von α-Synuclein (α-syn), die in PD gebracht wird, ist toxisch und mislocalizes cytoplasmatischen Aggregaten in Hefe 3 zu bilden. Diese Merkmale rekapitulieren Phänotypen bei Patienten mit diesen Erkrankungen 4,5. So, Hefe-Modelle bieten eine nützliche Plattform für das Screening für Proteine ​​oder kleine Moleküle, die verhindern oder rückgängig diese Phänotypen 2,6-13. Wir interessieren uns für die Entwicklung von Proteinen, die in der Lage umzukehren Aggregation und Toxizität durch TDP-43, FUS und α-syn sind. Wir konzentrieren uns auf Hsp104, ein AAA + Protein aus Hefe, die einzigartig in der Lage Disaggregieren Proteine ​​sowohl fr istom amorphen Aggregaten und Amyloid in Hefe, es hat noch keine menschliche Homolog 14,15. Hsp104 fein abgestimmt, um endogene Hefe Prionen zerlegen und hat nur begrenzte Fähigkeit, Substrate in menschlichen neurodegenerativen Erkrankungen in Verbindung gebracht, die es nie gewöhnlich begegnet 16,17 zerlegen. So wollen wir verbesserte Versionen von Hsp104, die in der Lage, wirksam zu zerlegen diese menschlichen Substrate sind Ingenieur. Dazu konstruieren wir große Bibliotheken Hsp104 Varianten mit fehleranfällige PCR; Diese Bibliotheken können mit den Hefe proteinopathy Modelle 17 zu sehen sein. Wir haben eine Domain-bezogenen Ansatz zur Konstruktion und Screening-Bibliotheken angenommen, wie Hsp104 ist sehr groß 17. Wir konzentrierte sich zunächst auf die Mitteldomäne (MD) von Hsp104 17, obwohl ähnliche Ansätze können verwendet werden, um andere Bereiche zu screenen. Diese Modelle ermöglichen für disaggregase Aktivität direkt Screening, im Gegensatz zu alternativen Techniken wie Oberflächen Display, das andere istricted zur Überwachung Bindungs ​​18 verwenden. Unser Protokoll auf zwei Screeningschritte (Abbildung 1). Zunächst werden Hsp104-Varianten, die die Toxizität der Krankheit Substrat in Hefe unterdrücken, ausgewählt ist. Um dies zu tun, Varianten der Hsp104 und krankheitsassoziierten Substrat in Δ hsp104 Hefe kotransformiert. Wir beschäftigen Δ hsp104 Hefe Hsp104 Sequenzraum in Abwesenheit von Wildtyp (WT) Hsp104 17 zu erkunden. Wichtig ist, dass das Löschen von Hsp104 nicht beeinträchtigt α-syn, FUS oder TDP-43 Toxizität in Hefe und die Expression von Hsp104 WT eine minimale Rettungs 1,13,17. Die Hefe wird dann auf induzierenden Medium, um die Expression der beiden Proteine ​​induzieren plattiert. Hefe beherbergen Hsp104 Varianten Toxizität der Krankheit assoziierten Substrat confer Wachstum der Kolonie zu unterdrücken. Diese Varianten werden zur weiteren Analyse ausgewählt, während Kolonien aufrechterhalten Varianten, die Toxizität sterben nicht unterdrücken müssen. JedochFehlalarme sind ein wesentliches Problem in diesem Bildschirm. Expression des TDP-43, FUS und α-syn hoch toxisch, was einen starken Selektionsdruck für das Auftreten von spontanen genetischen Suppressoren der Toxizität nicht mit dem Hsp104 Variante erstellt exprimiert. Somit haben wir einen zweiten Bildschirm, der auch relativ hohen Durchsatz, diese unspezifischen Toxizität Drücker 17 zu eliminieren. In diesem zweiten Bildschirm werden ausgewählte Hefe mit 5-Fluorootic Säure (5-FOA) behandelt, um entgegenzuwirken Select für den Hsp104 Plasmid 19. Die Stämme werden dann auf Substrat (TDP-43, FUS oder α-syn) Toxizität gegen Fleckentest, um sicherzustellen, dass die Toxizität des Substrats nach dem Verlust des Hsp104 Plasmid wieder bewertet. Somit Hefe toxisch ist in diesem Sekundärbild wiederhergestellt vermutlich ursprünglich angezeigte Toxizität Unterdrückung aufgrund der Anwesenheit des Hsp104-Variante. Diese Hefe werden als &quot;Hits&quot; bezeichnet, und die Hsp104 Plasmid sollte danngewonnen und sequenziert, um die Mutationen in der Hsp104-Gens 17 (Figur 1) zu identifizieren. Alle Treffer soll dann durch den Bau die Mutation unabhängig unter Verwendung ortsgerichtete Mutagenese und dann erneute Prüfung auf Toxizität Unterdrückung rückbestätigt werden. Die Anwendungsmöglichkeiten für dieses Protokoll sind breit. Mit Hilfe dieser Methoden können Bibliotheken von jeder Art von Proteinvarianten, die zur Toxizität von jedem Substrat Protein, das in Hefe toxisch ist drücken gescreent werden.

