Мы хотели бы поблагодарить д-ра Богатые Роджерс для оказания технической помощи с масс-спектрометрии анализа и полезные обсуждения, и д-ра. Роб-Мориц, Джефф Ranish, и Хамид Mirzai за полезные обсуждения. Эта работа финансировалась NIH Центры компетенции.

Клеточного роста и средств массовой информации <ol><li> Единственная колония BY4742Δarg4Δlys1 клетки на ночь в 100 мл богатых СМИ OD 600 на 1,0, то семя на две 1 л культур минимальной среде дрожжи (0,17% Дрожжи азотистого основания, без сульфата аммония или аминокислот, 0,5% сульфат аммония), содержащий полный набор аминокислот, дополнен 20 мг / л изотопически нормального или тяжелого аргинин (13 C 6 15 N 4; Isotec) и лизина (13 C 6 15 N 2; Isotec). </li><li> Клетки выращивались в течение 18 часов, чтобы OD 600 из 1,8. Очень важно, чтобы клетки пройти как минимум 9 поколений, чтобы добиться полного включения меченых изотопов. Свет образец осаждали, промывали стерильной водой, reseeded в олеат среде, содержащей (изотонически нормальной аргинин и лизин, 0,2% олеиновой кислоты (Sigma Chemicals) и 0,5% Твин 40 (Sigma Chemicals)) и стимулировали еще 85 минут. Это дает изотопно света, по отношению к аргинин и лизин, олеат-стимулированных образца и изотопно тяжелых глюкозы выращенных эталонного образца. </li></ol> Лизиса клеток, выделения и фракционирования пептиды <ol start="3"><li> Взвесьте центрифуги бутылку. Урожай образцы центрифугированием в течение 3 минут. Удалить СМИ и аспирации лишнюю жидкость. Взвесьте гранул и бутылки (как правило доходности между 1-2 грамма), то вспышка заморозить в жидком азоте. Добавить объеме, эквивалентном гранулы вес &quot;шлифовка&quot; буфер для замороженных гранул в жидком азоте (фосфатно-солевым буфером (PBS, Gibco), 10% глицерина, Ингибиторы протеазы (SigmaFAST Таблетки ингибитор протеазы, Sigma) и HALT фосфатазы ингибиторы (Thermo Scientific )). </li><li> Замораживание шлифовальные судна в жидком азоте. Подождите, пока жидкого азота перестает кипеть. Передача гранулы для шлифовальных судна, с замороженными шарикоподшипников. </li><li> Grind при 600 оборотах в минуту с 1 мин 20 сек цикл с направлением обращения в течение 3 минут. Замораживать шлифовальные судна в жидком азоте. Повторите 4 раза в течение 15 минут общего времени измельчения. </li><li> Сбор замороженных grindate в 50 мл Сокол трубки месте сухого льда. Хранить при температуре -80 ° С до готовности к использованию. </li><li> Сделать 10мл решение 8М мочевины, 0,1 М бикарбонат аммония, 0,1 М Трис рН 8,6. Добавить 3 объема мочевины буфера 1 объем замороженных grindate (например 3mls из мочевины буфера добавляется в 1 г гранул с 1 мл PBS буфера). Сразу же разрушать ультразвуком смеси с sonicator зонда (2 - 10 секунд импульсов). Держите наконечник в нижней части трубки для предотвращения вспенивания раствора. Grindate должны немедленно отправиться в раствор. </li><li> Очистить лизата центрифугированием в течение 5 минут при 4 ° C. </li><li> Передача супернатант на свежий труб. </li><li> Сделайте свежие аликвоту 0,5 М трис [2-карбоксиэтил] фосфин (TCEP). Добавить в образце при 1:100 для конечной концентрации 5 мМ. Инкубировать при 37 ° С в течение 1 ч. </li><li> Алкилирование снижается цистеина. Дать остыть до комнатной температуры. Сделайте свежие аликвоту 1M iodoacetamide. Хранить в темноте (мы обернуть трубу в фольге). Добавить в конечной концентрации 20 мМ. Инкубировать в темноте в течение 1 часа при комнатной температуре. </li><li> Quench iodoacetamide с 20 мМ DTT 1M от фондового при комнатной температуре в течение 1 ч. </li><li> Количественная белка со стандартным Брэдфорд или BCA анализа (не описаны). Возьмите образец для анализа с помощью SDS PAGE ниже. </li><li> Для проверки включения тяжелых изотопов, используйте 100 мкл снижается и алкилированные лизат. Развести 1:4 с дН 2 O, разрушать ультразвуком и добавить в трипсин 50:1 белка трипсином. Дайджест в течение минимум 4 часов, сухой образец вниз в вакууме скорость, а затем опреснения с C18 столбцов Ultramicrospin (гнездо Group). <ol><li> Гидратов колонку с 100 мкл ацетонитрила </li><li> Равновесие с 200 мкл 0,1% TFA. </li><li> Ресуспендируют сухого образца в 200 мкл 0,1% TFA. Нагрузка на колонну. Центрифуга при температуре ~ 200 мкг или минимальной скорости, необходимые для потока через колонку. </li><li> Промыть два раза с 200 мкл 0,1% TFA. </li><li> Элюируются в два раза с 50 мкл 60% ацетонитрила, 0,1% TFA. </li><li> Сухой образец в вакууме скорость. </li><li> Ресуспендируют образца в 20 мкл 0,1% муравьиной кислоты. Выполните 2 мкл на масс-спектрометре. Проверьте включение изотопно тяжелых аргинин и лизин сдвигом 10 и 8 Далтон в соответствующих т / г спектров. Включения для