Ashton Belew, Karen Jack Sharmishtha Musalga, Sergey Sulima Shivani Reddy ve onların yardım ve bu projenin giriş Michael Rhodin Dinman laboratuvar üyeleri, teşekkür etmek istiyoruz. Bu çalışma, Amerikan Kalp Derneği (AHA 0630163N), AM ve Ulusal Sağlık Enstitüleri (5R01 GM058859-12) JDD bağışları ile destek verdi. JAL, 2009 ek ana hibe bir Amerikan Yatırım ve Kurtarma Yasası (3R01GM058859-11S1) tarafından desteklenmiştir.

1. Bir sistein hazırlanması Sulfolink reçine ücret <ol><li> Oda sıcaklığında Sulfolink reçine (Pierce) hazırlayın. </li><li> Her bir şişenin içine dağıtılan 5 ml, 10 ml% 50 olarak iki adet 10 ml plastik şişeleri, üretici tarafından sağlanan Sulfolink kaplin jel bulamaç toplam yerleştirin. </li><li> Kısaca 850 xg&#39;de şişeleri santrifüj ve dikkatle süpernatantlar kaldırmak. </li><li> 5 ml boncuklar resuspending xg 850 kısaca santrifüj ve pipet ile süpernatantı çıkarmadan kaplin tamponu (50 mM Tris, pH 8.5, 5 mM EDTA) jel üç kez yıkayın. </li><li> Her tüp kaplin tamponu 50 mM L-sistein çözüm beş ml ekleyin ve 1 saat 25 bulamaç karıştırın ° C </li><li> Yukarıda adım 1.4 &#39;de açıklandığı gibi yıkama L-sistein kalan çıkarın. </li><li> Jel 10 ml bağlayıcı tampon yeniden süspanse [20 mM Hepes, pH 7.6, 5 mM Mg (oac) 2, 60 mM NH 4 Cl, 2 mM DTT] ve reçine bağlayıcı tampon 10 ml yıkama ile dengelenmesi3 kez adım 1.4 &#39;de açıklandığı gibi. Durusu reçine içine 10 ml santrifüj sütun, kap ve 4 saklanan ° C RNA bağlayıcı reçine kapasitesi ~ 20 ml başına 260 birimleri. </li></ol> 2. Sistein kullanarak ribozomların Kromatografik arıtma Sulfolink reçine ücret Not: Eğer proteaz aktivitesi bir sorun olduğunu kanıtlamaktadır bir yük ile çalışan, proteaz inhibisyonu kokteyller sonraki tüm tamponlar kullanılabilir. <ol><li> Buz üzerinde tüm bileşenlerini korumak ve RNaz-serbest su ve steril filtrasyon kullanan tüm çözümler hazırlıyoruz. </li><li> Büyümeye uygun ortam orta-log faz (1.2 OD 595 = 0.6) bir gecede maya hücreleri. Pelet hücreler santrifüj yoluyla, 4, 5 dakika boyunca 3700 xg bağlayıcı tampon ve santrifüj 1 gram hücreleri yıkayın ° C </li><li> Bağlayıcı tampon 1 ml yaklaşık toplam hacmi 2 ml ve 0,5 mm cam boncuklar 4 soğutulmuş bir eşit miktarda kullanarak bozabilir yeniden süspanse hücreler ° C uBiospec Mini-boncuk Bilo (Bartlesville, OK) şarkı. Örnekleri gerektiği gibi birden fazla tüp içine bölünebilir. </li><li> Beckman Coulter Optima Max E ultrasantrifüjdeki (Fullerton, CA) 30 dakika 30.000 xg&#39;de santrifüj kesintisiz hücrelerin, organellerin ve hücresel enkaz çıkarın. </li><li> Kullanımdan hemen önce, pelet, 1.000 az bir dakika boyunca reçine depolama çözümü kaldırmak için xg. İki ml reçine, hücre, her bir gram için kullanılır. </li><li> Doğrudan tahsil Sulfolink bulamaç haline lipid fraksiyonu ya en üst ya da hücre artıkları tüplerin alt kısmında kirlenme, yer ve en aza indirmek için özen, 30.000 xg dönüş (S30) hücre lizat süpernatantlar çıkarın. Lipid tabakasının ya kaldırılması ya da ucu süpernatant geçti sonra pipetlemeyin dışarı iterek önlenebilir. </li><li> S30/Sulfolink karışımı 15 dakika boyunca karıştırma olmadan buz üzerinde inkübe edin. </li><li> Sütun 15 ml konik tüpler içine yerleştirin ve 1000 xg&#39;de santrifüjr bir dakika. </li><li> Sütunlar, el ile karıştırın ve buz üzerinde bir 15 dakika daha inkübe flowthrough fraksiyonları içine geri yerleştirin. </li><li> Sütunlar, 15 ml konik tüpler içine yerleştirin ve bir dakika için 1000 xg&#39;de santrifüj. Flowthrough atın. </li><li> Kap sütun ve bağlayıcı tampon 5 ml ekleyin. Elle sütunları yeniden süspanse, kapakları kaldırmak ve bir dakika boyunca 1000 xg sütunları santrifüj santrifüj kadar karıştırın. Bu yıkama protokolü iki kez daha tekrarlayın. </li><li> 1.5 ml (20 mM Hepes-KOH, pH 7.6, 10 mM Mg (oac) 2, 500 mM KCl, 2mm DTT, 0.5 mg / ml heparin) elüsyon tampon her sütun ekleyin. Elle