我々は、プラスミド、酵母菌株を提供するためのS.ジンクスロバートソンとE ·ハイデンリッヒに感謝、P. PhamさんとMFグッドマンヘルプwiththe TLSアッセイのために。この作品は、エイジング総合研究助成のためのアメリカ連合によっておよびNIH AG20642、AG025135によって、部分的に、サポートされていました。

<ol>
	<li>Longo, V. D., Finch, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+medicine:+from+dwarf+model+systems+to+healthy+centenarians.">Evolutionary medicine: from dwarf model systems to healthy centenarians.</a> Science. 299, 1342-1346 (2003).</li><li>Fontana, L., Partridge, L., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+healthy+life+span--from+yeast+to+humans.">Extending healthy life span--from yeast to humans.</a> Science. 328, 321-326 (2010).</li><li>Mortimer, R. K., Johnston, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+span+of+individual+yeast+cells.">Life span of individual yeast cells.</a> Nature. 183, 1751-1752 (1959).</li><li>Muller, I., Zimmermann, M., Becker, D., Flomer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calendar+life+span+versus+budding+life+span+of+Saccharomyces+cerevisiae.">Calendar life span versus budding life span of Saccharomyces cerevisiae.</a> Mech. Ageing Dev. 12, 47-52 (1980).</li><li>Jazwinski, S. M., Egilmez, N. K., Chen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+control+and+cellular+life+span.">Replication control and cellular life span.</a> Exp. Gerontol. 24, 423-436 (1989).</li><li>Pohley, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+formal+mortality+analysis+for+populations+of+unicellular+organisms+(Saccharomyces+cerevisiae.">A formal mortality analysis for populations of unicellular organisms (Saccharomyces cerevisiae.</a> Mech Ageing Dev. 38, 231-243 (1987).</li><li>Longo, V. D., Gralla, E. B., Valentine, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+dismutase+activity+is+essential+for+stationary+phase+survival+in+Saccharomyces+cerevisiae.+Mitochondrial+production+of+toxic+oxygen+species+in+vivo.">Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae. Mitochondrial production of toxic oxygen species in vivo.</a> J Biol Chem. 271, 12275-12280 (1996).</li><li>Longo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The chronological life span of Saccharomyces cerevisiae. Studies of superoxide dismutase, Ras and Bcl-2 [dissertation]. , (1997).</li><li>Fabrizio, P., Pozza, F., Pletcher, S. D., Gendron, C. M., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+longevity+and+stress+resistance+by+Sch9+in+yeast.">Regulation of longevity and stress resistance by Sch9 in yeast.</a> Science. 292, 288-290 (2001).</li><li>Fabrizio, P., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chronological+life+span+of+Saccharomyces+cerevisiae.">The chronological life span of Saccharomyces cerevisiae.</a> Aging Cell. 2, 73-81 (2003).</li><li>Madia, F., Gattazzo, C., Fabrizio, P., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+model+system+for+age-dependent+DNA+damage+and+cancer.">A simple model system for age-dependent DNA damage and cancer.</a> Mech Ageing Dev. 128, 45-49 (2007).</li><li>Wei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+span+extension+by+calorie+restriction+depends+on+Rim15+and+transcription+factors+downstream+of+Ras/PKA,+Tor,+and+Sch9.">Life span extension by calorie restriction depends on Rim15 and transcription factors downstream of Ras/PKA, Tor, and Sch9.</a> PLoS Genet. 4, e13-e13 (2008).</li><li>Fabrizio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+is+a+mediator+of+an+altruistic+aging+program+in+Saccharomyces+cerevisiae.">Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae.</a> J Cell Biol. 166, 1055-1067 (2004).</li><li>Wei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tor1/Sch9-regulated+carbon+source+substitution+is+as+effective+as+calorie+restriction+in+life+span+extension.">Tor1/Sch9-regulated carbon source substitution is as effective as calorie restriction in life span extension.</a> PLoS Genet. 5, e1000467-e1000467 (2009).</li><li>Capizzi, R. L., Jameson, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+table+for+the+estimation+of+the+spontaneous+mutation+rate+of+cells+in+culture.">A table for the estimation of the spontaneous mutation rate of cells in culture.</a> Mutat Res. 17, 147-148 (1973).</li><li>Heidenreich, E., Novotny, R., Kneidinger, B., Holzmann, V., Wintersberger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-homologous+end+joining+as+an+important+mutagenic+process+in+cell+cycle-arrested+cells.">Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells.</a> Embo J. 22, 2274-2283 (2003).</li><li>Heidenreich, E., Wintersberger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-dependent+and+selection-induced+mutations+in+respiration-competent+and+respiration-deficient+strains+of+Saccharomyces+cerevisiae.">Replication-dependent and selection-induced mutations in respiration-competent and respiration-deficient strains of Saccharomyces cerevisiae.</a> Mol Gen Genet. 260, 395-400 (1998).</li><li>Chen, C., Kolodner, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gross+chromosomal+rearrangements+in+Saccharomyces+cerevisiae+replication+and+recombination+defective+mutants.">Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants.</a> Nat Genet. 23, 81-85 (1999).</li><li>Datta, A., Adjiri, A., New, L., Crouse, G. F., JinksRobertson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+crossovers+between+diverged+sequences+are+regulated+by+mismatch+repair+proteins+in+Saccaromyces+cerevisiae.">Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae.</a> Mol Cell Biol. 16, 1085-1093 (1996).</li><li>Madia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogene+homologue+Sch9+promotes+age-dependent+mutations+by+a+superoxide+and+Rev1/Polzeta-dependent+mechanism.">Oncogene homologue Sch9 promotes age-dependent mutations by a superoxide and Rev1/Polzeta-dependent mechanism.</a> J Cell Biol. 186, 509-523 (2009).</li><li>Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+recombinant+genes+and+preparation+of+cell-free+extracts+in+yeast.">Controlled expression of recombinant genes and preparation of cell-free extracts in yeast.</a> Methods Mol Biol. 313, 317-331 (2006).</li><li>Fabrizio, P., Pletcher, S. D., Minois, N., Vaupel, J. W., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronological+aging-independent+replicative+life+span+regulation+by+Msn2/Msn4+and+Sod2+in+Saccharomyces+cerevisiae.">Chronological aging-independent replicative life span regulation by Msn2/Msn4 and Sod2 in Saccharomyces cerevisiae.</a> FEBS Lett. 557, 136-142 (2004).</li><li>Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+quiescent+and+nonquiescent+cells+from+yeast+stationary-phase+cultures.">Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures.</a> J Cell Biol. 174, 89-100 (2006).</li><li>Madia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+mutation+in+SCH9+prevents+recombination+errors+and+premature+genomic+instability+in+a+Werner/Bloom+model+system.">Longevity mutation in SCH9 prevents recombination errors and premature genomic instability in a Werner/Bloom model system.</a> J Cell Biol. 180, 67-81 (2008).</li></ol>

