Bu çalışma İnsan Frontier Bilim Programı hibe RGP0024 tarafından finanse edildi.

1. Şablon DNA hazırlanması ve in vitro transkripsiyonu sonucunda <ol><li> Ilgi genindeki T7 ve / veya örneğin SP6 transkripsiyonel promotörü altında klonlanmış edilir. </li><li> In vitro transkripsiyonu sonucunda için DNA ORF durdur kodonu ve / veya mRNA 3 &#39;end of aşağı kesme uygun sınırlama enzimi ile lineerleştirilebilir edilir. Bir agaroz jel elektroforez ile ilgili sınırlama sindirim ürünü çalıştırarak plazmid DNA tam doğrusallaştırma doğrulamak için gereklidir. </li><li> Lineerize plazmid vitro transkripsiyon reaksiyonunda kullanılmıştır. Şablon DNA, farklı konsantrasyonda in vitro transkripsiyonu sonucunda için gereken optimal DNA konsantrasyonu tespit etmek test edilebilir. Genellikle, Ambion Kullanıcı MEGAscript Yüksek verim Transkripsiyon Seti (Ambion / Life Technologies, Grand Island, NY) ile, 1 mikrogram lineerleştirilebilir DNA mRNA 40-60 mikrogram verir. In vitro transkripsiyon olarak üreticinin talimat (Ambion / Life Technologies, Grand Islan takip yapılırd, NY). </li><li> In vitro transkripsiyonu sonucunda takiben mRNA üreticinin talimat (Ambion en MEGAscript yüksek verim Transkripsiyon Kit) uygun olarak lityum klorürle çökeltme ile arıtılır. </li><li> mRNA bütünlüğünü daha da akrilamid veya agaroz jel elektroforez tarafından doğrulanır. </li></ol> 2. In vitro Hücre Bedava Çeviri Aşağıdaki adımları izleyin R abbit R eticulocyte Lysate, RRL (Promega, Madison, WI) kullanılarak in vitro çeviri için: <ol><li> In vitro çeviri reaksiyon aşağıdaki üreticinin talimat 100 ul hazırlayın. Kısaca, nükleaz serbest su içinde 2 ul amino asit karışımı eksi metiyonin (1 mM), RNaz önleyici (Invitrogen / Life Technologies, Grand Island, NY), radyoaktif 20 μCi [35S]-Metionin (MP Biomedicals 10 birim, ekleme ) OH, Solon, tavşan Retikülosit Lizat (Promega, Madison, WI) ve 70 ul. </li><li> Th inkübeSıcak için önceden reaksiyon için 5 dakika süreyle 30 ° C&#39;de e reaksiyon. 5 dakika boyunca 30 ° C&#39;de inkübe çevirisi ve reaksiyon için mRNA 2 ug ekleyin. </li><li> Buz üzerinde çeviri tepki koyarak reaksiyonu durdurun. </li></ol> 3. In vitro Çeviri Reaksiyonunda gelen Doğan Polipeptitler İzolasyonu <ol><li> Ribozom (polysome) bağlı olgunlaşmamış bir polipeptid izole etmek için, reaksiyon için 100 mM KCI, 10 mM 2 MgCI içeren 10 mM Tris-HCl tamponu, pH 7.6 içinde% 30 gliserol 4.5 ml üst katmanlar, 100,000 ve X g&#39;de santrifüje tabi tutulur 4 ° C de 1 saat süre ile, bir TLA-110 rotor (Beckman Coulter, Inc, Brea, CA) içinde </li><li> Süpernatant daha dikkatli bir şekilde çıkarılır. </li><li> Olgunlaşmamış polipeptidlerin içeren polysomal pelet, 0.5 mg / ml bir ribonükleaz (Invitrogen / Life Technologies, Grand Island, NY), ve daha inkübe içeren, 1 mM Tris-HCl tampon pH 7.6 küçük bir hacim (10-15 ul) içinde tekrar süspanse edilir 37, 30 dakika ° C. Sıraylapeptidil-tRNA ester bağı hidroliz geliştirmek için, NaOH 10 mM nihai bir konsantrasyona ilave edildi ve inkübasyon 30 dakika daha devam edilir. </li></ol> In vitro çeviri reaksiyon mRNA olmadan monte edilmiş ve aynı koşullarda (tarif edildiği gibi doğmakta olan peptidler izolasyonu takiben) altında yapılan büyük bir kontrol olarak hizmet verecek. Yoğunluk gradyanı santrifüjleme ile ekstre tabi önce puromycin ile için reaksiyon (lar) tedavisinde ek bir kontrol olarak hizmet edebilir. 