çamurlar karıştırın ve 2 dakika boyunca buz üzerinde inkübe </li><li> Yeni 15 ml konik boru ve santrifüj içine koyun 1 dakika boyunca 1000 xg sütunlar. Eluat toplayın. Bir kez daha final kombine örnek hacmi ~ 3 ml olduğunu elüsyon adımı yineleyin. İkinci el reçine, heparin olmadan elüsyon tamponu ile yıkanır ve bağlayıcı Buffe ile dengelemer ve yeniden daha sonra kullanmak için bağlayıcı tampon 4 10 ml ° C saklanır. </li></ol> 3. Puromycin tedavi Not: tRNA türlerinin kirlenmesine neden kaldırmak için alternatif bir yöntem ribozom ikinci tur teşvik etmek için glikoz tükenmiş medya zengin bir glikoz geçmek için. Ancak, bu ribozom fonksiyonu ve konsantrasyonu etkileyebilir maya hücrelerinin metabolik durumunu değiştirmek. Endojen peptidil-tRNA, puromycin yapılan bir tedavi ribozomlar şerit için. <ol><li> Oda sıcaklığında 1M KOH damla damla ekleyerek pH 7.5 100 mM puromycin çözüm nötralize. </li><li> ~ 1mm eluat nihai konsantrasyonu nötralize puromycin çözüm ekleyin. </li><li> 1 mM nihai konsantrasyonu 100 mM GTP ekleyin. </li><li> 30 dakika boyunca inkübe 30 ° C </li></ol> 4. Gliserol yastıklar üzerinden sedimantasyon ribozomlar Saflaştırılması <ol><li> Bir ml4 ml&#39;lik hacim, polikarbonat ultrasantrifüjdeki tüp içine minder tamponu (20 mM Hepes, pH 7.6, 10 mM Mg (oac) 2, 500 mM KCl, 2 mM DTT,% 25 gliserol). </li><li> Katmanı puromycin yavaşça 4 geceleme xg yastık üstüne elüsyon kesirler ve santrifüj örnekleri 100.000 ° C tedavi </li><li> Santrifüj rotor tüpleri çıkarın ve süpernatantlar aspire. </li><li> Pelet saflaştırılmış ribozom içeren yastık tampon 1 ml iki kez yıkayın. </li><li> Cam bir çubuk kullanarak nazik bozulma yastık tampon 100 ul ribozomlar tekrar </li><li> Kesinti sonra, parafilm tüpler kapak ve bir saat boyunca soğuk bir odaya girdap içinde ılımlı bir hızda sallamak. </li><li> Mikrosantrifüj tüp, maksimum hızda 5 dakika santrifüj 4 ° C, içeriği aktarın ve taze tüplerine süpernatantlar kaldırmak. </li><li> 4,1-4,7 o 100 ul depolama tampon (50 mM HEPES-KOH pH 7.6, 50 mM NH 4 Cl, 5 mM Mg hariç 2 yastık tampon ml ve adımları tekrarlayın.(Oac) 2, 1 mM DTT,% 25 gliserol) yastık tampon yerine adımları 4.4 ve 4.5 kullanılır. </li><li> Sayısal olarak saflaştırılmış ribozomlar spektrofotometrik (1 A 260 = 20 maya ribozomların pmoles) ve -80 mağaza ° C 1 gram maya tipik sonuçları ribozom 300-400 pmoles. </li></ol> 5. Temsilcisi sonuçları: RNA türlerinin her protokol üç önemli adımlar çıkarılan bir örnek Şekil 2&#39;de gösterilmiştir. RRNA olmasına rağmen en önemli türler toplam hücre lizatları bulunan (T), bu diğer RNA türlerinin çok sayıda da içerir. Sulfolink sütun (SL) arınmış ribozomlar da, bu türler için de sütun yatağın yüksek afinite nedeniyle tRNAs büyük miktarda içerir. Peptidil-tRNAs peptidler hidroliz puromycin sonuçları ile bu fraksiyonun Tedavi ve ribozomlar bu türlerin ayrılma teşvik etmektedir. Puromycin tedavi örnekleri (Pm) eksikliği co-purifyinböylece g tRNA türleri ve tamamen saf ribozomlar temsil eder. Daha önce 8 bildirildiği gibi, bu ribozom detaylı fonksiyonel ve yapısal analizler için ideal substratlar, son derece sağlam ve biyokimyasal olarak aktif. <img src="/files/ftp_upload/3214/3214fig1.jpg" alt="Şekil 1"/> Şekil 1. Yöntemi Akış Şeması sulfolink reçine bağlı sistein kullanarak ribozomların Kromatografik arıtma tasvir edilmiştir. <img src="/files/ftp_upload/3214/3214fig2.jpg" alt="Şekil 2"/> Şekil 2. Ribozom hazırlıkları Temsilcisi analiz Total RNA türleri, daha sonra puromycin ile tedavi edilen toplam hücre lizatları (T), Sulfolink saflaştırılmış ribozomlar (SL) ve Sulfolink saflaştırılmış ribozomlar çıkarılan ve gliserol yastık (Pm) aracılığıyla tortulaşmış edildi . Lane M RNA boyutu belirteçler temsil eder. 25S ve 18S rRNA, tRNAs ve diğer RNA türlerinin yanı sıra temsil Gruplar gösterilir. Tümşeritli RNA 2 mg ile yüklendi.

