研究是由美国国立卫生研究院1R15GM094770。

1。殖民地隔离和固定<ol><li>指定的时间孵育300琼脂培养基上菌落（一个孤立的殖民地应在直径1-2毫米）。 </li><li>删除殖民地（正面朝上）及相关的媒介，使用一个狭窄的铲。 </li><li>将数滴2％琼脂42 ° C的载玻片，使用琼脂面对1毫升pipetman提示，并立即将殖民地之前巩固。 </li><li>置于42℃菌落数滴2％琼脂，并允许以巩固。 </li><li>穿手套的所有步骤，通过本协议的其余部分</li><li>修剪块刀片，并将其放置在一个3.5毫升硼硅螺丝帽7天的2％多聚甲醛/ 2％戊二醛固定液的小瓶4 ° C。 </li></ol> 2。清洗和锇治疗<ol><li>约1周后，洗下孵育15分钟，两次不够0.15米的钠cacodylate（pH 7.2）中，同体积的完全覆盖殖民地（约1.5毫升），然后5分钟两次化学通风柜在冰上的琼脂块1X操作系统缓冲区（100毫米KH 2 PO 4 10毫米氯化镁2，pH值6.0）。 </li><li>加入相同体积的1％OSO 4 [2 OSO 4毫升（2％）+ 2毫升2个操作系统的缓冲区]小瓶（冰）覆盖琼脂块化学通风柜下1小时，然后洗两次相同数额1X操作系统的缓冲区，每次10分钟。 </li><li>离开瓶过夜，在4 ° C的1X操作系统的缓冲区。 </li><li>固定液，并在此步骤中的所有洗作为危险废物处置。所有含有OSO 4的移液器冲洗用植物油的三倍。 </li></ol> 3。清洗和脱水<ol><li>第二天早晨洗两次冰琼脂块相同体积冷水以​​上，每次10分钟使用巴斯德吸管。 </li><li>顺序相同体积的25％，50％，75％，95％乙醇10分钟，每2 100％乙醇10分钟洗涤，冲洗。离开一夜之间在4 ° C的100％的乙醇。 </li><li>这一步洗处置危险废物。 </li></ol> 4。 Spurr的准备<ol><li>第二天早晨，（尽快）权衡成一次性塑料烧杯中的下列解决方案下通风柜准备Spurr试剂（EM）。使用这种试剂对于所有后续工作，继续使用通风柜。 （广深港高速铁路4221解决方案5克） （DER 736解决方案4克） （NSA的解决方案，13克） </li><li>搅拌20分钟罩，使用搅拌棒的这些解决方案（慢慢） </li><li>添加0.15克的DMAE的解决方案，并搅拌20分钟以上。 </li><li>德加罩在1-2小时以上的混合物。 </li></ol> 5。 Spurr的渗透<ol><li>只要Spurr的是洗琼脂块，与同体积的5倍以上100％的空间，每次10分钟的温度乙醇。在最后的乙醇洗涤，除去乙醇，用巴斯德吸管，使剩余的乙醇只是涵盖的琼脂块。 </li><li>新增约等量Spurr的试剂（约0.5毫升）和旋转为15分钟，在室温下对车轮的小瓶，然后让站在30分钟。 30分钟后删除的解决方案，完全使用巴斯德吸管，添加Spurr的覆盖琼脂块（约1.5毫升），对轮旋转瓶在室温下15分钟，静置30分钟。重复Spurr的更换，旋转和孵化3次以上。 </li><li>最后30分钟后，取出Spurr的试剂，并补充新鲜Spurr的覆盖块。允许琼脂块，在室温下静置4小时。 </li><li>更换Spurr的旋转过夜。 </li><li>在早上取代Spurr和旋转，直到次日离开。 </li><li>第二天早晨，地方Spurr的编号硅胶模具（尔科技），只是覆盖模具的底部，然后加入琼脂块的模具。在60℃孵育4小时</li><li> 4小时后，关闭模具的顶部，Spurr的，在60 ° C过夜孵育。 </li><li>如果取出后从模具块的块还是稍微灵活的，返回的模具，并培育一个额外的2天</li></ol> 6。切片<ol><li>徕卡Ultracut小号切片机是用于切割0.5μ厚切片光镜和0.1μ薄片传输的EM。 </li><li>光镜，部分被切断，他们被安置在生署2下降，并在52℃加热块干燥。干节，然后沾上下降1.0％甲苯胺蓝，1％硼酸钠为5-15秒。染色后，部分被立即下的水流洗净，晒干，在安装媒体（KPL）覆盖，并盖玻片样品密封。 </li></ol>酵母和其他微生物的测量模式形成的广泛的相关性已审查以前1。特别值得注意的是增加功能相对混乱的社区组织的微生物菌落。 所描述的方法中嵌入较大的专门殖民地2的方法修改的简短描述了我们的修改已出版了3 。 野生S.殖民地中心的一个部分，通过光显微镜的一个例子酵母酵母殖民地如图1所示。 ASCI，假菌丝及卵圆形的酵母很容易分辨，和入侵的底层琼脂的殖民地地区也很明显。 电子显微镜实验室的酵母株（W303背景）的一个例子是如图2所示。该图像是从一个区域含有孢子细胞的高频率的殖民地。 <img src="/files/ftp_upload/2510/2510fig1.jpg" alt="图1" /> 图1。野生酵母殖民地的光镜。这酵母（YPS133）最近被隔离树分泌物4。培养6天YNA中等5到切片前殖民地。打开的箭头指示代表ASCI，充满箭头指示代表卵形营养细胞，充满箭头指示链拉长细胞（假菌丝）和ASCI。 <img src="/files/ftp_upload/2510/2510fig2.jpg" alt="图2" /> 图2电子显微镜实验室酵母菌株。 SH1020（W303背景）菌落培养6天YNA中等，前切片 3 。图像显示了与高频率的ASCI部分地区。箭头指示的孢子壁的双向层结构。 <img src="/files/ftp_upload/2510/2510fig3.jpg" alt="图3" /> 图3。在殖民地孢子细胞分布的定量分析 ：一）包含沿其长边堆放的15个毗邻的矩形网格叠加在中部地区的一个殖民地部分的形象。网格缩放，同时保持其比例不变，只是涵盖的顶部和底部的殖民地。 （B＆C）在每个矩形在该矩形的细胞总数相对孢子细胞的一小部分是图像的目视检查，为期4天的老（B）和8日龄（三）殖民地决定。例如，在殖民地上方的矩形（标有“1”）对应的图形的左侧。平均4殖民地显示错误栏显示STD。错误。

