在作者的实验室工作的加拿大卫生研究院的创新，自然科学和工程研究理事会和康考迪亚大学，加拿大基金会的资助。

建设二倍体菌株 通过交配生产二倍体议定书“ <ol><li>最初的菌株，应取得YPD，或选择性培养基上，如果必要，产生单菌落。两个单倍体菌株应相反交配型（即MATA和MATα）。 </li><li>使用无菌接种环挑一个殖民地的一小部分，从两个单倍体菌株之一。在一个新的YPD板连胜循环一次，在整个板块的直线。标记板底部的箭头方向连胜。 </li><li>重复上相同的YPD板第二应变。这一次连胜垂直殖民地的第一，过路的第一与第二连胜。马克第二连胜的方向，并注意该地区的第二连胜跨越第一。这个区域将是会产生二倍体。 </li><li>在30℃过夜孵育YPD板。 </li><li>副本板到选择性培养基YPD板，将有选择地允许新成立的二倍体增长。 </li><li>选择相同的选择性培养基上单个菌落，如果可能的话，或一个二倍体连胜和连败的一小部分，产生单菌落。 </li></ol>产孢和消化的ASCI 协议<ol><li>从上述板块中挑选单菌落，使小（1厘米x 1厘米），孢子（SPO）板的补丁，并在30 ° C孵育3-7天，以孢子。 </li><li>产孢可以在从每一个补丁细胞，光镜下检查。位置细胞，在显微镜幻灯片5-10μL水液滴上放置盖玻片，查找存在的ASCI。如果ASCI不容易被发现，然后放回孵化器。 </li><li>如果有一个补丁的有效产孢，准备与四分汁100μL无菌15ML锥形管。 </li><li>挑选从这个产孢修补细胞无菌的3mm接种环（细胞应包括循环的四分之一）和四分体汁。轻轻摇动循环，使细胞脱落与溶液的循环和​​混合。 </li><li>留在四分汁（含zymolyase），4 - 10分钟（取决于zymolyase和应变本身的浓度和活性）。允许子囊孢子壁细胞消化。 </li><li>标记在YPD板上接种区域的顶部和底部。 </li><li>可选。加入1ML冰冷的无菌DH 2 O反应，可以停止允许细胞为5分钟解决。从上清液取出1毫升，然后用手轻轻混合置于冰上。 </li><li>从第5步消化细胞5μL​​，轻轻地滴在接种前显着的YPD板区域。 </li><li>使用无菌的3mm接种环轻轻地分布在一条线下降1-3倍，而勉强用琼脂接触。 </li><li>让液体吸收到琼脂（如果省略第7步，这是当外层的细胞壁的消化将减少或停止）。继续解剖镜下。 </li></ol>剖析ASCI 协议请注意，下面的协议是一个歌手的MSM系统200显微显微镜。该协议可以遵循与轻微的修改，如果使用手动显微操纵器，如蔡司显微致辞。 <ol><li>专注于一个地区的板块，在该领域的一半包含细胞，一半是空的。针慢慢地将成为关注的焦点，倒是该领域的空白区域。这将确保针头不包含从以前使用的细胞。针和琼脂之间的联系被看作是一个暗黑色圆圈，慢慢地成为关注的焦点。 </li><li>转到位置A1矩阵，并再次刺入琼脂商标。这是为了帮助确定方向的板块，如果它是在完成之前，在显微镜。 </li><li>切换搜索模式。找到一个ASCI。最好选择在细胞伸长地区的ASCI。避免细胞簇的地区。四个子囊孢子从一个ASCI仍然应该重视对方。两个子囊孢子可能会比其他两个稍微大一点。避免采摘ASCI一个子囊孢子是松散的，而不是连接到其余的子囊。 </li><li>拿起针子囊。慢慢地将针板。当看到微针的影子，此行与ASCUS最多被拾起。同样的方法针，直到看到一个圆圈（针尖）的黑色轮廓。触摸这个圈子里的四分体，然后迅速拉针。确保所有四个子囊孢子从子囊坚持针和微针，是不是这ASCUS的一部分，不需要额外的细胞聚集。 </li><li>远离板的微针拉矩阵（第一子囊，去位置A1）。请注意每一个子囊搬迁的位置。将子囊接触与微针板和拉远，直到所有四个子囊孢子脱落的微针。温和攻针手臂或控股YPD板的平台往往是有用的，在这一步。 </li><li>重复步骤3-5，在矩阵上的连续位置ASCI下来，直到所需数量的ASCI已经回升。 </li><li>返回到每个子囊和戏弄针，攻丝轻轻平台或两者的结合，打破四个单元。拿起细胞留在矩阵中的一个新的位置，后面四排分开，直到每个子囊孢子在每个子囊。 </li><li> YPD板孵化器放置在30℃，几天来观察经济增长方式。 </li><li>副本板解剖细胞选择性培养基如有必要。 </li></ol>搬迁的受精卵 协议<ol><li>数出两个在适当的介质，以获得单菌落相反交配型的单倍体菌株。 </li><li>伴侣这两个菌株如上所述，并允许将从4〜6小时（或过夜，如果必要的话）。 </li><li>光显微镜下检查受精卵。受精卵会出现一芽带或不带哑铃状细胞。 </li><li>匹克细胞3mm的接种环（细胞应包括循环的四分之一），轻轻放置在无菌DH 2 O 100μL </li><li>标记在YPD板上接种区域的顶部和底部。 </li><li>取5μL，轻轻地滴在接种前显着的YPD板区域。 </li><li>使用3mm的接种环，非常轻柔而勉强用琼脂接触传播滴线。 </li><li>让液体吸收到琼脂。继续解剖镜下。 </li><li>专注于一个地区的板块，在该领域的一半包含细胞，一半是空的。针慢慢地将成为关注的焦点，倒是该领域的空白区域。这将确保针头不包含从以前使用的细胞。针和琼脂之间的联系被看作是一个暗黑色圆圈，慢慢地成为关注的焦点。 </li><li>转到位置A1矩阵，并再次刺入琼脂商标。这是为了帮助确定方向的板块，如果它是在完成之前，在显微镜。 </li><li>切换搜索模式。找到一个受精卵。 </li><li>拿起针受精卵。 </li><li>地方受精卵单独的矩阵。 </li><li>允许受精卵成长为几天。 </li><li>副本板的受精卵到适当的选择性培养基。 </li></ol>代表性的成果<img src="/files/ftp_upload/1146/1146fig1.jpg" alt="" /> 图1：两个相反的交配型的单倍体菌株交配产生的二倍体菌株。 <img src="/files/ftp_upload/1146/1146fig2.jpg" alt="" /> 图2：（A）之间的温度敏感株（用LEU2营养缺陷型）和野生型菌株（LEU2）交配THRE结果YPD板。消化细胞分布在接种地区上的两条黑线之间的厚厚的增长被认为是左。 ASCI被解剖的增长均匀分布在右边的矩阵。 （二）板（一）复制到SD -亮氨酸的辍学板。注意2:2增长LEU2表示经过验证的子囊孢子解剖的标记。 （三）板（一）被复制到YPD板，并在37 ° C孵育注意2:2的增长，进一步验证了一个子囊孢子解剖。

