우리는 질량 분석계 분석과 도움이 토론과 기술 지원 박사 서식 로저스 감사하고, DRS 것입니다. 롭 모리츠, 제프 Ranish, 그리고 도움이 토론에 대한 하미드 Mirzai. 이 작품은 우수의 NIH 센터 추진하는 사업.

세포 성장 및 미디어 <ol><li> 최소한의 효모 매체의 두 1리터 문화 (질산 황산이나 아미노산없이 0.17 %의 효모 질소베이스, 0.5 % 질산 황산)에 1.0 다음의 씨앗 OD 600-100 ML 리치 미디어에서 밤새 BY4742Δarg4Δlys1 세포의 단일 콜로니를 포함 (; Isotec 13 C 6 15 N 4)과 라이신 (13 C 6 15 N 2; Isotec) 20mg / isotopically 정상 또는 무거운 아르기닌의 L과 보충 아미노산의 완전한 보완합니다. </li><li> 세포는 1.8 OD 600, 18 시간 동안 성장했다. 이것은 세포가 표시된 동위 원소의 전체 결합을 달성하기 위해 최소한 9 세대를 통과 것이 중요합니다. 가벼운 예제 매체 (isotopically 정상 아르기닌과 리신, 0.2 % Oleate (시그마 화학)와 0.5 % 트윈 40 (시그마 화학))가 포함된 oleate에 reseeded 다른 85분에 대한 자극, 멸균 물을 씻어, pelleted했다. 이것은 아르기닌과 리신과 관련하여, isotopically 빛 수율, 샘플 및 isotopically 무거운 포도당 - 재배 참조 샘플을 oleate 자극. </li></ol> 세포 용해, 분리 및 펩티드의 분류 <ol start="3"><li> 원심 분리기 병의 무게를. 3 분 원심 분리하여 수확 샘플. 미디어를 제거하고 여분의 액체를 대기음. 펠렛 및 병 (일반적으로 1~2그램 간의 수율됩니다) 액화 질소에 다음 플래시 동결 나가는거야. 액체 질소 (인산 10% 글리세롤, 프로 테아제 억제제가 (SigmaFAST 프로 테아제 억제제의 알약, 시그마 (PBS, Gibco) 살린 버퍼)의 냉동 알약의 &#39;그라인딩&#39;버퍼의 펠렛 중량에 볼륨 상당을 추가하고 (써모 과학 인산 가수 분해 효소 억제제를 중단 )). </li><li> 액체 질소의 분쇄 용기를 고정. 액체 질소가 끓는 중단 때까지 기다리십시오. 냉동 볼 베어링과 함께 분쇄 용기에 펠렛을 전송합니다. </li><li> 3 분 방향 반전과 함께 1 분 20 초주기와 600 rpm으로 분쇄. 액체 질소의 연삭 함선을 Refreeze. 십오분 총 분쇄 시간을 4 번 더 반복합니다. </li><li> 50ml 팰컨 튜브 장소 드라이 아이스로 냉동 grindate를 수집합니다. 사용하기 -80에서 보관 ° C까지 준비. </li><li> 8M의 요소, 0.1M의 탄산수 소 암모늄, 0.1M 트리스 산도 8.6의 10ml 솔루션을 확인하십시오. 냉동 grindate 1 볼륨 (우레아 버퍼의 예제 3mls을위한 PBS 버퍼 1 ML과 1g 펠렛에 추가)에 요소 버퍼 3 볼륨을 추가합니다. 즉시 프로브 팁 sonicator (- 10초 펄스 2)와 혼합 sonicate. 솔루션의 거품 방지하기 위해 튜브의 아래쪽 끝 보관하십시오. grindate은 즉시 솔루션으로 가야한다. </li><li> 4 5 분 원심 분리하여 lysate을 해제 ° C. </li><li> 신선한 튜브에 뜨는 전송합니다. </li><li> 0.5M 트리스의 신선한 나누어지는 만들기 [2 - carboxyethyl] phosphine (TCEP). 5mM 최종 농도에 대한 1:100에서 샘플을 추가합니다. 37 품어 ° C를 한 H.위한 </li><li> 감소 cysteine​​s의 알킬화. 실온으로 냉각하도록 허용합니다. 