وأيد هذا العمل عن طريق البحث الاسترالي غرانت DP0986937 المجلس.

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	<li>Levine, B., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+the+pathogenesis+of+disease.">Autophagy in the pathogenesis of disease.</a> Cell. 132, 27-42 (2008).</li><li>Mizushima, N., Levine, B., Cuervo, A. M., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+fights+disease+through+cellular+self-digestion.">Autophagy fights disease through cellular self-digestion.</a> Nature. 451, 1069-1075 (2008).</li><li>He, C., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+mechanisms+and+signaling+pathways+of+autophagy.">Regulation mechanisms and signaling pathways of autophagy.</a> Annu. Rev. Genet. 43, 67-93 (2009).</li><li>Kroemer, G., Mari&#241;o, G., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+the+integrated+stress+response.">Autophagy and the integrated stress response.</a> Mol Cell. 40, 280-293 (2010).</li><li>Camougrand, N., Kissov&#225;, I., Salin, B., Devenish, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+mitophagy+in+yeast.">Monitoring mitophagy in yeast.</a> Methods Enzymol. 451, 89-107 (2008).</li><li>Yen, W. L., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+live+long+and+prosper:+autophagy,+mitochondria,+and+aging.">How to live long and prosper: autophagy, mitochondria, and aging.</a> Physiology (Bethesda). 23, 248-262 (2008).</li><li>Tolkovsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy.">Mitophagy.</a> Biochim. Biophys. Acta. 1793, 1508-1515 (2009).</li><li>Bhatia-Kissov&#225;, I., Camougrand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+in+yeast:+actors+and+physiological+roles.">Mitophagy in yeast: actors and physiological roles.</a> FEMS Yeast Res. 10, 1023-1034 (2010).</li><li>Kanki, T., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanism+of+mitochondria+autophagy+in+yeast.">The molecular mechanism of mitochondria autophagy in yeast.</a> Mol. Microbiol. 75, 795-800 (2010).</li><li>Nowikovsky, K., Reipert, S., Devenish, R. J., Schweyen, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdm38+protein+depletion+causes+loss+of+mitochondrial+K+/H++exchange+activity,+osmotic+swelling+and+mitophagy.">Mdm38 protein depletion causes loss of mitochondrial K+/H+ exchange activity, osmotic swelling and mitophagy.</a> Cell Death Differ. 14, 1647-1656 (2007).</li><li>Devenish, R. J., Prescott, M., Turcic, K., Mijaljica, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+organelle+turnover+in+yeast+using+fluorescent+protein+tags.">Monitoring organelle turnover in yeast using fluorescent protein tags.</a> Methods Enzymol. 451, 109-131 (2008).</li><li>Rosado, C. J., Mijaljica, D., Hatzinisiriou, I., Prescott, M., Devenish, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosella:+a+fluorescent+pH-biosensor+for+reporting+vacuolar+turnover+of+cytosol+and+organelles+in+yeast.">Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast.</a> Autophagy. 4, 205-213 (2008).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+yeast+by+lithium+acetate/single-stranded+carrier+DNA/polyethylene+glycol+method.">Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.</a> Methods Enzymol. 350, 87-96 (2002).</li></ol>

الإعلان عن أي تضارب في المصالح.