4. Tris-trişin SDS PAGE üzerine Doğan ekleşimlerin çözümleniyor <ol><li> Izole doğmakta olan polipeptidler giderilmiş ve Tris-trişin SDS-PAGE analiz edilir. Materyalin miktarı protein etiketleme, protein çevirisi ve diğerleri parametreleri üzerindeki etkinliğini verimi üzerinde olacak bağımlı jel üzerinde, yüklenen dikkat ediniz. Yüklenen materyalin miktarı bes izin verecek şekilde ampirik olarak ayarlanması gerekmektedirt görselleştirme ve bireysel doğmakta olan polipeptid zincirlerinin ayrılması. </li><li> Elektroforez jeller sabitlenir sonra, vakum jel kullanılarak Dyer ve otoradyografi tabi kurutuldu. Doğmakta olan polipeptidlerin dağılımı phosphoimaging kullanarak gözlenmiştir. </li></ol> 5.. Temsilcisi Sonuçlar Sığır Gamma-B kristalen yanı E. olduğu gibi Tavşan Retikülosit Lysate tercüme edildi coli S30 Ekstrakt Sistemi (Promega, Madison, WI) olgunlaşmamış bir polipeptid zincirleri izole edilmesi, ardından. Şekil 1 ribozom bağlı olgunlaşmamış zincirlerinin izolasyonu dahil adımları tasvir etmektedir. TRNAs (dolayı organizmalar arasında kodon yanlılığı farklılıklar önemli ölçüde memeli farklı (RRL) ve prokaryotik (E. coli) sistemleri olarak da bilinir) çevirisi yani repertuarı çevre değişiyor eğer Bu çalışmada amaç, test oldu mRNA üzerinde ribozom hareketi değiştirmek ve çeviri duraklama sitelerin dağılımı etkileyebilir.Altered ribozom hareketi çeviri yanı sıra onların göreceli yoğunlukları sırasında toplanan olgunlaşmamış polipeptidlerin boyutlarda dağılımında değişikliklere yol açacaktır. Bantların Bağıl yoğunluğu duraklama süresini yansıtmaktadır. Doğmakta olan polipeptidlerin izole edilmiş ve% 16.5 T,% 6 C Tris-trişin SDS PAGE (Şekil 2) çözüldü. Gamma-B kristalen bir 20 kDa bir proteindir. RRL ve E. içinde, özdeş olmayan uzunlukları ve yoğunlukları doğmakta polipeptidlerin gözlem coli sistemleri, bu iki sistemlerinde mRNA boyunca ribozom hareketi için farklı kinetik aşağıdaki olduğunu gösterir. Örneğin, E. 17.7 kDa arasında belirgin bir bant gözlenen Bu E&#39;de çevrildiğinde mRNA 3&#39;-ucu bölgesi (gama B kristalen bir kodlama C-ucu bölgesi), ek bir duraklama translasyon sitesi olduğunu düşündürmektedir RRL sisteminde coli lizat değil, coli sistemi. Biz unutmayın Gamma-B kristalen limanlar tandem Arg kodonlar (AGG 1Memeli sistemlerinde sık sık, fakat E&#39;de çok nadir ve yavaşça çevrilmelidir bilinmektedir ve 53 AGA 154) coli. E. biriken bir roman doğmakta olan peptid coli sistemi (~ 17.7 kDa Şekil 2.2) muhtemelen bu tandem nadir kodonlar az duraklatarak Ribozom neden olur. Hiçbir bantları mRNA yokluğunda (Şekil 2.3) gözlendi ve bu puromycin tedavi polysomes (Şekil 2.3) den doğmakta zincirlerinin en kaldırır olabilir unutmayın. Puromycin (&gt; 15 dk) ile uzun süreli tedavi tüm doğmakta olan zincirleri (veriler gösterilmemiştir) kaldırır. Bu gözlenen polipeptid ürünler yerine polysomes ile cosedimenting mRNPs daha ribozom ilgili doğmakta olan zincirleri, olduğunu