1M iodoacetamide의 신선한 나누어지는하십시오. (우리는 호일에 튜브를 줄 바꿈) 어둠 속에 보관. 20mM의​​ 최종 농도에 추가합니다. 실온에서 1 H에 대한 어둠 속에 품어. </li><li> 1 H. 위해 실온에서 1M 주식에서 20mM DTT와 iodoacetamide을 끄다 </li><li> 표준 브래드 또는 BCA 분석 (설명하지 않음)로 단백질을 정량. 아래 SDS 페이지로 분석을 위해 샘플을 가져가라. </li><li> 무거운 동위 원소의 결합을 확인하려면, 감소 및 alkylated lysate 100 μl를 사용합니다. DH 2 O와 1시 4분을 희석, sonicate 및 트립신에 50:1 단백질의 트립신을 추가합니다. C18 Ultramicrospin 열 (네스트 그룹)과 VAC 후 탈염하다 속도 다운 4 시간 최소 건조 시료에 대한 다이제스트. <ol><li> Acetonitrile 100 μl와 수화물 칼럼 </li><li> 200 μl 0.1 % TFA와 평형. </li><li> 200 μl 0.1 % TFA의 건조 표본을 Resuspend. 칼럼에로드합니다. ~ 200 XG 또는 열의를 통해 흐름에 필요한 최소한의 속도로 원심 분리기. </li><li> 200 μl 0.1 % TFA로 두 번 씻으십시오. </li><li> 50 μl 60 % Acetonitrile, 0.1 % TFA로 두 번과 Elute. </li><li> VAC 속도 건조 샘플. </li><li> 20 μl 0.1 %의 개미의 산이 Resuspend 샘플. 질량 분석계 2 μl를 실행합니다. isotopically 무거운 아르기닌과 각 M / Z 스펙트럼에서 10 8 돌턴 변화에 의해 라이신의 법인을 확인합니다. 98% - 각 아미노산에 대한 설립 96 사이 여야합니다. </li></ol></li><li> isotopically 무겁고 가벼운 샘플 이에 상응하는 금액을 (단백질의 MG) 섞는다. </li><li> 20 % 메탄올로 샘플 1시 4분을 희석. </li><li> 1:200에​​서 트립신을 추가합니다. 37 밤새 품어 ° C. </li><li> 그 소화는 SDS 페이지로 완료 갔어 확인하십시오. 2 4 predigestion 샘플의 μl 8를 실행게시물 트립신 다이제스트의 D 16 μl. 소화가 완료되는 쿠매시의 얼룩으로 시각화. 그것이 완료되지 않은 경우, 프로브 팁 sonicator의 잠시와 샘플을 sonicate하고 트립신로 다시 소화. </li><li> VAC 속도 샘플을 건조시킵니다. 건조 펠렛이 솔루션에 들어가기까지 필터링 DH 2 O와 와동 200 μl을 추가하면. VAC 속도 아래로 드라이 샘플. DH 2 O.로 다시 Resuspend 다시 샘플을 건조시킵니다. </li><li> 0.1 % Trifluoracetic 산이 Resuspend 펠릿 (TFA, 시그마). 산도가 산도 스트립을 사용하여 3시 경이되었는지 확인합니다. 10 % TFA와 시게하다 필요하면. </li><li> 어떤 침전물 밖으로 펠렛. </li><li> 워터스 9 월 박 VAC 500 MG C18 컬럼에서 샘플을 탈염하다. 이러한 열 과부하하지 마십시오. 최대 금액은 5 MG이지만 그것은 덜로드하는 것이 좋습니다. 필요한 경우 여러 열 사이의 샘플을 나눕니다. <ol><li> 5 ML 100 % acetonitrile와 하이드 열. ~ 200 XG 또는 열의를 통해 흐름에 필요한 최소한의 속도로 원심 분리기. </li><li> 5ml 0.1 % TFA와 열 평형. 샘플을로드합니다. 흐름을 수집하고 다시로드합니다. </li><li> 5 ML 0.1 % TFA로 씻으십시오. 