وأيد هذا العمل من جانب صناديق من المعاهد الوطنية للصحة والبحوث معهد بيكمان في مدينة الأمل. نود أن نشكر المقيمين على تعليقاتهم البناءة التي الوضوح وأضاف أن المخطوطة.

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direction+of+chromosome+rearrangements+in+Saccharomyces+cerevisiae+by+use+of+his3+recombinational+substrates.">Direction of chromosome rearrangements in Saccharomyces cerevisiae by use of his3 recombinational substrates.</a> Mol. Cell. Biol. 8, 4370-4380 (1988).</li><li>Manthey, G. M., Naik, N., Bailis, A. 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الإعلان عن أي تضارب في المصالح.

جرعات عالية من الإشعاعات المؤينة تمثل مخاطر عدم الاستقرار المتأصلة في الجينوم من خلال توليد عدد كبير من DSBs 19. الجينوم حقيقية النواة تزخر تسلسل المتكررة التي هي ركائز ممتازة لتوليد translocations وإعادة ترتيب الجينوم أخرى 20،21. ويلاحظ في كثير من الأحيان عن طريق الموارد البشرية translocations الكروموسومات عند إدخال DSBs بين متواليات تكرارية 12،21،22. أدلة دامغة تشير إلى أن الكثير من عدم الاستقرار الجيني المرتبطة اللوكيميا والأورام اللمفاوية ويمكن أن يعزى إلى translocations الكروموسومات ، مسلطا الضوء على أهمية فهم كيفية حدوث هذه الآلية في حقيقيات النوى 22،23. قمنا بتطوير نظام الخميرة في مهدها لدراسة تشكيل الصبغيات إزفاء بواسطة جهاز تسوية المنازعات الناجمة عن الموارد البشرية بين المناطق قصيرة من التماثل على الكروموسومات المختلفة التي تشبه في الحجم العناصر المتكررة في جميع أنحاء متفرقة الخميرة والجينوم البشري. في مقايسة ، ويقع على الركيزة his3 - Δ3 &#39;إزفاء على نسخة واحدة من الكروموسوم الخامس عشر. الآخر his3 أليل (his3 - Δ200) قد حذف من 1KB ~ المروج HIS3 وتسلسل الترميز الذي يمنع هذا التسلسل من أن تستخدم كنموذج لإصلاح 24. يقع على الركيزة his3 - Δ5 &quot;في موضع LEU2 على نسخة واحدة من الكروموسوم الثالث ، مع نسخة أخرى من كروموسوم الثالث يحتوي على LEU2 أليل دون تغيير (الشكل 1). أدرجت نوكلياز داخلية HO - الغالاكتوز محرض الكاسيت التعبير ملحوظ مع KAN - MX في الموضع الرابع TRP1 الصبغي (trp1 : GAL - HO - KAN - MX). ويحيط كل ركيزة من خلال تسلسل إزفاء الاعتراف HO - نوكلياز داخلية يمكن أن تكون مستهدفة من قبل لإحداث انشقاق التعبير عن الجينات HO من خلال إضافة اللبن إلى المتوسط. بعد الانقسام الناجم HO - نوكلياز داخلية في his3 - Δ3 &#39;وhis3 - Δ5&#39; ركائز ، يمكن استخدام الخلايا كفاءة الجهاز المشتركة قصيرة من تسلسل تناظر HIS3 (311 سنة مضت أو غليان 60) لإصلاح الكروموزومات تخللتها الموارد البشرية ، وتوليد كروموسوم إزفاء مع أليل HIS3 12-14،25 سليمة. لأن الأصل عدم الخلايا نسخة سليمة من الجين HIS3 ، فهي غير قادرة على النمو على صاحب المتوسط. يمكن تحديد الخلايا الوحيدة التي خضعت لهذا الحدث إزفاء على الحامض الاميني المتوسطة معدومة. لذا ، يمكن حساب تواتر الصبغيات translocations بقسمة عدد من المستعمرات بدئية الحامض الاميني على العدد الكلي للخلايا قابلة للحياة مطلي ، والتي تحددها الطلاء على YPD. ويمكن بعد ذلك الحمض النووي الجينومية والكروموسومات سليمة يمكن عزلها عن المستعمرات + ممثل له ، وجود كروموسوم إزفاء التحقق من جنوب الجينومية وتحليلات لطخة الصبغي. وقد سمح لنا تحليلا دقيقا لجمع معلومات مهمة إضافية حول مقايسة 12. وقد وفرت جنوب لطخة تحليل الجينوم أدلة على أن هناك عدة قطاعات كاملة تقريبا من الصبغيات الخامس عشر والثالث بعد 30 دقيقة من HO - نوكلياز داخلية الاستقراء ، وبالتالي ليس هناك أي خلفية هامة من ركائز الكروموسومات غير المصقول في عدد السكان (G. مانثي Bailis &amp; A. ، لم تنشر النتائج). الجينومية تحليلات لطخة الجنوبية وصاحب كروموسوم -- الناجين تشير إلى أن تفقد الخلايا بشكل متكرر واحد ، وأخرى ، أو كليهما الكروموسومات قص وتبقى قابلة للحياة (L. A. Bailis ويديل ، ونتائج غير منشورة). الأهم من ذلك ، الكفاءات الطلاء يعادل تقريبا على غير انتقائية المتوسط ​​قبل وبعد تحريض من التعبير HO - نوكلياز داخلية تشير إلى أن أيا من الفشل في إصلاح الكروموزومات مكسورة ، أو فشل في الاحتفاظ الكروموسومات إزفاء يؤثر على القدرة على البقاء على قيد الحياة تشكيل جهاز تسوية المنازعات. تمشيا مع هذا ، tXV : لقد ثبت الثالث كروموسوم إزفاء أن تكون غير مستقرة في الخلايا الإنقسامية في غياب التحديد. وقد تجلى ذلك من خلال تنامي tXV : الثالث الذي يشتمل recombinants + بلده بين عشية وضحاها غير انتقائي ، والطلاء واحد على المستعمرات غير انتقائية لوحات ، والطلاء على طبق متوسط ​​انتقائية تفتقر الحامض الاميني. وكان عشرة إلى 70 ٪ من المستعمرات الناتجة عن هذه اللوحات فقدت tXV : كروموسوم إزفاء الثالث (N. Pannunzio Bailis &amp; A. ، نتائج غير منشورة). إزفاء الكروموسومات التي تم إنشاؤها بواسطة الأشعة تحت الحمراء التعرض في البشر مماثلة يحمل عدم الاستقرار 26. هذا يشير إلى أن تشكيل إزفاء قد تسهم في احداث تكون الأورام في وقت مبكر عن طريق تعزيز فقدان تغاير الزيجوت. الثانية ، تحليلات واسعة النطاق الوراثية والجزيئية تشير إلى أن منطقة أفريقيا جنوب الصحراء ، وإنشاء آلية فعالة وغير المحافظ اجباريا للموارد البشرية ، هو الآلية الأساسية لتكوين الموارد البشرية إزفاء التي أعقبت إنشاء في وقت واحد على اثنين من الكروموزومات DSBs 12،27،28. ويشترك هذاnsistent مع الحقائق أن جرعات كبيرة من الأشعة تحت الحمراء في نتيجة كثافة DSBs كافية لإنشاء فواصل المتاخمة لمتواليات تكرارية متعددة في الجينوم الخميرة ، وارتفاع وتيرة تشكيل إزفاء بواسطة الموارد البشرية. معا ، هذه الملاحظات تشير إلى أن تأثير الأشعة تحت الحمراء أنكجنيك التعرض في البشر ، قد جزئيا ، نتيجة لإصلاح DSBs بواسطة آلية فعالة للموارد البشرية التي تولد translocations أنه ، من خلال عدم الاستقرار الكامنة ، وتعزيز التغيرات الجينية التي تكون الأورام الاطلاق. لأنه غالبا ما تستخدم لعلاج الإشعاع سرطان إعادة ترتيب الجينوم ، التي تنتج عن إصلاح الناجمة عن الاشعاع DSBs يمكن أن تسهم في توليد السرطانات الثانوية التي تنشأ في كثير من الأحيان المرضى. وبالتالي ، قد يكون هذا النموذج يساعدنا على اكتساب فهم أفضل للأساس الوراثية والجزيئية للاستجابة للعلاج السريري مهم الأشعة تحت الحمراء.