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	<li>Palade, G. E., Siekevitz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+microsomes;+an+integrated+morphological+and+biochemical+study.">Liver microsomes; an integrated morphological and biochemical study.</a> J. Biophys. Biochem. Cytol. 2, 171-200 (1956).</li><li>Fabry, M., Kalvoda, L., Rychlik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interactions+of+Escherichia+coli+ribosomes.">Hydrophobic interactions of Escherichia coli ribosomes.</a> Biochim. Biophys. Acta. 652, 139-150 (1981).</li><li>Jelenc, P. 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Çıkar çatışması ilan etti.

Ribozom arıtma protokolleri temelde düşük hızda spin sitozolik bir fraksiyonu hasat parçalama hücreleri içerir ve daha sonra yüksek hızda santrifüj 1 ribozomlar peletleme . Bakteri ribozomları temizlemek için bir kaç yeni yöntemler kullanılmış olsa da, aynı ökaryotlar 2-4 için doğru değildi. Maya ribozomlar biyokimyasal ve yapısal çalışmalar ek adımlar yaş, örneğin tuz yıkama ve gliserol minderler (örneğin: 5) boyunca eklenmiş olmasına rağmen, büyük...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Sulfolink Kaplin reçine </td><td> Delmek </td><td> 20402 </td></tr><tr><td> Mini boncuk-çırpıcı 16 </td><td> Biospec </td><td> 607 </td></tr><tr><td> Optima Max E ultrasantrifüjdeki </td><td> Beckman Coulter </td><td></td></tr><tr><td> Legend RT </td><td> Sorvall </td><td></td></tr><tr><td> 0,5 mm Cam boncuklar </td><td> Biospec </td><td> 11079105 </td></tr><tr><td> Tris </td><td> Sigma-Aldrich </td><td> 252859 </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E9884 </td></tr><tr><td> DTT </td><td> Sigma-Aldrich </td><td> 43815 </td></tr><tr><td> Hepes </td><td> Sigma-Aldrich </td><td> 54459 </td></tr><tr><td> NH 4 Cl </td><td> Sigma-Aldrich </td><td> A9434 </td></tr><tr><td> KCl </td><td> Sigma-Aldrich </td><td> 60128 </td></tr><tr><td> Mg (oac) 2 </td><td> Sigma-Aldrich </td><td> M5661 </td></tr><tr><td> KOH </td><td> Sigma-Aldrich </td><td> 221473 </td></tr><tr><td> Gliserin </td><td> Sigma-Aldrich </td><td> G5516 </td></tr><tr><td> Puromycin </td><td> Sigma-Aldrich </td><td> P7255 </td></tr><tr><td> GTP </td><td> Fermentas </td><td> R0461 </td></tr></tbody></table>

