Forschung wurde durch die NIH 1R15GM094770 finanziert.

1. Colony Isolierung und Fixierung <ol><li> Inkubieren 300 Kolonien auf Agar-Medium für die angegebene Zeit (eine isolierte Kolonie sollte 1-2 mm im Durchmesser). </li><li> Entfernen Kolonie (face up) und die zugrunde liegenden Medium mit einem schmalen Spachtel. </li><li> Einige Tropfen von 2% Agar 42 ° C auf einem Objektträger mit 1 mL Pipetman Spitze und sofort Platz Kolonie auf Agar Gesicht, bevor es erstarrt. </li><li> Einige Tropfen von 2% Agar 42 ° C auf Kolonie und damit zu verfestigen. </li><li> Tragen Sie Handschuhe für alle Schritte für den Rest dieses Protokolls </li><li> Trimmblock mit Rasierklinge und legen Sie sie in einem 3,5 ml Borosilikatglas Schraubverschluss Fläschchen mit 2% Paraformaldehyd / 2% Glutaraldehyd Fixativ für 7 Tage bei 4 ° C. </li></ol> 2. Wäscht und Osmium-Behandlung <ol><li> Nach ca. 1 Woche, waschen Agarblöcken auf Eis unter Laborabzug durch Inkubation zweimal für 15 Minuten mit genügend 0,15 Natriumcacodylat (pH 7,2), um vollständig zu bedecken die Kolonie (ca. 1,5 mL), dann noch zweimal für 5 Minuten mit dem gleichen Volumen 1X OS-Puffer (100 mM KH 2 PO 4 10 mM MgCl 2, pH 6,0). </li><li> Fügen Sie das gleiche Volumen von 1% OsO 4 [2 mL OsO 4 (2%) + 2 mL 2x OS Buffer], um Fläschchen (auf Eis) auf die Agar-Blöcke unter Laborabzug für 1 Stunde decken dann zweimal waschen mit der gleichen Menge 1X OS-Puffer für jeweils 10 Minuten. </li><li> Lassen Sie Vials über Nacht bei 4 ° C in 1X OS-Puffer. </li><li> Fixativ und alle Wäschen in diesem Schritt sind als gefährlicher Abfall zu entsorgen. Alle Pipetten mit OsO 4 gespült 3X mit Pflanzenöl. </li></ol> 3. Waschen und Dehydration <ol><li> Am nächsten Morgen waschen Agarblöcken zweimal auf dem Eis mit dem gleichen Volumen wie oben mit kaltem Wasser für jeweils 10 Minuten mit Pasteurpipette. </li><li> Wash nacheinander mit dem gleichen Volumen von 25%, 50%, 75%, 95% Ethanol für jeweils 10 Minuten um 2 Waschgänge mit 100% Ethanol für 10 Minuten. Lassen Sie über Nacht bei 4 ° C in 100% Ethanol. </li><li> Alle Wäschen in diesem Schritt sind als gefährlicher Abfall zu entsorgen. </li></ol> 4. Spurr der Vorbereitung <ol><li> Am nächsten Morgen (so früh wie möglich) wiegen die folgenden Lösungen (EM Sciences) in einen Einweg-Plastikbecher unter einer Abzugshaube zu Spurr-Reagenz vorbereiten. Für alle weiteren Arbeiten mit diesem Reagens, weiterhin einen Abzug verwenden. (ERL 4221-Lösung 5 Gramm) (DER 736-Lösung 4 Gramm) (NSA Lösung 13 Gramm) </li><li> Mischung dieser Lösungen (langsam) für 20 Minuten in der Kapuze mit einem Rührstab </li><li> Add DMAE-Lösung 0,15 Gramm und rühren 20 Minuten wie oben. </li><li> Degas obigen Mischung für 1-2 Stunden in der Kapuze. </li></ol> 5. Spurr die Infiltration <ol><li> Sobald Spurr ist gemacht waschen Agarblöcken 5 mal mit gleichem Volumen wie oben von 100% Raumtemp Ethanol für jeweils 10 Minuten. Nach dem letzten Ethanol waschen entfernen Sie den Ethanol mit einer Pasteurpipette, so dass die restliche Ethanol deckt gerade die Agar-Blöcke. </li><li> Fügen Sie etwa die gleiche Menge Spurr-Reagenz (ca. 0,5 mL) und drehen am Rad Fläschchen für 15 Minuten bei Raumtemperatur und dann lassen Sie es 30 Minuten stehen. Nach 30 Minuten entfernen Sie die Lösung vollständig mit einer Pasteurpipette, fügen Spurr auf Agar-Block (ca. 1,5 ml) Deckel ist, drehen Fläschchen auf Rad für 15 Minuten bei Raumtemperatur und lassen Sie es 30 Minuten stehen. Wiederholen Sie die Spurr Nachfolger, Rotation und Inkubation 3 weitere Male. </li><li> Nach der letzten 30 Minuten, entfernen Spurr-Reagenz und fügen frische Spurr ist, um die Blöcke zu decken. Lassen Sie die Agar-Blöcke, um bei Raumtemperatur für 4 Stunden stehen. </li><li> Ersetzen Sie die Spurr und drehen über Nacht. </li><li> Am Morgen ersetzen Spurr und verlassen