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液体老化の文化はいつか再生/ CLS分析を複雑にあえぎ表現型13を 、示す。人口以上の90から99パーセントが生存能力を失ったときに再生が一般的に発生します。この表現型は、多くの場合、酸化ストレスおよび/または細胞の減少の保護に関連付けられています。例えば、この表現型の頻度は、より多くの細胞質スーパーオキシドジスムターゼを欠く細胞で2倍以上と大幅に長寿命変異体で減少します（例えば、sch9ΔまたはRAS2Δ）または変異体を過剰発現してスーパーオキシドは、22ジスムターゼ。実際には、我々は、経時的老化時の高死亡率の段階で3つの連続した​​サンプル用実行可能性の実現可能性または安定化の増加として再成長を定義する。 異なるDNAの変異の年齢に依存する周波数が大幅にひずみ背景、遺伝子操作、培養条件、ならびに再生/あえぎと非常に低い生存率に応じて異なります。プレ実験はこのようなperformingtheフルスケールの寿命解析の前に変異の周波数範囲を決定するために突然変異試験のための様々な播種密度と平均値と最大生存などの時点で行ってください。野生型の菌株は、常に、任意の治療や遺伝的変異体と平行に寿命や突然変異の頻度の研究に含まれるべきである間実験-ばらつきが考慮できるように。複数の生物学的なレプリカが研究に含まれるべき、と、液体の両方とその場可能性/突然変異アッセイの結果を裏付けるために実施すべきである。 代わりに組み合わせて複数のアッセイを用いて年齢依存性ゲノム不安定性のプロファイリング、DNAの突然変異の特定のタイプに焦点を当ててから、年齢依存性のゲノム不安定性に寄与する特定のDNA損傷とDNA損傷修復システム（秒）に光を当てることができる。例えば、他のDNAの突然変異のものに比べて総染色体再配列（GCRs）の有意な増加が、、elevationof二重鎖切断および/または酵母中の接合端（NHEJ）非相同の減損を示唆する野生型酵母で観察される経時的老化（表2）。野生型エージング酵母で得られたCAN1突然変異のスペクトルは、（CAN1遺伝子の塩基配列決定）経時的老化とエラーが発生しやすいDNAの修復中に酸化的損傷の増加を示唆し、一方、長寿命のsch9Δ変異体では、以下の酸化的DNA損傷と程度に下げてエラーを起こしやすいtranslesionの合成は、20を観察した。 ここで説明する方法は、さらに年齢依存性のゲノム不安定性を研究するために費やさすることができます。例えば、静止及び非静止期の細胞は、Allenらによって記述された密度勾配法を用いて酵母固定相培養細胞から分離することができます。23。ここで説明する変異のアッセイと組み合わせることで、我々は、年齢依存性の変異の大部分は静止細胞ではなく、分割し、損傷やアポトーシス細胞20,24から生じることが報告されている。