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.

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

في مقايسة إزفاء HO - حفز مراقبين منفردة الجديلة الصلب بعد إنشاء فواصل المزدوج حبلا الحمض النووي في مواضع متعددة في ضعفاني<em&gt; السكيراء الجعوية</em&gt;. قد يكون هذا النموذج آلية إعادة ترتيب الجينوم في الخلايا الجسمية العالي حقيقيات النوى عقب التعرض لجرعات عالية من الإشعاعات المؤينة.

حفز الكميات وتحليل تشكيل HO - نوكلياز داخلية Translocations الصبغية التي ستراند ، واحدة في التخمير السكيراء الجعوية</em

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

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Biology

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

14.6K Views.  Irell & Manella Graduate School of Biological Sciences. في مقايسة إزفاء HO - حفز مراقبين منفردة الجديلة الصلب بعد إنشاء فواصل المزدوج حبلا الحمض النووي في مواضع متعددة في ضعفاني السكيراء الجعوية. قد يكون هذا النموذج آلية إعادة ترتيب الجينوم في الخلايا الجسمية العالي حقيقيات النوى عقب التعرض لجرعات عالية من ال...

Video: Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in 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 ...

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 ...

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.

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 ...

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

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

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

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

التعبير وتنقية عبر الغشاء التليف الكيسي بروتين منظم المواصلة في خميرة الخباز

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

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 ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

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

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

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

EPR رصدت الأكسدة والاختزال المعايرة من العوامل المساعدة لل<em&gt; خميرة الخباز</em&gt; Nar1

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

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

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

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

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

قياس مرنا تسوس الأسعار<em&gt; خميرة الخباز</em&gt; استخدام<em&gt; rpb1-1</em&gt; سلالات

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