rotierenden bis zum nächsten Tag. </li><li> Am nächsten Morgen statt Spurr ist in nummerierte Silikonformen (Fisher Scientific) nur auf den Boden der Formen bedecken dann add Agarblöcken die Formen. Inkubation bei 60 ° C für 4 Stunden </li><li> Nach 4 Std., runden die Formen, mit mehr Spurr und Inkubieren bei 60 ° C über Nacht. </li><li> Wenn nach dem Entfernen der Blöcke aus den Formen der Blöcke noch leicht flexibel, um die Formen wieder und inkubieren eine zusätzliche 2 Tage </li></ol> 6. Sectioning <ol><li> Ein Leica Ultracut S Mikrotom wurde auf 0,5 μ dicke Schnitte für die Lichtmikroskopie und 0,1 μ dünne Schnitte für die Übertragung EM schneiden. </li><li> Für die Lichtmikroskopie, wie Abschnitte geschnitten wurden, wurden sie auf einen Rückgang von dH 2 O gelegt und getrocknet auf einer 52 ° C Wärme zu blockieren. Getrocknete Schnitte wurden dann mit einem Rückgang um 1,0% Toluidinblau, 1% Sodium Borate für 5-15 Sekunden gefärbt. Nach der Färbung wurden die Schnitte sofort unter einem Strom von Wasser gewaschen, getrocknet, bedeckt in Eindeckmittel (KPL) und ein Deckglas über die Probe versiegelt. </li></ol> Die breite Relevanz der Messung von Musterbildung in der Hefe und anderen Mikroorganismen wurden bisher 1 Bewertung. Besonders hervorzuheben ist die verbesserte Funktionalität der organisierten Mikrobenkolonien relativ unorganisiert Gemeinden. Das beschriebene Verfahren ist eine Abwandlung einer Methode zur Einbettung von größeren spezialisierten Kolonien 2 und eine kurze Beschreibung unserer Modifikationen wurde 3 veröffentlicht. Ein Beispiel für eine lichtmikroskopische Aufnahme eines Schnitts durch das Zentrum einer Kolonie von wilden S. cerevisiae Hefe Kolonie ist in Abbildung 1 dargestellt. Asci, Pseudohyphen und eiförmig Hefe sind leicht zu unterscheiden, und die Region der Kolonie Eindringen in die zugrunde liegenden Agar ist auch offensichtlich. Ein Beispiel für eine elektronenmikroskopische Aufnahme eines Labor-Hefestamm (W303 Hintergrund) ist in Abbildung 2 dargestellt. Das Bild ist aus einer Region der Kolonie, die eine hohe Frequenz von sporenbildenden Zellen. <img src="/files/ftp_upload/2510/2510fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Lichtmikroskopie einer wilden Hefe Kolonie. Diese Hefe (YPS133) wurde vor kurzem von Baum Exsudate 4 isolierte. Die Kolonie wurde für 6 Tage auf YNA Medium 5 vor dem Schneiden inkubiert. Offene Pfeile zeigen repräsentative Asci, gefüllt Pfeile zeigen repräsentative eiförmig vegetativen Zellen, gefüllt Pfeilspitzen zeigen Ketten von langgestreckten Zellen (Pseudohyphen) und Asci. <img src="/files/ftp_upload/2510/2510fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Elektronenmikroskopie eines Labor-Hefestamm. Die Kolonie der SH1020 (W303 Hintergrund) war für 6 Tage auf YNA Medium vor dem Schneiden 3 inkubiert. Bild zeigt einen Bereich des Abschnitts mit einer hohen Frequenz von Asci. Ein Pfeil zeigt die bi-Schicht-Struktur der Spore Wand. <img src="/files/ftp_upload/2510/2510fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Die Quantifizierung der Verteilung der sporenbildenden Zellen in Kolonien: A) ein Raster mit 15 angrenzenden Rechtecke entlang ihrer Längsseite gestapelt wird auf den zentralen Bereich eines Bildes von einer Kolonie Abschnitt überlagert. Das Gitter wird so skaliert, ohne dabei die Proportionen konstant, nur deckt den oberen und unteren Rand der Kolonie. (B &amp; C) Der Anteil der sporenbildenden Zellen in jedes Rechteck bezogen auf das Gesamtgewicht Zellen in diesem Rechteck wird durch visuelle Inspektion von Bildern aus 4-Tage alte (B) und 8-Tage-alten (C) Kolonien bestimmt. Zum Beispiel entspricht das Rechteck am oberen Rand der Kolonie (mit &quot;1&quot;) auf der linken Seite des Diagramms. Mittel von 4 Kolonien gezeigt; die Fehlerbalken zeigen die std. Fehler.