каждой аминокислоты должны быть в пределах от 96 - 98%. </li></ol></li><li> Смешайте эквивалентные суммы (мг белков) изотопически тяжелые и легкие образцы. </li><li> Разбавьте образцы 1:04 с 20% метанола. </li><li> Добавить трипсина на 1:200. Выдержите в течение ночи при температуре 37 ° C. </li><li> Убедитесь, что пищеварение отправился в завершение СТРАНИЦА SDS. Выполните 2 и 4 мкл предварительное переваривание образца и 8D 16 мкл сообщение трипсина переварить. Визуализируйте окрашиванием Кумасси, что пищеварение завершена. Если он не является полным, разрушать ультразвуком образца с sonicator кратко кончика зонда и переварить снова трипсином. </li><li> Сухой образец вниз в скорости вакцины. При сухой добавить 200 мкл с фильтром дН 2 O и вихревые до гранул переходит в раствор. Сухой образец вниз в скорости вакцины. Ресуспендируют снова дН 2 O. Сухой образец снова. </li><li> Ресуспендируют гранулы в 0,1% трифторуксусной кислоты (ТФУ; Sigma). Убедитесь, что рН составляет около 3 с помощью рН полоски. При необходимости подкисляют 10% TFA. </li><li> Гранул из любой осадок. </li><li> Опреснения образцов на водах Sep-Pak Vac 500 мг C18 колонке. Не перегружайте этих столбцов. Максимальная сумма составляет 5 мг, но лучше, чтобы загрузить немного меньше. Разделить между образцом несколько столбцов, если необходимо. <ol><li> Гидратов колонки с 5 мл на 100% ацетонитрила. Центрифуга при температуре ~ 200 мкг или минимальной скорости, необходимые для потока через колонку. </li><li> Равновесия колонка с 5 мл 0,1% TFA. Нагрузка образца. Сбор потока и загрузить снова. </li><li> Промыть 5 мл 0,1% TFA. Повторить. </li><li> Элюции с 2 мл 60% ацетонитрила, 0,1% TFA. </li><li> Сухой образец вниз в скорости вакцины. </li></ol></li></ol> Пептидов Фракционирование и выделение фосфопептидов <ol><li> Пептиды фракционируют на HILIC колонке. Мы используем Tosoh TSK-гель Амид-80 4,6 мм х 25 см аналитическую колонку с охраной колонке. Не торопитесь с этим шагом, запустить колонку в течение 2 часов при 90% запустить две пустые градиенты до загрузки вашего образца. Максимальная нагрузка на колонки такого размера, как считается, около XX, мы обычно не загружайте более 5 мг в перспективе. </li><li> Растворитель - 98% ACN/0.1% TFA Растворитель B - 2% ACN/0.1% TFA Нагрузка в 90% в течение 20 &#39;, 90% до 85% в течение 5, 85% до 60% в течение 40&#39;, 60% до 0% в течение 5 &#39;, 0% до 90% в течение 5 &#39;. </li><li> Расход 1ml/min и фракций были собраны на 2 минуты интервалы, начиная с 85% до 60% градиент. </li><li> Комбинат фракций уменьшить количество фракций до 10. Увеличение фракций, скорее всего, принесет больше охват phosphoproteome. </li></ol> Обогащение фосфопептидов <ol><li> Фосфопептиды обогащенного использованием PHOS-Select Железный Affinity лари. Мы аликвоту нашей IMAC смолы, чтобы избежать повторного замораживания оттаивания. Сделайте 50 мл 250 мМ уксусной кислоты, 30% раствор АКС нагрузки. Довести рН до 2,7 с HCl. Ресуспендируют гранул в 500 мкл нагрузка решение. Проверьте рН, отрегулируйте с HCl или NaOH по мере необходимости. </li><li> Предварительная стирка 200 мкл IMAC бисером в соответствующую графу. Мы используем Molbiotec столбцов спина. Добавить 15 мкл 50% раствора (гель для загрузки раствора) в каждой фракции, и инкубировать образцы для 30 &#39;с конца по конец вращения. Держите потока для анализа. </li><li> Вымойте образцы 3 раза с грузом решение и один раз с 500 мкл фильтруется дН 2 O. </li><li> Элюции пептидов с 2 - 3 мин при 400 мкл 50 мМ Na 2 HPO 4 (рН 8,4) буфера. Трансфер в стеклянный пузырек. </li><li> Подкисляют до рН 3 с 5 мкл 100% муравьиной кислоты, флэш заморозить в жидком азоте, и высушенного образца вниз в скорости вакцины. </li><li> Ресуспендируют фосфопептидов в 200 мкл 0,1% TFA и C18 чист, как описано выше. Сухой вниз элюировали образцов в вакууме скорость. </li><li> Ресуспендируют пептидов в 20 мкл 0,1% муравьиной кислоты. Используйте 2 мкл на масс-спектрометре анализа или по мере необходимости. </li></ol> Примечания На протяжении большей части этого протокола вы будете работать «слепым». Лизиса клеток можно контролировать с помощью западных промокательной и Брэдфорда или BCA анализы, и пищеварение трипсина также может быть легко оценена. HILIC столбец дал след, подобный этому, или, как показано в других работах [1, 2] в зависимости от жидкой хроматографии машина, которая используется. Образцы контролируются масс-спектрометрии; тестирование образцов на недорогих машин, таких как LCQs или Мальдис перед отходом ко высокой анализ стоимости на высокой точностью массы, высокий машина разрешения. Наконец, если вы вниз сушки образца с кислотой в нем, использование стекла.

