作者感谢勒Scriwanek与图编制的援助。 FR是由ECHO（700.59.003）的支持，ALW打开程序（821.02.017和822.02.014），DFG-NWO合作（DN82-303）和ZonMW VICI（016.130.606）资助。

1，原生质球的制备<ol><li>过夜孵育的酵母在30℃下在10毫升的实验设计来确定适当的介质中。 </li><li>一天后，用光度计测定培养物的光密度在600nm（OD 600），在相同的培养基稀释细胞至OD 600为0.2-0.4，并将它们生长至指数生长期除非的设计实验需要不同的条件。细胞处于指数生长期时，培养具有OD 600为1-2。 </li><li>收集离心10 OD 600细胞等价物，在3,500 xg离心5分钟，在50毫升的试管。离心后，弃上清。 </li><li>重悬细胞沉淀在5毫升100毫摩尔PIPES（pH 9.6）中，10 mM的二硫苏糖醇温育于30℃下10分钟。 </li><li>通过离心再次收集细胞，在3,500 xg离心5分钟。弃上清。 </li><li>悬浮细胞我ñ5毫升培养基（由实验的​​设计确定）含1M山梨醇和5mg溶解酶，并在30℃孵育混合物，轻轻摇动30分钟。 </li><li>离心细胞悬浮液以300×g离心5分钟，收集沉淀级分，其对应于球状体。弃上清。 </li><li>重悬原生质球在960微升冰冷的培养基（培养基由实验的设计确定）含1M山梨糖醇，将混合物转移至冰冷的2 ml离心管。 </li></ol> 2，吸收纳米金<ol><li>使用移液管，轻轻混合的原生质球用4纳摩尔再悬浮于40μl水中带正电的纳米金颗粒。该混合物的终体积必须为1毫升。 </li><li>放置在冰上将得到的细胞悬浮液15分钟。 </li><li>转移的悬浮液在室温下孵育它为所需要的时间，以允许内化纳米金通过电子邮件ndocytosis。 注：一个5分钟的摄取将主要导致内吞囊泡和早期内涵体室的标签，而15分钟的摄取将允许标记整个内体系统（早期和晚期内涵体）。潜伏期较长时间（超过30分钟）也将允许标注液泡。 注：脉冲追踪标记实验中不能用这种方法进行的。因此，LE的分析标记时，纳米金也将在下午和INEE发现。 </li></ol> 3，固定和切片<ol><li>加入1 ml的双重强度固定液停止纳米金摄取[4％多聚甲醛（PFA），0.4％的戊二醛（GA）在0.1M PHEM缓冲液（20mM PIPES，50mM的HEPES，pH值6.9，20mM的EGTA，4毫摩尔MgCl 2）]含1M山梨醇在细胞悬浮液。保持在管中于室温下进行。 </li><li>在30分钟轻轻手动反转micocentrifuge管数次（这将允许保持在原生质球悬浮液），然后离心两次在1700×g离心25秒。 </li><li>通过丢弃上清，加入1ml新鲜的标准强度固定液（2％PFA中，0.2％GA在0.1M PHEM缓冲液）含有1M山梨糖醇，孵育2小时，在室温下在缓慢旋转轮更换固定液。 </li><li>如前面所述6处理细胞冷冻切片。注：对于吸收纳米金，银增强程序，在指定的协议描述的周期性酸处理并没有被执行。周期性酸处理是为了更好地透化细胞壁26，但这种结构也球状体的生成过程中被消除。 </li><li>切50nm的薄的冷冻切片在-120℃下干燥钻石刀使用UCT超薄切片机如前所述8，并将其放置在涂福尔瓦碳50啮合镍网格。 </li></ol> 4，银增强的纳米金粒子Visualization 注意：要准备反应混合物中的程序基本上是由制造商所指示的之一。使用此协议的实际适应性，使得银增强程序，有效和可靠的冰冻切片。 <ol><li>从冰箱中取出银增强套件和解冻它在37℃培养箱或水浴。解冻时，将试剂盒在24℃培养箱放置到一个黑暗的房间里，直到使用（见4.7）。 </li><li>将加热板在黑暗的房间，并加热到24℃，终温</li><li>覆盖加热板的顶部表面的保护，有光泽的一面朝上，并用胶带将其固定。 </li><li>将封口膜对Benchkote并标记边缘用黑色标记，以能够看到的封口膜边在黑暗中。 </li><li>磁带上的​​Benchkote顶部的温度计监测加热板的温度。逐步调整温度，如果不是24°C。 </li><li>解放军CE上的50ml管中用双蒸水和内部的银增强试剂盒。 </li><li>填充小培养皿用双蒸水，预加热至37℃。放置在网格中的水，试样面朝下，持续30分钟。 </li><li>重复此4.7步一个更多的时间。 </li><li>传送网格，储物箱，将他们的黑暗的房间。 </li><li>由上几滴双蒸水在24℃下放置于封口膜已被固定在加热板传递它们（与试样的一面朝下）再次冲洗的网格。 </li><li>确保9个附加双蒸水滴准备上的封口膜，用于冲洗的银增强反应后。 </li><li>关灯，并确保所有灯都熄灭。打开红灯。 </li><li>取出从24℃培养箱中银增强试剂盒的A和B的解决方案。 </li><li>把第一个6滴溶液中，然后加入6滴乙溶液在1.5米升微量离心管中，并用玻璃巴斯德吸管，避免使气泡拌匀。该解决方案是很厚，它有手动，最后才涡旋它简单地用吸管混合。 </li><li>把A和B的解决方案早在24℃培养箱中培养，并采取了C溶液。 </li><li>加入6滴了C解决了A / B混合。然后先混合再一次用巴氏吸管，然后通过涡旋，避免气泡。 </li><li>通过将滴（〜20微升/滴）上的封口膜立即使用最终混合物。 </li><li>用干净，防磁镊子，将网格的混合物（ 如 。，银增强反应）为6〜15 min，取决于希望得到增强（ 即 ，大小的金粒）的。 </li><li>从混合物中取出电网，并通过他们的双蒸水的顶部下降在24℃下洗涤。首先，在使用快速连续6滴，然后3滴，每滴7分钟孵育时间，开灯之前。 </li><li>继续下一个步骤，以可视化的结果为EM调查或者免疫金标或膜染色27。 </li></ol>