chromatographic purification of highly active yeast ribosomes

Ökaryotik ribozomlar, böylece araştırmacılar için önemli bir meydan okuma poz eubacterial ve archael muadillerine göre çok daha dayanıksız. Özellikle sorunlu hücrelerin parçalanması, proteaz ve ribozomlar düşürebilir nükleazlar çok sayıda serbest olduğu bir gerçektir. Bu nedenle, mümkün olduğunca çabuk bu enzimler ribozom ayırmak için önemlidir. Ne yazık ki, geleneksel diferansiyel Ultrasantrifügasyon yöntemleri, yapısal bütünlüğünü ve işlevselliğini etkileyen bu enzimler zaman kabul edilemeyecek kadar uzun süre maruz ribozomlar bırakır. Bu sorunu çözmek için, bir sistein ücret Sulfolink reçine kullanarak kromatografik yöntem kullanmaktadır. Bu basit ve hızlı uygulama, sağlam, yüksek biyokimyasal aktif maya ribozomların yüksek verim üreten ortak arıtma proteolitik ve nucleolytic faaliyetleri, önemli ölçüde azaltır. Bu yöntem aynı zamanda memeli ribozomlar için geçerli olması gerektiğini düşündürmektedir. Sadeliğiyöntemi ve ökaryotik ribozomlar çalışma için önemli bir teknik ilerleme, kromatografik saflaştırılmış ribozom gelişmiş saflık ve aktivite gösterir.

Watch this Scientific Journal Video about Chromatographic Purification of Highly Active Yeast Ribosomes at JoVE.com

Chromatographic Purification of Highly Active Yeast Ribosomes

Ortak arıtma nükleazlar ve proteazlar tarafından geleneksel yöntemlerle saflaştırılmış ökaryotik ribozomlar hazırlıkları kontaminasyonu mansap biyokimyasal ve yapısal analizleri olumsuz etkiler. Maya ribozomlar bir model sistem olarak kullanarak bu sorunu çözmek için basit ve hızlı bir kromatografik saflaştırma yöntemi kullanılır.

Yüksek Aktif Maya Ribozomlar Kromatografik Arıtma

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

chromatographic-purification-of-highly-active-yeast-ribosomes

Research

JoVE Journal

Biology

16.7K Görüntüleme.  University of Maryland. Ortak arıtma nükleazlar ve proteazlar tarafından geleneksel yöntemlerle saflaştırılmış ökaryotik ribozomlar hazırlıkları kontaminasyonu mansap biyokimyasal ve yapısal analizleri olumsuz etkiler. Maya ribozomlar bir model sistem olarak kullanarak bu sorunu çözmek için basit ve hızlı bir kromatografik saflaştırma yöntemi kullanılır.

Video: Chromatographic Purification of Highly Active Yeast Ribosomes

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Biyoloji

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Maya hücreleri Mitokondri saflaştırılması

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Mitokondri Yüksek Coupled Sıçan Kalp hazırlanması

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Fonksiyonel ve Yapısal Analizler için Peptidil tRNAs İçeren Çeviri Ribozomlar izolasyonu

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Maya Colony Gömme Yöntemi

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Active T. in vitro Sulandırma castaneum Telomeraz

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Yüksek verim Maya Plazmid overekspresyonu Ekran

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Fonksiyon Çalışma, Etkileşimler, ve Post-translasyonel değişiklikler için tomurcuklanan Maya Protein Ekstraksiyonu ve Saflaştırılması

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Peptidler ve proteinler ile rekombinant Elastin gibi Polipeptitlerin ve Fusions olmayan kromatografik saflaştırma<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...