<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>

方法揭示了殖民地的内部结构。由于该方法是有效S 的范围内确定的细胞类型的模式酵母株不同的菌落形态，而且在一个相关的物种S. paradoxus 5，该方法也有可能工作的真菌和其他微生物的广泛。 方法的成功的一个关键的步骤是，以确保整个殖民地，包括殖民地的顶部，是在整个协议的琼脂包裹。无论是殖民地完全包裹在琼脂可以切片光镜和甲苯胺蓝染?...

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		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Osmium tetroxide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19152</td>
<td>&nbsp;</td>
<tr>
<td>Silicone embedding molds</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>NC 9975029</td>
<td>&nbsp;</td>
<tr>
<td>Cycloaliphatic epoxide resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 15004</td>
<td>ERL 4221</td>
</tr>
<tr>
<td>Epoxy resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13000</td>
<td>DER 736</td>
</tr>
<tr>
<td>Nonenyl Succinic Anhydride</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19050</td>
<td>NSA</td>
</tr>
<tr>
<td>2-Dimethyl aminoethanol	</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13300</td>
<td>DMAE</td>
</tr>
<tr>
<td>Mounting media</td>
<td>&nbsp;</td>
<td>KPL</td>
<td>71-00-16</td>
<td>&nbsp;</td>
<tr>
<td>Rotating wheel</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>Pelco 1055</td>
<td>&nbsp;</td>
<tr>
<td>Microtome</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>Ultracut S</td>
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yeast colony embedding method

不同类型的细胞在胚胎的图案是一个后生发展的关键机制。殖民地和生物膜的微生物，如社区也显示细胞类型的模式。例如，在酵母S.的酵母 ，孢子细胞和假菌丝细胞不是均匀分布在菌落。图案和背后的分子机制，这些模式的功能的重要性仍然知之甚少。 调查真菌菌落的细胞类型模式的一个挑战是，不像后生动物的组织，细胞在殖民地比较弱连接到另一个。特别是，真菌菌落不包含在大多数组织中发现的细胞外基质的广泛的同一水平。在这里，我们报告上嵌入和切片酵母殖民地，揭示了在这些殖民地的细胞类型的内部模式的方法。该方法可用于准备厚切片，光镜和超薄切片（0.1μ）适合透射电子显微镜（0.5μ）有用。 ASCI和假菌丝细胞可以很容易地区别于卵形酵母细胞，通过光镜，而这些细胞的内部结构可以由EM观察。 该方法是基于对菌落周围的琼脂，渗透Spurr的培养基，然后切片。菌落直径在1-2毫米的范围内适用于本议定书。此外，以可视化的内部殖民地，该方法允许侵入底层琼脂的殖民地地区的可视化。

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

Yeast Colony Embedding Method

嵌入酵母菌菌落，允许光学和电子显微镜切片的一种方法。此协议允许孢子细胞的分布和假菌丝细胞内提供了一个新的工具，对理解组织类型的细胞内一种真菌社会的殖民地的决心。

酵母殖民地嵌入方法

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 次观看.  University of Missouri - Kansas City. 嵌入酵母菌菌落，允许光学和电子显微镜切片的一种方法。此协议允许孢子细胞的分布和假菌丝细胞内提供了一个新的工具，对理解组织类型的细胞内一种真菌社会的殖民地的决心。

视频: 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.

在芽殖酵母中的自我复制寿命测量

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

科研

生物学

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.

从酵母细胞线粒体的纯化

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

High-throughput Yeast Plasmid Overexpression Screen

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

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

高通量酵母质粒过表达屏幕

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

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

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

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.

一个定量测定胰岛素表达细胞集落形成先觉

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.

高活性酵母核糖体的色谱分离纯化

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

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

检查荧光模式Cryosectioning酵母社区

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

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.

软琼脂集落形成分析

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.

表面扩散和酵母染色体免疫染色

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.

裂殖酵母性生命周期的显微镜

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