<ol>
	<li>Kimmel, A. R., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+symmetries:+regulation+of+Dictyostelium+development+through+chemoattractant+and+morphogen+signal-response.">Breaking symmetries: regulation of Dictyostelium development through chemoattractant and morphogen signal-response.</a> Curr Opin Genet Dev. 14, 540-540 (2004).</li><li>Palkova, Z., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+within+a+community:+benefit+to+yeast+long-term+survival.">Life within a community: benefit to yeast long-term survival.</a> FEMS Microbiol Rev. 30, 806-806 (2006).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significances+of+bacterial+colony+patterns.">The significances of bacterial colony patterns.</a> Bioessays. 17, 597-597 (1995).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+bacterial+populations+as+multicellular+organisms.">Thinking about bacterial populations as multicellular organisms.</a> Annu Rev Microbiol. 52, 81-81 (1998).</li><li>Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+stalk-like+structures+in+Saccharomyces+cerevisiae.">Multicellular stalk-like structures in Saccharomyces cerevisiae.</a> J Bacteriol. 180, 3992-3992 (1998).</li><li>Scherz, R., Shinder, V., Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+analysis+of+Saccharomyces+cerevisiae+stalk-like+structures+reveals+spatial+organization+and+cell+specialization.">Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization.</a> J Bacteriol. 183, 5402-5402 (2001).</li><li>Piccirillo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rim101p/PacC+pathway+and+alkaline+pH+regulate+pattern+formation+in+yeast+colonies.">The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.</a> Genetics. 184, 707-707 (2010).</li><li>Sniegowski, P. D., Dombrowski, P. G., Fingerman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+and+Saccharomyces+paradoxus+coexist+in+a+natural+woodland+site+in+North+America+and+display+different+levels+of+reproductive+isolation+from+European+conspecifics.">Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation from European conspecifics.</a> FEMS Yeast Res. 1, 299-299 (2002).</li><li>Piccirillo, S., Honigberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+patterning+and+invasive+growth+in+wild+and+domesticated+yeast+colonies.">Sporulation patterning and invasive growth in wild and domesticated yeast colonies.</a> Res Microbiol. 161, 390-390 (2002).</li><li>Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environ Microbiol. , (2009).</li></ol>

Die vorgestellte Methode zeigt, die inneren Strukturen der Kolonien. Da die Methode ist effektiv bei der Bestimmung Muster von Zelltypen in einer Reihe von S. cerevisiae-Stämme mit unterschiedlichen Morphologien Kolonie, und auch in einer verwandten Spezies S. paradoxus 5, ist das Verfahren auch am ehesten auf eine breite Palette von Pilzen und anderen Mikroorganismen zu arbeiten. Ein entscheidender Schritt für den Erfolg der Methode ist, um sicherzustellen, dass die ge...

