PA Laval Üniversitesi Tıp Fakültesi bursu &quot;Pierre Durand&quot; bir alıcı. Bu çalışma Polizom parçalama RMR M yeni CIHR araştırmacı maaş ödülü tutar için inovasyon hibe Kanadalı Vakfı (MOP-GF091050) yoluyla elde edilmiş RM Doğa Bilimleri ve Kanada&#39;nın Mühendislik Araştırma Konseyi (MOP-CG095386) tarafından desteklenmiştir. Biz Dr minnettarız. Yararlı tavsiyeler E. Khancıyan, I. Gallouzi, S. Di-Marco ve A. Cammas.

Protokol kuralları takip Laval Etik Değerlendirme Kurulu tarafından onaylanmıştır. 1.. Hücre Kültürlerinin Hazırlanması ve Beyin Manipülasyon <ol><li> Memeli hücreleri ve Drosophila <ol><li> American Type Culture Collection tarafından tavsiye edilen, HeLa servikal kanser hücreleri ve Schneider Drosophila embriyonik hücreler büyür. Düşük bir geçit hücreleri ile çalışmak. </li><li> % 80 izdiham Deneyin gün ulaşmak için Plaka hücreler. En iyi sonuçları elde etmek için, her bir deney için hücreler 12 x 10 ~ 6 kullanın. Polizom analizi için özler yapmadan önce, en az 90 dakika boyunca, taze tam orta ekleyerek çeviri uyarır. </li></ol></li><li> Beyin izolasyon: doğumdan sonra 9 ve 16 gün arasında değişen farelerin beyinleri kesin. Ötenazi anestezi ve servikal dislokasyon sonra CO 2 solumaktan içerir. Kurban sonra, bütün beyin ve iki yerde izole it -80 ° C ya da doğrudan doğruya hemen kullanım için soğuk PBS 1X içine aktarın. </li></ol> 2.. Yoğunluk hazırlanması Gradyan Bölünmesi Sistemi <ol><li> 5 dakika için% 0.1 SDS ile boru sistemi yıkayın. </li><li> DEPC su ile yıkamadan önce, 5 dakika boyunca RNaseZAP Çözüm ile, daha sonra 5 dakika için% 70 etanol ile boru sistemi yıkayın. </li><li> Aparatın boru sistemi kurutmak için hava pompası. </li></ol> Sakroz Gradyanların 3. Hazırlanması <ol><li> 20 mM Tris-HCl, pH 7.4, 1.25 mM MgCI2, 150 mM NaCI, ve 1 mM DTT,% 15 ve% 55 sükroz cevap verir. </li><li> Oluşturmak üreticinin talimatlarına göre tarif edildiği gibi, eski bir Isco Model 160 kullanarak lineer gradyan% 15-55 sukroz gradyanı. Bununla birlikte, bu geçişlerini kabarcıklar veya türbülanslar oluşmasını önlemek için de TRIS peristaltik pompa hızını kontrol etmek önemlidir. Kullanılana kadar 4 ° C&#39;de gradyanlar tutun. </li><li> Degrade yapımcısı programı çalıştığı süre boyunca, 4 ° C&#39;ye ultrasantrifüjdeki serin </li></ol> 4. Hücre hazırlanması ve Fare Beyin özler <ol><li> Hücre ekstrelerinin hazırlanması <ol><li> Buz ve yıkama hücreleri üzerinde plaka (lar) soğuk PBS 1X ile 3x. </li><li> Hasat hücreleri, pH 7.4, 1.25 mM MgCI2, 150 mM NaCI, 1 mM DTT,% 1 Nonidet P40, 5 U / ml RNase inhibitör, takviye edilmiş, 1 ml liziz tamponu (20 mM Tris-HCI (12 x 10 ~ 6) tam Mini EDTA&#39;sız proteaz inhibitörü kokteyl tabletleri ile birlikte), bir Eppendorf tüpüne transfer ve 1 cc U100 insülin şırınga 28 G 1/2 ile olanağına 15x geçirilerek iyice karıştırın. Hücre lizatı 15 dakika boyunca buz üzerinde bekletin. </li><li> 10X objektif kullanarak bir faz kontrast mikroskop ile gözleyerek hücre lizizi değerlendirmek için bir slayt üzerine hücre lisatı birkaç damla koyun. Sadece çekirdekleri görünür olmalıdır ve