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			<td height="20" style="height:20px;">Name</td>
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			<td height="20" style="height:20px;">GeneMorphII EZClone Domain Mutagenesis Kit</td>
			<td>Agilent</td>
			<td>200552</td>
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			<td height="20" style="height:20px;">150mm Petri dishes</td>
			<td>Falcon</td>
			<td>351058</td>
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			<td height="20" style="height:20px;">5-Fluorootic Acid</td>
			<td>Research Products International</td>
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			<td height="20" style="height:20px;">96-DeepWell 2mL Plates</td>
			<td>Eppendorf</td>
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			<td height="20" style="height:20px;">96 bold replicator tool</td>
			<td>V&#38;P Scientific</td>
			<td>vp-404</td>
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			<td height="20" style="height:20px;">ExoSAP-IT</td>
			<td>Affymetrix</td>
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isolating potentiated hsp104 variants using yeast proteinopathy models

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

Wir haben eine Bibliothek von Hsp104 Varianten in der Mitteldomäne randomisiert konstruiert und gesiebt es zur Unterdrückung von TDP-43 Toxizität. Die Bibliothek wurde transformiert und auf Glucose und Galactose-Platten (2) überzogen, um die Stringenz der Bildschirm zu bewerten. Einzelne Kolonien wurden ausgewählt und die Stämme wurden Zähler mit 5-FOA die Hsp104 Plasmid beseitigen ausgewählt. Diese Stämme wurden dann bewertet, diese Toxizität zu bestätigen war auf TDP-43 allein, ohne den Hsp...

Watch this Scientific Journal Video about Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models at JoVE.com

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

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

Isolieren Potenzierte Hsp104 Varianten unter Verwendung von Hefe Proteinopathy Models

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

isolating-potentiated-hsp104-variants-using-yeast-proteinopathy-models

Research

JoVE Journal

Biology

8.1K Aufrufe.  Perelman School of Medicine at the University of Pennsylvania. Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Serie: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Eine quantitative Bewertung der Hefe Lipidome mit Elektrospray-Ionisation-Massenspektrometrie

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Forschung

Biologie

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

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

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

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

Genetic Studies of Human DNA-Reparatur-Proteinen unter Verwendung von Hefe als Modellsystem

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

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Hefekolonie Embedding-Methode

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

Principles of Surgery Nager für die New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Manuelle Restraint und Common Compound Verabreichungswege bei Mäusen und Ratten

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

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 Hefeplasmid Überexpression Bildschirm

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

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Reinigung von Hsp104, ein Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Maus Sperma Kryokonservierung und Recovery mit der I · Cryo-Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

Nachweis seltener Varianten von Genomic Pooled-Sequenzierung mit SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Screening für Funktionelle nichtkodierenden genetischen Varianten Mit elektrophoretische Mobilitäts-Shift-Assay (EMSA) und DNA-Affinitätspräzipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...