<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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+complex+protein+mixtures+using+isotope-coded+affinity+tags.">Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.</a> Nat Biotechnol. 17 (10), 994-999 (1999).</li><li>Ross, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol Cell Proteomics. 3 (12), 1154-1169 (2004).</li><li>Gruhler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteomics+applied+to+the+yeast+pheromone+signaling+pathway.">Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.</a> Mol Cell Proteomics. 4 (3), 310-327 (2005).</li><li>Wilson-Grady, J. T., Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+analysis+of+fission+yeast.">Phosphoproteome analysis of fission yeast.</a> J Proteome Res. 7 (3), 1088-1097 (2008).</li><li>Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SCX/IMAC+enrichment+approach+for+global+phosphorylation+analysis+by+mass+spectrometry.">The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.</a> Nat Protoc. 3 (10), 1630-1638 (2008).</li><li>Jensen, S. S., Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+impact+of+some+experimental+procedures+on+different+phosphopeptide+enrichment+techniques.">Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.</a> Rapid Commun Mass Spectrom. 21 (22), 3635-3645 (2007).</li><li>Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+enrichment+of+phosphorylated+peptides+from+peptide+mixtures+using+titanium+dioxide+microcolumns.">Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.</a> Mol Cell Proteomics. 4 (7), 873-886 (2005).</li><li>Pinkse, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isolation+at+the+femtomole+level+of+phosphopeptides+from+proteolytic+digests+using+2D-NanoLC-ESI-MS/MS+and+titanium+oxide+precolumns.">Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.</a> Anal Chem. 76 (14), 3935-3943 (2004).</li><li>Tao, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteome+analysis+using+a+dendrimer+conjugation+chemistry+and+tandem+mass+spectrometry.">Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.</a> Nat Methods. 2 (8), 591-598 (2005).</li><li>Zhou, H., Watts, J. D., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+approach+to+the+analysis+of+protein+phosphorylation.">A systematic approach to the analysis of protein phosphorylation.</a> Nat Biotechnol. 19 (4), 375-378 (2001).</li><li>Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+isolation+of+distinct,+overlapping+segments+of+the+phosphoproteome.">Reproducible isolation of distinct, overlapping segments of the phosphoproteome.</a> Nat Methods. 4 (3), 231-237 (2007).</li></ol>