في الصور المعروضة في شكل تمثيلية. ويمكن 2B 2C يمكن ان ينظر اليها بشكل واضح أن استخدام عضية محددة للصحفيين الفلورسنت ومضان المجهري توفر أدلة على توطين الميتوكوندريا / التوزيع في كل من النمو والخلايا النيتروجين المتعطشة ، ويسمح mitophagy أن تصور. فمن ينبغي التأكيد على أن لا يقتصر هذا الأسلوب لmitophagy التالية. يمكن رصد الالتهام الذاتي من المكونات الخلوية الأخرى أو العضيات (على سبيل المثال ، ريبوسوم ، قطرات الدهون ، peroxisomes ، شبكية هيولي باطني والنواة) مع وضع العلامات المناسبة. وقد ذكرت تفاصيل عن بناء جهاز الاستشعار البيولوجي روزيلا ، والظروف والنتائج خلية ثقافة نموذجية لدوران النواة (nucleophagy) 11،12 توضح التوسع في تطبيق الأسلوب. استخدام عضية محددة للصحفيين الفلورسنت لرصد الالتهام الذاتي يتطلب أن تبقى عدة اعتبارات تصميم بناء في الاعتبار. وهذه تشمل : (1) اختيار والانصهار في إشارة إلى مناسبة تستهدف تحقيق عضية محددة الاستهداف ؛ (2) اختيار البروتين الفلوري المناسبة (ق) ، (3) الانبعاثات مضان قوية ؛ (4) توطين عضية الصحيح و / أو التوزيع داخل الخلية. بمجرد مراسل عضية محددة أعرب بنجاح في استضافة الخميرة اختيار سلالة المجموعة الثانية من الاعتبارات يصبح : (1) ظروف نمو الخميرة ، (2) إعداد نموذج الخلية ؛ (3) معلمات التصوير كما أنه من المهم أن المعلمات المختارة لCLSM (على سبيل المثال ، وكثافة الليزر ، وفحص معدل واسطة ، قيمة ذات الثقب ، وقيمة التكبير وأهدافها المستخدمة أو التعرض للكاميرا CCD) تمسك طوال تجربة ضمان إمكانية إجراء مقارنات بين السيطرة (خلايا النمو) وعينات جوعا ؛ ( 3) الحث الالتهام الذاتي وناجحة تسليم الصحفي إلى فجوة. عموما ، هذا الأسلوب المجهري الفلورسنت سهلة وسريعة نسبيا ، والتطبيق المحتمل لفحص إنتاجية عالية على المخدرات الرواية التي تعزز أو تعيق الالتهام الذاتي ، وأيضا عن الجينات التي تنظم أو تعدل الالتهام الذاتي.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف / المعدات </th><th> شركة </th><th> فهرس العدد </th></tr><tr><td> السكيراء الجعوية EasyComp تحويل كيت </td><td> Invitrogen </td><td> K5050 - 01 </td></tr><tr><td> الخميرة قاعدة النيتروجين دون الأحماض الأمينية وكبريتات الامونيوم </td><td> دينار بحريني Difco </td><td> 200-0030 </td></tr><tr><td> انخفاض ذوبان agarose </td><td> PROGEN العلوم البيولوجية </td><td> 200-0030 </td></tr><tr><td> شرائح مجهرية (76 × 26 ملم) </td><td> منزل - جلاسر </td><td></td></tr><tr><td> coverslips المجهر (22 × 22 ملم) </td><td> منزل - جلاسر </td><td> BB022022A1 </td></tr><tr><td> ليزر متحد البؤر المسح الضوئي مجهر Fluoview FV500 </td><td> اوليمبوس </td><td></td></tr><tr><td> الارجنتين تواجه المركز. </td><td> Moleular مجسات - Invitrogen </td><td> Y - 7531 </td></tr></tbody></table> جميع التركيزات المعطاة ث / ت ، ما لم يرد خلاف ذلك. متوسط ​​الحد الأدنى من الخميرة (YMM) : 0.67 ٪ النيتروجين قاعدة الخميرة دون الأحماض الأمينية والغلوكوز 2 ٪ ، 1.5 ٪ أجار (المضافة فقط عندما جعل وسائل الاعلام الصلبة) وأية ملاحق عونية التغذي المطلوبة (0.01 ٪)]. السكيراء الأملاح (SS) : (NH 4) 2 S0 4 ، 0.12 ٪ ؛ KH 2 PO 4 ، 0.10 ٪ ، 0.6 MgC1 2 H2O ، 0.07 ٪ ؛ NaC1 ، 0.05 ٪ ؛ CaCl2 ، 0.01 ٪ ؛ FeC1 3 ، 0،0005 ٪. متوسطة النمو : SS والإيثانول 2 ٪ (V / V) والمكملات عونية التغذي المناسبة. النيتروجين التجويع المتوسط ​​: 0.17 ٪ النيتروجين قاعدة الخميرة دون الأحماض الأمينية وكبريتات الامونيوم (Difco) ، والإيثانول 2 ٪ (V / V).

a fluorescence microscopy assay for monitoring mitophagy in the yeast saccharomyces cerevisiae