onaylar. Yukarıda bahsedildiği gibi, kodon önyargı organizmanın gibi doku özgüdür. Temsili örnek, burada açıklandığı şekilde tRNAs / kodon çeviri yani repertuarı ortamı değiştirerek gösterirönyargı mRNA boyunca ribozomunun değiştirilmiş bir harekete neden olabilir. Besbelli, bu basit tekniği sadece mRNA boyunca öteleme duraklama sitelerinin kimlik izin veremeyiz, ama aynı zamanda farklı sistemler arasında çeviri duraklama sitelerin hızlı karşılaştırma izin verir. <img alt="Şekil 1" src="/files/ftp_upload/4026/4026fig1.jpg" /> Şekil 1.. Şematik ribozom bağlı olgunlaşmamış polipeptidlerin yalıtılmasının yer adımları açıklayan. Sığır Gamma-B kristalen ORF sırası (528 baz çifti) pBSKS içinde aşağı T7 promotörünün klonlandı +. In vitro transkripsiyonu sonucunda için DNA EcoRI ile işleme tabi tutulmasıyla lineerleştirilebilir edildi. Lineerize şablon in vitro transkripsiyonu sonucunda için kullanılmıştır. DNA şablonu T7 promotor aşağı Gamma-B kristalen gen transkripsiyonun T7 RNA polimeraz tarafından tanınır. mRNA lityum klorür çöktürme yöntemi kullanılarak saflaştırıldı. MRNA arıtılmış vitro çeviri için kullanılır. Ardındanİnkübasyondan sonra, reaksiyon için% 30 gliserol çözeltisi üstüne katmanlı ve 4, 1 saat boyunca santrifüje tabi tutulur ribozom bağlı olgunlaşmamış polipeptidlerin izole etmek için 100,000 x g ° C&#39;de. <img alt="Şekil 2" src="/files/ftp_upload/4026/4026fig2.jpg" /> Şekil 2. Tavşan Retikülosit Lysate (RRL) ve E. tercüme Sığır Gamma-B kristalen doğmakta polipeptidlerin İzolasyonu Lizat coli. 2 ug mRNA RRL veya E. içeren 100 ul reaksiyon içinde karıştırılmıştır 5 dakika süreyle 20 μCi [35S]-metionin ve 30 ° C&#39;de olan üreticinin talimatlarına uygun olarak coli S30 Ekstrakt Sistemi (Promega, Madison, WI). Aşağıdaki reaksiyon için inkübasyon 100 mM KCI, 10 mM 2 MgCI içeren, 10 mM Tris-HCl tamponu, pH 7.6 içinde% 30 gliserol 4.5 ml üstüne katmanlı ve 4 ° C&#39;de 100,000 ve X g&#39;de 1 saat boyunca santrifüj edildi Bir TLA-110 rotor (Beckman Coulter, Inc, Brea, CA). Izole polyribosome pelet resu olduA (Invitrogen / Life Technologies, Grand Island, NY) NaOH (olarak protokolü bölümünde açıklandığı) içinde gerçekleştirilmiş ve ardından 0.5 mg / ml ribonükleaz içeren, 1 mM Tris-HCl, pH 7.6 ile 20 ul içerisinde harcanan. Doğan polipeptidlerin% 16.5 T,% 6 C Tris-trişin SDS PAGE jel çözüldü. Jel, kurutulur ve otoradyografi için maruz bırakıldı. Maruziyet sonrasında jel GE Healthcare&#39;in Typhoon Görüntüleme Tarayıcı ile taranmıştır. Şekil 2.1 çeviri reaksiyonları iki farklı sistem (RRL ve E. coli) yapılır sonra izole ve çözülmesi doğmakta polipeptidlerin ile jel gösterir. Şekil 2.2 jel Görüntü Quant TL ile analiz edildi . çeviri sonrasında SDS PAGE izole ve çözülmesi doğmakta polipeptidlerin: V2005 yazılım ve iki sistem biriken doğmakta polipeptidlerin molekül ağırlığı ve yoğunluğu kolayca analiz ve mukayese edilebilir olduğunu göstermek için burada örnek olarak kullanılan Şekil 2.3 kontroller iki set gösterir reaksiyonlar monte edilmiştirReaksiyon santrifüjleme tabi tutuldu ve daha önce mRNA ve / veya sonra olmadan için reaksiyonlara puromycin (5 dakika, nihai kons. 1 mM) ile muamele edildi.