<ol>
	<li>Sherman, F., Hicks, J., Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+and+Dissection+of+Asci.">Micromanipulation and Dissection of Asci.</a> Guide to Yeast Genetics and Molecular Biology. , 21-37 (1991).</li></ol>

酵母夹层是一个有用的工具，选择所需的标记新菌株。当确定四个子囊孢子的理论增长模式，重要的是看的营养细胞的基因型。如果一个质粒是存在的，人们必须知道，如果它是单一的，高或低复制，因为这会影响子囊孢子（S）接收的质粒，并会影响预测和结论，就被操纵的菌株。 某些菌株可以比别人在很短的时间内生产的许多子囊孢子的更好。其他菌株可能产生的子囊孢?...

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<td>Singer MSM System 200</td>
<td>Microscope</td>
<td>Singer Instruments</td>
<td>NA</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
<td>SOR508</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
<td>TRS001</td>
<td>&nbsp;</td>
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<td>Zymolyase 100T</td>
<td>Reagent</td>
<td>Seikagaku Corporation</td>
<td>120493</td>
<td>&nbsp;</td>
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<td>Yeast extract</td>
<td>Reagent</td>
<td>BioShop</td>
<td>YEX401</td>
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<td>PEP 403</td>
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<td>YNB406</td>
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<td>TRP402</td>
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dissection of saccharomyces cerevisiae asci

酵母是一种非常听话的模型系统，用于研究许多不同的细胞过程。常见的实验室应变<em&gt;酿酒酵母</em单倍体或二倍体状态存在。能够结合两个单倍体的等位基因和能力引入到基因组的修改要求ASCI生产和清扫。 ASCI单倍体细胞开始生产与兼容的交配型酵母单倍体菌株产生二倍体菌株交配。这可以是固体或液体介质的方式来完成。这是有利的，选择的媒介，选择性促进其生长的单倍体菌株相比，二倍体。然后让贫营养培养基中的孢子，形成子囊，捆绑四个单倍体的女儿从减数分裂繁殖的二倍体细胞的二倍体。植物细胞和ASCI的混合物，然后处理与酶zymolyase消化掉膜囊周围ASCI子囊。使用显微操作与微针下解剖镜下可以拿起个人ASCI和分离和重新定位的四个ascopores。解剖ASCI长大了好几天，和到适当的选择性培养基的标记或等位基因副本电镀兴趣测试。

Watch this Scientific Journal Video about Dissection of Saccharomyces Cerevisiae Asci at JoVE.com

Dissection of Saccharomyces Cerevisiae Asci

酵母细胞减数分裂的遗传分析，或选择二倍体受精卵显微操作是需要。这些micromanipulations都是开展使用了解剖镜下微针。微针是用于搬迁细胞的，由显微不同程度的自动化控制。

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

dissection-of-saccharomyces-cerevisiae-asci

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JoVE Journal

Biology

Dissection of Saccharomyces Cerevisiae Asci

18.4K 次观看.  Concordia University. 酵母细胞减数分裂的遗传分析，或选择二倍体受精卵显微操作是需要。这些micromanipulations都是开展使用了解剖镜下微针。微针是用于搬迁细胞的，由显微不同程度的自动化控制。

视频: Dissection of Saccharomyces Cerevisiae Asci

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

科研

生物学

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPR监控的辅助因子氧化还原滴定<em&gt;酿酒酵母</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA的衰变率的测量<em&gt;酿酒酵母</em&gt;使用<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

定向进化法<em&gt;酿酒酵母</em&gt;：突变体库的创建和筛选

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