반복합니다. </li><li> 2ml 60 % Acetonitrile, 0.1 % TFA로 Elute. </li><li> VAC 속도 아래로 드라이 샘플. </li></ol></li></ol> Phosphopeptides의 펩타이드 분류 및 절연 <ol><li> 펩티드는 HILIC 컬럼에 fractionated 있습니다. 우리는 TOSOH 쯧 - 젤 아미드 - 80 4.6 mm X 25cm 경비원 컬럼 분석 컬럼.를 사용하여 이 단계를 사용하여 시간을 가지고, 다음 실행을 두 개의 빈 그라디언트는 샘플을 로딩하기 전에 90%에서 2 시간 동안 컬럼을 실행합니다. 이 크기는 XX 주변에있을 것으로 생각되는 열을 최대 부하, 우리는 일반적으로 실행 당 5 개 이상 MG를 로드할 수 없습니다. </li><li> 솔벤트 - 98% ACN/0.1 % TFA 솔벤트 B - 2퍼센트 ACN/0.1 % TFA 90%의 하중 20 &#39;, 90 % A 85%에 이상 5&#39;, 85 % A 60 % 40 &#39;, 60 % 0 % 5 이상&#39;, 0 % A 90 % 이상 5 &#39;를 보자. </li><li> 유속은 1ml/min이며 분수는 85% 60 % 기울기 시작하여 2 분 간격으로 수집되었다. </li><li> 10 분수의 수를 줄이기 위해 분수를 결합합니다. 증가 분수는 가능성 phosphoproteome의 큰 혜택을 얻을 것입니다. </li></ol> phosphopeptides의 농축 <ol><li> Phosphopeptides는 PHOS의 선택 철 선호도 젤을 사용하여 풍부하게하고 있습니다. 우리는 나누어지는 반복 동결 융해가되지 않도록하기 위해 아이맥 수지. 250 MM 초산, 30 % ACN 부하 솔루션 50 MLS하십시오. HCL로 산도 2.7 가져와. 부하 솔루션 500 μl의 알약을 Resuspend. 필요에 따라 HCL 또는 NaOH로 조정, 산도를 확인합니다. </li><li> 해당 항목에 아이맥 구슬 Prewash 200 μl. 우리는 Molbiotec 스핀 컬럼을 사용합니다. 각 분획에 50 % 슬러리 (솔루션을 로드할 젤) 15 μl를 추가하고, 최종 회전을 통해 최종 30에 대한 샘플을 &#39;품다. 분석을위한 흐름 유지. </li><li> 부하 솔루션 샘플 3 번 씻고 번 500 μl 필터 DH 2 O. </li><li> 2 Elute 펩티드 - 50 MM 나 2 HPO 4 (산도 8.4) 버퍼 400 μl로 3 분. 유리관에 전송. </li><li> 5 μl 100 % 개미의 산, 액체 질소에 플래시 동결하고, VAC 속도 건조 샘플 아래와 산도 3 시어지다. </li><li> 0.1 % TFA하고 위에 설명된대로 깨끗한 C18 200 μl에 Resuspend phosphopeptides. VAC 속도 eluted 샘플을 건조시킵니다. </li><li> 0.1 % 개미 산성 20 μl의 펩티드를 Resuspend. 이 질량 분석계 분석 μl 당 또는 필요에 따라 사용하십시오. </li></ol> 노트 많이 본 프로토콜의 여러분은 &#39;장님&#39;을 작동됩니다. 세포 용해는 서양의 모래 바닥과 브래드 또는 BCA assays, 그리고 트립신의 소화도 쉽게 평가 수에 의해 모니터할 수 있습니다. HILIC 컬럼이 하나와 유사한 추적을 준, 또는 다른 사람 [1, 2] 사용되는 액체 크로마 토그래피 시스템에 따라가 볼 것이다. 샘플은 대량 분석법에 의해 모니터링되며 이러한 높은 질량 정확도, 높은 해상도 머신에 높은 비용 분석 가기 전에 LCQs 또는 MALDIs로 저렴 컴퓨터에 샘플을 테스트합니다. 당신이 거기에 산이있는 샘플을 다운 건조 마지막 경우, 유리를 사용합니다.