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Çıkar çatışması ilan etti.

In vitro transkripsiyonu ve translasyon reaksiyonlarda kullanılan bileşenlerin tekrarlanabilir sonuçlar, kalite ve konsantrasyonu için kritiktir. Mevcut çalışmada dikkatli kullanılırsa, yüksek oranda tekrarlanabilir veri sağlamak ticari kitler ve özleri kullanmışlardır. Gerekirse Bununla birlikte, çeviri-yetkin özler, birinin tercih edilen hücre ile hazırlanabilir. MRNA Kalite çeviri etkileyebilir, bu nedenle in vitro çeviri sırasında kullanmadan önce mRNA&#39;ların b?...

<table border="1"><tbody><tr><td> Reaktif / Kit Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> MEGAscript T7 Yüksek verim Transkripsiyon Kiti </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> Ribonükleaz İnhibitör </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Trans [35 S]-Etiket </td><td> MP Biomedicals </td><td> 0151006 </td></tr><tr><td> Ribonükleaz-A </td><td> Invitrogen </td><td> 12091 </td></tr><tr><td> Tavşan Retikülosit Lysate Sistemi, Nükleaz Tedavi </td><td> Promega </td><td> L4960 </td></tr><tr><td> Doğrusal Şablonlar için E.coli S30 Özü Sistemi </td><td> Promega </td><td> L1030 </td></tr><tr><td> Santrifüj </td><td> Beckman Coulter </td><td> Optima TLX ultrasantrifüjdeki </td></tr><tr><td> Depolama fosfor autoradiography </td><td> GE Healthcare </td><td> Typhoon 9410 değişken modunu görüntüleme </td></tr><tr><td> Doğmakta olan polipeptid analizi için yazılım </td><td> GE Healthcare </td><td> Görüntü Quant TL, V2005 </td></tr></tbody></table>

isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna

Translasyonel uzama oranı non-üniform. ikincil yapı, kodon kullanım mRNA ve 1 görmek ilişkili proteinlerin inceleme için mesaj üzerinde ribozom hareketini değiştirebilir mRNA&#39;ya. Ancak, günümüzde yaygın eşanlamlı kodon kullanımı düzgün olmayan translasyonal uzama oranları 1 birincil nedeni olduğu kabul edilmektedir. Eşanlamlıdır kodonlar aynı frekansı ile kullanılmaz. Bir önyargı diğerleri 2 daha sık kullanılan bazı kodonlar ile eşanlamlı kodon kullanımı var. Kodon önyargı organizma gibi 2,3 spesifik bir dokudur. Dahası, kodon kullanımına sıklığı soydaş tRNAs 4 konsantrasyonları ile doğrudan orantılıdır. Böylece, sık kullanılan bir kodon daha sık bir kodon hızlı bir seyrek bir daha tercüme edilecektir ima ilgili tRNAs yüksek sayıda sahip olacak. Böylece, nadir kodonlar (potansiyel duraklama siteleri) zenginleştirilmiş mRNA üzerindeki bölgelerde kural olarak Messa üzerinde ribozom hareketi yavaşlatacakilgili boyutları 5-8 doğmakta peptidlerin ge ve neden birikimi. Bu duraklama siteler protein ekspresyonu fonksiyonel etkisi olabilir, mRNA stabilitesi ve inceleme için protein katlanması 9&#39;a bakın. Gerçekten de, bu tür duraklama siteleri hafifletilmesi mRNA üzerinde ribozom hareketi değiştirebilir ve daha sonra co-translasyon (in vivo) protein katlama 1,7,10,11 etkinliğini etkileyebilir olduğu gösterilmiştir. Sonuçta o translasyonel uzama sırasında mRNA boyunca ribozomların hareketine kodon kullanım / tRNA içerik etkisi içine kapsamlı bilgiler edinmek için gerekli olan protein sentezinin sürecine birleşen hücre, in vivo protein katlanması süreci anlamak için . Burada çeşitli hücre içermeyen sistemlerde 6-8 tercüme belirli bir mRNA için büyük çevirisi duraklama yerleri bulmak için kullanılabilecek basit bir teknik açıklar. Bu prosedür, olgunlaşmamış polipeptidlerin accumulati izolasyonu dayanmaktadırbir hedef mRNA in vitro çeviri sırasında ribozomlar üzerinde ng. Mantığı düşük frekanslı kodonlar az, karşılık gelen boyutlarda olgunlaşmamış peptid artan miktarlarda ribozom sonuçların kalma süresi de artar. In vitro transkripsiyonu mRNA olarak radyoaktif olarak etiketlenmiş amino asitlerin varlığında in vitro translasyon reaksiyonlar için kullanılan olmasıdır doğmakta olan zincirlerinin algılanmasına izin vermek için. Ribozom bağlı olgunlaşmamış bir polipeptid kompleksleri izole etmek için, reaksiyon için santrifüjleme ardından% 30 gliserol çözeltisi üstüne katmanlı edilir. Polysomal pelet yılında Doğan polipeptidlerin daha ribonükleaz A ile tedavi edilen ve SDS PAGE ile çözümlenir. Bu teknik potansiyel olarak herhangi bir protein için kullanılan ve ribozom mRNA boyunca hareket ve büyük duraklama sitelerin tespiti analizi sağlar edilebilir. Ayrıca, bu protokol ribozom hareketi değiştirebilir ve böylece potansiyel olarak da inci değiştirebilir faktörleri ve koşulları incelemek için adapte edilebilire fonksiyon / proteinin konformasyon.

Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

MRNA üzerindeki translasyonel duraklama siteleri tanımlamak için bir teknik tarif edilmiştir. Bu prosedür bir denatüre jel elektroforez ile olgunlaşmamış zincirlerinin boyutu analizi ve ardından bir hedef mRNA in vitro için, içerisinde sırasında ribozom üzerinde biriken olgunlaşmamış polipeptidlerin izolasyonu dayanmaktadır.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

isolation-ribosome-bound-nascent-polypeptides-vitro-to-identify

Research

JoVE Journal

Biology

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

16.2K Görüntüleme.  Cleveland State University. MRNA üzerindeki translasyonel duraklama siteleri tanımlamak için bir teknik tarif edilmiştir. Bu prosedür bir denatüre jel elektroforez ile olgunlaşmamış zincirlerinin boyutu analizi ve ardından bir hedef mRNA in vitro için, içerisinde sırasında ribozom üzerinde biriken olgunlaşmamış polipeptidlerin izolasyonu dayanmaktadır.

Video: Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Klip - RNA Cilt Proteinler Transcriptome geniş Cilt Siteleri Belirlemek Amacıyla Bir Yöntem

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biyoloji

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

İzolasyon ve In vitro Etkinleştirme Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Coregulated Genler Potansiyel Düzenleme Motifler belirlenmesi için KAPSAMI

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa Hücre Transfeksiyon Ardından Gaussia Lusiferaz mRNA in vitro transkripsiyon ve Kapatma yılında

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribozom Ciltli Polipeptitler izole bir aracı olarak SECM Tutuklanma sırası kullanılarak

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

In vitro sentezi insan hücrelerinde protein sentezlenmesinin uyarımından için mRNA&#39;nın Değiştirilmiş

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Oksidatif Stres Direnci Ölçüm<em&gt; Caenorhabditis elegans</em&gt; 96 oyuklu mikrotiter plakalarında

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Memeli Hücrelerde Nascent DNA Bound Proteinlerin incelenmesi BrdU ChIP-Slot-Batı Tekniği

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Gen kullanmak için bir protokol translasyonel araştırma için uygun hayvan modeli belirlemek için zenginleştirme çözümlemesi ayarla

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Vitro SUMOylation SUMO E3 ligaz etkinlik eğitim için tahlil

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...