الالتهام الذاتي المهم للدوران من المكونات الخلوية في ظل مجموعة من الظروف المختلفة. أنها تخدم وظيفة أساسية استتبابي فضلا عن آلية لمراقبة الجودة التي يمكن أن تستهدف بشكل انتقائي وتتحلل المادة الخلوية بما في ذلك 4 - organelles1. على سبيل المثال ، تضررت أو زائدة الميتوكوندريا (الشكل 1) ، وليس التخلص منها عن طريق الالتهام الذاتي يمكن أن تمثل تهديدا للتوازن الخلوية وبقاء الخلية. في الخميرة ، خميرة يمكن السكيراء ، والحرمان من المواد الغذائية (مثل المجاعة النيتروجين) أو الضرر تعزيز دوران الانتقائي للالميتوكوندريا التي الالتهام الذاتي في عملية تسمى mitophagy 5-9. وصفنا بسيطة نهج المجهري مضان لتقييم الالتهام الذاتي. من أجل الوضوح ونحن لدينا وصف تقييد هنا لاظهار كيف يمكن استخدام نهج لرصد mitophagy في خلايا الخميرة. ويجعل من استخدام مقايسة مراسل الفلورسنت ، روزيلا ، وهو جهاز الاستشعار البيولوجي المزدوجة الانبعاثات يضم الحموضة مستقرة نسبيا بروتين فلوري الأحمر مرتبطة بروتين درجة الحموضة حساسة الفلورية الخضراء. تشغيل هذا المراسل تعتمد على الاختلافات في درجة الحموضة بين (5،0-5،5 درجة الحموضة ~) فجوة والميتوكوندريا (درجة الحموضة ~ 8.2) في الخلايا الحية. تحت ظروف النمو ، وخلايا النوع البري يحمل كل مضان أحمر وأخضر وزعت في طريقة مميزة في الميتوكوندريا. لا يرتبط مضان الانبعاثات مع فجوة. عندما تتعرض لمجاعة النيتروجين ، وهو الشرط الذي يدفع mitophagy ، بالإضافة إلى مضان أحمر وأخضر وصفها الميتوكوندريا ، والخلايا يحمل تراكم الحمراء ، ولكن لا مضان أخضر ، في التجويف وفجوي الحمضية يمثل تسليم الميتوكوندريا إلى فجوة. احرز مع الخلايا الحمراء ، ولكن يمكن استخدام الفجوات الفلورية الخضراء لا كمقياس للنشاط في الخلايا mitophagic 5،10-12.

Watch this Scientific Journal Video about A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae at JoVE.com

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

ووصف نهج قوي لمراقبة تسليم العضيات لمعة الحامضية للفجوة الخميرة للتحلل وإعادة التدوير. الأسلوب يعتمد على وضع العلامات المحددة الهدف العضيات مع مضان المزدوجة الانبعاثات المرمزة وراثيا الحموضة ، جهاز الاستشعار البيولوجي ، والتصور من الخلايا الفردية باستخدام المجهر مضان.

والفحص المجهري لالإسفار Mitophagy الرصد في الخميرة السكيراء الجعوية

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

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

25.4K Views.  Monash University. ووصف نهج قوي لمراقبة تسليم العضيات لمعة الحامضية للفجوة الخميرة للتحلل وإعادة التدوير. الأسلوب يعتمد على وضع العلامات المحددة الهدف العضيات مع مضان المزدوجة الانبعاثات المرمزة وراثيا الحموضة ، جهاز الاستشعار البيولوجي ، والتصور من الخلايا الفردية باستخدام ا?...

Video: A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast 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.

تشريح السكيراء الجعوية زقاق

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

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

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

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

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

حفز Phosphoproteomics الكمية في الأحماض الدهنية السكيراء الجعوية</em

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

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

الوقت الفاصل بين التصوير من الانقسام الميتوزي بعد ترنسفكأيشن سيرنا

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

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.

تحليل لميكروأري السكيراء الجعوية</em

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

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

الكمية خلية حية الإسفار - المجهري تحليل الخميرة الانشطار

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

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.

التصور والتحليل من جزيئات الرنا المرسال عن طريق الإسفار<em&gt; في الموضع</em&gt; التهجين في<em&gt; خميرة الخباز</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.

فحوصات بالإضافة لرصد طوي ثانية بروتين في<em&gt; خميرة الخباز</em

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

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

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

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

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

تنقية التليف الكيسي عبر الغشاء المواصلة منظم البروتين المعبر عنه في<em&gt; خميرة الخباز</em

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

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.

تحديد سرعة التفاعلات الكيميائية الوراثية في<em&gt; خميرة الخباز</em

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

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).