Этот метод даст обогащение фосфопептидов, которые могут быть количественный анализ. Число параметров может быть изменен в данном протоколе, но самый важный аспект помнить, чтобы сохранить свои фосфорилирования. Подготовка клеток для количественной может быть достигнуто в несколько р...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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

Этот протокол описывает роста и стимулирования, с жирной олеиновой кислоты, изотопно-тяжелой и легкой клетки С. CEREVISIAE. Клетки местности с помощью криодеструкции процедура в мясорубку шаровой мельнице и в результате grindate приведены в растворе мочевины солюбилизации. Эта процедура позволяет лизис клетки в метаболически неактивное состояние, сохраняя фосфорилирования и предотвращения переориентации phosphoproteome во время клеточного лизиса. После сокращения, алкилирования, трипсина переваривание белков, образцы обессоленной на C18 колонны и образец сложности сократились на фракционирования использованием гидрофильной хроматографии взаимодействия (HILIC). HILIC колонн преимущественно сохраняют гидрофильных молекул, которые хорошо подходят для phosphoproteomics. Фосфорилированных пептиды, как правило, элюируются позднее в хроматографического профиля, чем не фосфорилированных аналогов. После фракционирования, фосфопептиды обогащенного использованием иммобилизованных металла хроматографии, которая опирается на заряд основе сродство к фосфопептидов обогащения. В конце этой процедуры образцы готовы к количественному анализу методом масс-спектрометрии.

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

Описание процедуры количественного фосфорилирования использованием криодеструкция, мочевина solubilziation, HILIC фракционирования и IMAC обогащения фосфорилированных пептиды.

Количественные Phosphoproteomics в жирные кислоты Вынужденное 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

Research

JoVE Journal

Biology

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

10.0K Views.  Institute for Systems Biology. Описание процедуры количественного фосфорилирования использованием криодеструкция, мочевина solubilziation, HILIC фракционирования и IMAC обогащения фосфорилированных пептиды.

Video: 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.

Препарирование Saccharomyces CEREVISIAE Сумки

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

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 анализа для 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.

Флуоресценция Пробирной микроскопии для мониторинга Mitophagy в Дрожжи 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.

Количественный и анализ формирования HO-эндонуклеаза Вынужденное Хромосомные транслокации по однонитевые Отжиг в 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.

Визуализации и анализа молекул мРНК с помощью флуоресцентной<em&gt; В Ситу</em&gt; Гибридизация в<em&gt; 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.

В сочетании Анализы для мониторинга рефолдинг Белок в<em&gt; Saccharomyces CEREVISIAE</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.

Измерение мРНК темпы разложения в<em&gt; Saccharomyces Cerevisiae</em&gt; Использование<em&gt; Rpb1-1</em&gt; Штаммы

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.

Быстрая идентификация химических генетических взаимодействий в<em&gt; Saccharomyces Cerevisiae</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.

Направленный метод Эволюция<em&gt; Saccharomyces CEREVISIAE</em&gt;: Mutant Создание и скрининг библиотеки

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).