<ol>
	<li>Albuquerque, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multidimensional+chromatography+technology+for+in-depth+phosphoproteome+analysis.">A multidimensional chromatography technology for in-depth phosphoproteome analysis.</a> Mol Cell Proteomics. 7 (7), 1389-1396 (2008).</li><li>McNulty, D. E., Annan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+interaction+chromatography+reduces+the+complexity+of+the+phosphoproteome+and+improves+global+phosphopeptide+isolation+and+detection.">Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.</a> Mol Cell Proteomics. 7 (5), 971-980 (2008).</li><li>Gygi, S. 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이 방법은 양적 분석 수 phosphopeptides의 농축을 얻을 것입니다. 매개 변수의 숫자는이 프로토콜의 변경,하지만 기억에 가장 중요한 부분은 인산화를 보존하는 것입니다 수 있습니다. 당신이 그토록 ICAT와 같은 생체내, 태그에 전지를 라벨 수없는 경우 부량에 대한 세포의 준비는 예를 들어, 다른 여러 가지 방법으로 얻을 수 [3] 또는 ITRAC [4] differentially 부량 두 문화를 라벨에 게시물을 추출을 ...

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<td>Isotec&trade; L-Arginine-13C6,15N4</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>608033</td>
<td>&nbsp;</td>
<tr>
<td>Isotec&trade; L-Lysine-15N2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>609021</td>
<td>&nbsp;</td>
<tr>
<td>GIBCO&#x2122; Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>70011044</td>
<td>&nbsp;</td>
<tr>
<td>SigmaFAST Protease Inhibitor Tablets</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Pierce Halt Phosphatase Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>78420</td>
<td>&nbsp;</td>
<tr>
<td>Retsch PM100 Ball Mill Grinder </td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>20.540.0001</td>
<td>&nbsp;</td>
<tr>
<td>Retsch 125 ml Stainless Steel Grinding Jar</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>01.462.0148</td>
<td>&nbsp;</td>
<tr>
<td>Ultramicrospin C18 column</td>
<td>&nbsp;</td>
<td>The Nest Group</td>
<td>SUM SS10</td>
<td>&nbsp;</td>
<tr>
<td>Sep-Pak Vac 500 mg C18</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT043395</td>
<td>&nbsp;</td>
<tr>
<td>TSK-Gel Amide-80 4.6 mm x 25 cm analytical column</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>13071</td>
<td>This is the HILIC chromatographic column.</td>
</tr>
<tr>
<td>Guard column 4.6 mm ID x1 cm</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>19021</td>
<td>We recommend this guard column to extend the life of your HILIC column.</td>
</tr>
<tr>
<td>PHOS-Select&trade; Iron Affinity Gel</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P9740</td>
<td>This is the IMAC resin. Aliquot and store.</td>
</tr>		</tbody>
		</table>
		</div>

quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae

이 프로토콜은 isotopically 무거운 가벼운 S. cerevisiae의 세포의 지방산 oleate와, 성장과 자극을 설명합니다. 세포는 볼 밀 분쇄기 및 요소의 solubilization에 의해 솔루션으로 가져온 결과 grindate에 cryolysis 절차를 사용하여 접지합니다. 이 절차는 인산화을 보존하고 세포 용해 동안 phosphoproteome의 reorientation 방지, metabolically 비활성 상태에있는 세포의 용해를 위해 수 있습니다. 감소, 알킬화, 단백질의 트립신의 소화에 따라, 샘플은 C18 컬럼과 친수성 ​​상호 작용 크로마 토그래피 (HILIC)을 사용 분별화 감소 샘플 복잡 desalted 있습니다. HILIC 컬럼은 우선적으로 잘 phosphoproteomics 적합합니다 친수성 ​​분자를 유지합니다. Phosphorylated 펩티드가 아닌 phosphorylated 대응보다 색층 프로필 나중에 elute하는 경향이있다. 분별화 후, phosphopeptides은 인산 농축에 대해 요금을 부과 기반의 동질성에 의존 고정화 금속 크로마 토그래피를 사용하여 풍부하게하고 있습니다. 이 절차의 뒷부분에있는 샘플은 양적 질량 분광법으로 분석 준비가 된 것입니다.

Watch this Scientific Journal Video about Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae at JoVE.com

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

cryolysis, 요소의 solubilziation, HILIC 분류 및 phosphorylated 펩티드의 아이맥 농축을 사용하여 양적 인산화 과정에 대한 설명.

지방산의 정량 Phosphoproteomics은 자극 Saccharomyces cerevisiae</em

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

quantitative-phosphoproteomics-fatty-acid-stimulated-saccharomyces

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Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

10.0K 조회수.  Institute for Systems Biology. cryolysis, 요소의 solubilziation, HILIC 분류 및 phosphorylated 펩티드의 아이맥 농축을 사용하여 양적 인산화 과정에 대한 설명.

비디오: Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

의 해부 Saccharomyces Cerevisiae Asci

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

연구

생물학

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.

Microarray 분석을위한 Saccharomyces cerevisiae</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.

효모에서 모니터링 Mitophagy을위한 형광 현미경 분석 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 ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

HO - Endonuclease의 형성의 Quantitation과 분석에서 어닐링 단일 스트랜드에 의해 염색체 Translocations을 자극 Saccharomyces cerevisiae</em

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

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.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</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.

의 모니터링 단백질 접힘에 대한 결합 분석 실험<em&gt; 사카 cerevisiae의</em

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

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.

mRNA의 붕괴 요금의 측정에<em&gt; 사카로 미세스 세 레비 시아</em사용&gt;<em&gt; rpb1-1</em&gt; 균주

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.

화학 유전자 상호 작용의 신속한 식별<em&gt; 사카로 미세스 세 레비 시아</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.

진화 방법에 감독<em&gt; 사카로 마이 세스 세레 비지에</em&gt; : 돌연변이 라이브러리 생성 및 검사

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

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).