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<td>Osmium tetroxide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19152</td>
<td>&nbsp;</td>
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<td>Silicone embedding molds</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>NC 9975029</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 15004</td>
<td>ERL 4221</td>
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<td>Epoxy resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
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<td>Nonenyl Succinic Anhydride</td>
<td>&nbsp;</td>
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yeast colony embedding method

Strukturierung von verschiedenen Zelltypen im Embryo ist ein wichtiger Mechanismus in Metazoen Entwicklung. Gemeinschaften von Mikroorganismen, wie Kolonien und Biofilmen zeigen auch Muster von Zelltypen. Zum Beispiel in der Hefe S. cerevisiae, sind sporenbildenden Zellen und pseudohyphal Zellen nicht gleichmäßig in Kolonien verteilt. Die funktionelle Bedeutung der Strukturierung und der molekularen Mechanismen, die diese Muster zugrunde liegen, sind noch weitgehend unverstanden. Eine Herausforderung in Bezug auf die Untersuchung Muster von Zelltypen in Pilzkolonien ist, dass im Gegensatz zu vielzelligen Gewebe, Zellen in Kolonien relativ schwach miteinander verbunden. Insbesondere sollten Sie Pilzkolonien nicht die gleichen umfangreichen Ebene der extrazellulären Matrix in den meisten Geweben gefunden. Hier berichten wir über eine Methode zum Einbetten und Schneiden Hefekolonien, dass das Innere Muster von Zelltypen zeigt in diesen Kolonien. Die Methode kann verwendet werden, um dicke Abschnitte vorbereiten (0,5 μ) sinnvoll für die Lichtmikroskopie und dünne Schnitte (0,1 μ) für Transmissions-Elektronenmikroskopie werden. Asci und pseudohyphal Zellen können leicht von eiförmige Hefezellen mittels Lichtmikroskopie unterschieden werden, während die innere Struktur dieser Zellen können mit Hilfe von EM. Die Methode basiert auf umliegenden Kolonien mit Agar-Agar, infiltrieren sie mit Spurr das Medium, und dann Schnitte basieren. Kolonien mit einem Durchmesser im Bereich von 1-2 mm eignen sich für dieses Protokoll. Zusätzlich zu visualisieren das Innere der Kolonien, erlaubt das Verfahren die Visualisierung der Region der Kolonie, dass die zugrunde liegenden Agar eindringt.

Watch this Scientific Journal Video about Yeast Colony Embedding Method at JoVE.com

Yeast Colony Embedding Method

Eine Methode zur Einbettung von Hefe-Kolonien ermöglicht Schnitte für Licht-und Elektronenmikroskopie. Dieses Protokoll erlaubt die Bestimmung der Verteilung der sporenbildenden Zellen und pseudohyphal Zellen innerhalb Kolonien bietet ein neues Werkzeug zum Verständnis der Organisation von Zelltypen innerhalb eines Pilz-Gemeinschaft.

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 ...

Research

JoVE Journal

Biology

11.4K Aufrufe.  University of Missouri - Kansas City. Eine Methode zur Einbettung von Hefe-Kolonien ermöglicht Schnitte für Licht-und Elektronenmikroskopie. Dieses Protokoll erlaubt die Bestimmung der Verteilung der sporenbildenden Zellen und pseudohyphal Zellen innerhalb Kolonien bietet ein neues Werkzeug zum Verständnis der Organisation von Zelltypen innerhalb eines Pilz-Gemeinschaft.

Serie: Yeast Colony Embedding Method

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Mess-replikative Lebensspanne in der Hefe

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Forschung

Biologie

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Reinigung von Mitochondrien aus Hefezellen

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video bioinformatischen Analyse von humanen embryonalen Stammzellen Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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, ...

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.

Competitive Genomic Screens von Barcoded Hefe Bibliotheken

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

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

A Quantitative Assay für Insulin-exprimierenden Koloniebildende Vorläufern

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Chromatographische Reinigung von hoch aktiven Hefe Ribosomen

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Die Soft-Agar Koloniebildungstest

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Oberfläche Verbreitung und Immunfärbung von Hefechromosomen

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

Mikroskopie von Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...