studying age-dependent genomic instability using the s. cerevisiae chronological lifespan model

Saccharomyces cerevisiaeの老化モデルを用いた研究は、部分的に高等真核生物1-2に保存されている寿命調節経路を明らかにしました。酵母老化モデルのシンプルさとパワーは、DNA損傷とゲノム維持だけでなく、老化の間に病気への貢献を研究するために調べることができます。ここで、我々は単純なDNAで酵母の時系列の寿命を組み合わせることにより、塩基置換、フレームシフト変異、総染色体再配列、および相同/ homeologous組換えだけでなく、核DNA修復活性を含め、年齢依存性のDNA変異を研究するためにシステムを説明損傷と突然変異試験。ここで説明する方法は、ゲノムの不安定性と哺乳類では年齢依存性DNAの変異と癌の根底にあるメカニズムを制御する遺伝子/経路の同定を容易にしてください。

Watch this Scientific Journal Video about Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model at JoVE.com

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

ここでは、エージング中規制またはゲノムDNAの不安定性に寄与する遺伝子/経路を研究するために酵母の時系列の寿命モデルと組み合わせることができるDNAの突然変異試験のセットを説明します。

使用年齢に依存するゲノム不安定性を研究して S.出芽酵母時系列寿命モデル

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

studying-age-dependent-genomic-instability-using-s-cerevisiae

Research

JoVE Journal

Biology

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

15.5K ビュー.  University of Southern California, Los Angeles. ここでは、エージング中規制またはゲノムDNAの不安定性に寄与する遺伝子/経路を研究するために酵母の時系列の寿命モデルと組み合わせることができるDNAの突然変異試験のセットを説明します。

ビデオ: Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

S. cerevisiaeの接着と寒天の侵略のためのアッセイ

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

生物学

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiaeの定量的プロテオミクスとタンパク質の複合体の同定

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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.

マイクロアレイ解析のためのサッカロマイセスセレビシエ</em

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

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

ゲノムMRI - ゲノムDNA内の配列パターンを研究するためのパブリックリソース

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

のフローサイトメトリーベースの精製<em&gt; S.セレビ</em&gt;受精卵

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

蛍光クリアランスアッセイを用いてゼブラフィッシュ腎臓機能の評価

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

シングル出芽酵母細胞での寿命と細胞表現型を測定するためのマイクロ流体デバイスを使用しました

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

出芽酵母におけるタンパク質ベース継承の高速スクリーニング

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

腰椎不安定性のマウスモデル

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...