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The authors have nothing to disclose.

免疫电子显微术是一种技术，它允许结合蛋白的定位与在这些蛋白质所在的载体和细胞器的超微结构分辨率。研究了酵母核内体系统时，因为它的车厢出现点状结构，在荧光显微镜中，这一点尤其重要。因此它是难以区分它们。由于这个原因，用一个探针通过EM检测并输入的内吞途径中一个时间依赖的方式是非常重要的特异性标记不同内涵体。这种类型的探针是有价值的，以评估细胞内吞作用...

内体系统中起着至关重要的多种细胞的作用包括贩运溶酶体酶分选受体和质膜（PM）的回收受体1,2的主要膜分拣设备。内涵体被划分在三个不同的车厢， 即 。在早期内涵体（EE），晚期内涵体（LE）和回收内涵体。这种分类是基于它需要的内吞物质到达他们，对特定的标记蛋白，并且在其形态的时间。膜， 即蛋白质和脂质双层，从PM内在设计可以通过内体输送到溶酶体中降解或再循环回。膜也被输送到内涵体从高尔基体，同样，要么继续溶酶体或检索返回到高尔基体。此外，蛋白质可以分为管腔囊泡从内体界膜，这一过程导致形成向内出芽子类别LE时，多泡体。 酵母胞内体系统是相对较不复杂的较高的真核细胞中的一个。酵母胞内体分为EE和LE。相反，哺乳动物细胞中不含有回收内涵体，而且组织特异性溶酶体相关细胞器。因此，他们有一个不太复杂的内体的贩运路线3,4的网络。因此，酵母曾代表仍然表示一个有利的实验系统，研究一些基础原则膜交通的内体系统。这个优点是通过以下事实，涉及该内体途径的许多基因已被初步分离与遗传筛选在酵母5强调。 而酵母胞内体系统中的野生型和突变的细胞已被广泛采用的生化和荧光显微镜方法的研究，其在超微结构水平调查至今只有最小的。形态分析是特别相关的酵母，因为大部分核内体的细胞器被检测为通过荧光显微镜圈点结构，这使得他们难以明确识别6。不幸的是抗血清仅有限数量识别酵母胞内蛋白标志物是工作在免疫电子显微镜（IEM）制剂7-10。对于一些蛋白质，这个问题已经绕过由感兴趣的基因的内源性标记和使用的抗体识别，检测它7,11,12和标签。然而，通常蛋白是因为它们的低表达水平的检测不到的IEM。他们的表达是不是一个解决方案，因为这种方法可以诱导误本地化和/或改建中的细胞器形态/功能。因此，内吞区室中与探针可检测通过电子显微镜EM标签是一种有效的选择。这是特别是如果一个最优解的公关OBE是进入细胞内吞途径中以时间依赖性方式，它允许知道什么时候会标记一个特定的细胞器6。 带正电荷的纳米级金的酵母原生质球（ 即 ，酵母，其中细胞壁已酶促去除）的摄取已经被成功地用于识别酵母胞内隔室10。这些颗粒牢固结合到带负电荷的脂质构成的生物膜。因此，带正电荷的纳米级金的联系人与PM，穿透细胞通过内吞作用和通过EE和LE到达液泡之前。这些小的金颗粒，但是，没有一个合适的尺寸由EM待观察。以使它们可见，它们的大小可通过化学反应，导致银或金的周围的黄金探测器13-15的沉积被放大。我们已经开发并成功应用的IEM方法的基础上，得安方法来执行的亚细胞定位研究8,16。这种方法允许进行酵母制剂免疫标记与形态8,17-24一个出色的分辨率。我们还建立了一个程序结合本IEM协议与酵母核内体系统的纳米金标记舱6。使用这种方法，我们已形态学特征内涵体的不同亚类和超微结构研究突变体与内体运输缺陷6,25。此外，我们已经证明，这种纳米金标记可以与免疫标号提供探索对不同亚群内体目标蛋白的分布的可能性相结合。在这里，我们介绍如何在酵母核内体系统的带正电的纳米金标记实际上是执行。

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nanogold labeling of the yeast endosomal system for ultrastructural analyses

内涵体是主要的膜排序在真核细胞中的检查点中的一个和它们调节回收的蛋白质或毁坏大多从质膜和高尔基体。其结果是内体系统在维持细胞稳态中发挥中心作用，并在属于该网络的细胞器通过小泡运输相互连接的基因的突变，引起严重的病症，包括癌症和神经生物学障碍。因此，黄金的相关性是理解内涵体系统的生物合成和组织相关的机制。 酿酒酵母中一直举足轻重的完成这项任务。要明确标示，并在超微结构水平这一​​模式生物的内体系统的分析，我们在座的带正电荷的纳米金摄取原生质其次是这些粒子通过银增强反应可视化的详细协议。这种方法也是一种宝贵的太l适用于与内体运输缺陷突变体的形态学检查。此外，它不仅适用于超微结构检查，但它也可与免疫标号用于蛋白质定位的调查相结合。

根据所提出的协议，酵母核内体系统的形态可以是由接入传输的EM。 图1显示了不同类型的内体区室中已达成，因此，标记的纳米金。银增强纳米金可以清楚地看到作为电子致密颗粒。建立了银增强反应的最佳设置允许具有在5至15纳米，不与酵母细胞的超微结构的干扰之间的均匀粒度范围的金颗粒。质膜以及早期内涵体车厢都可以访问的纳米金5分钟孵化在24°C（ 图1A）后?...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

酵母， 酿酒酵母 ，一直是一个关键模式生物识别和研究基因调节内体系统的生物合成和功能。在这里，我们提出了内体车厢的超微结构研究特定的标签一个详细的协议。

酵母核内体系统的超微结构分析的纳米金标记

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

nanogold-labeling-yeast-endosomal-system-for-ultrastructural

Research

JoVE Journal

Biology

10.2K 次观看.  University Medical Center Utrecht. 酵母， 酿酒酵母 ，一直是一个关键模式生物识别和研究基因调节内体系统的生物合成和功能。在这里，我们提出了内体车厢的超微结构研究特定的标签一个详细的协议。 

视频: Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

被截肢者的智能植入种植体设计系统的生物电分析

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

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.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

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

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...