herhangi bir hücre zarları hücre lizizi görebilir doğrulayan olmalıdır. Bir kontrol olarak, gözlemlemekLizinlerle parçalanmayan hücreler. </li><li> 4 ° C&#39;de 20 dakika boyunca 11,000 x g&#39;de santrifüj ile hücre lizat açıklığa kavuşturulması ve poliribozomların içeren çözünebilir lizat tutun. </li></ol></li><li> Fare Beyin ekstrelerinin hazırlanması <ol><li> Liziz tamponu 2 ml buz gibi soğuk bir Dounce homojenizatör içinde 10 vuruş ile izole edilmiş bütün beyin (~ 500 mg) homojen ve 4 ° C&#39;de 15 dakika boyunca 11,000 x g&#39;de santrifüj ile Homojenat açıklamak </li><li> % 50 sakroz yastığı üzerine elde edilen süpernatan yükleyin ve 4 ° C&#39;de TH-641, bir ultra santrifüj rotor kullanılarak 200,000 xg&#39;de 2 saat boyunca devam sedimantasyon </li><li> Liziz tamponu içinde 1 ml poliribozomların içeren şeffaf pelet yeniden süspanse edin ve aşağı ve yukarı pipetlenerek ile iyice karıştırın. Geçişlerini üzerine yüklenmeden önce 30 dakika için buz üzerinde kalan süspansiyon olsun. </li></ol></li></ol> 5.. Sakaroz Gradyanların ve Ultrasantrifügasyon üzerine ayıklar yükleniyor <ol><li> Konsantrasyonunu ölçmekBir spektrofotometre kullanılarak, sitoplazmik ekstre içinde mevcut RNA. Dikkatli ve yavaş yavaş% 15 ila% 55 sukroz gradyanı üzerine ~ ekstrenin 20 OD 260 adet yükleyin. Kullanılabilir alan 2-3 mm santrifüj sırasında tüpler taşan önlemek için borunun yüzeyinde olduğundan emin olun. </li><li> 4 ° C&#39;de TH-641, bir ultra santrifüj rotor kullanılarak 230,000 x g, 2 saat 30 dakika için ultra-santrifüj kullanarak gradyanlara santrifüj Santrifüj sona erdiğinde, tüpleri çıkarın ve geçişlerini rahatsız etmemek için dikkatli bir şekilde buz koyun. </li></ol> Polisomlann profilleme için Sitoplazmik Ekstraktlarının 6. Fraksiyonu <ol><li> Üretici tarafından tarif edildiği gibi, (0.5 ml her biri) Otomatik Yoğunluk Fraksiyonu Sistemi sakaroz gradyanları yerleştirin ve fraksiyonların otomatik fraksiyonasyonu ve toplama ile devam edin. </li><li> 254 nm&#39;de absorbans sürekli olarak izlenmesi ile ayrı Eppendorf tüpleri içine her fraksiyonu (~ 500 | il) toplayın. Parall yılındagradyan ayrıştırma işleminin el, polyribosomal profil grafik kağıt üzerinde düzenleme olacaktır. Seçenek olarak ise, polisom profilinin bir elektronik satın için grafik kaydedicisinde bağlanmış bir veri toplama birimi kullanır. </li><li> Her çalışma sonunda, kuru buz üzerinde toplanan Eppendorf şişesi aktarın. Şu anda, mağaza ya da -80 ° C&#39;de kısımlar toplandı ve aşağıdaki gibi direkt protein-RNA kompleksleri hızlandırabilir. </li></ol> 7. Protein Ekstraksiyon ve Analiz <ol><li> Sukroz gradyanı her toplanan fraksiyon için,% 100 soğuk 2 hacim etanol ekleyin ve RNA-protein kompleksleri, -20 ° C sıcaklıkta gece boyunca çökelmeye sağlar. </li><li> Santrifüj 4 ° C&#39;de 20 dakika boyunca 11,000 x g, her bir RNA-protein çökelti daha sonra% 70 etanol ile yıkayın. Kuru ve (bkz. Şekil 2) özel antikorlar kullanılarak Batı beneği ile devam etmeden önce, SDS-PAGE numune tampon maddesi içinde tekrar süspansiyon çökelti. </li></ol> 8. RNA Ekstraksiyon ve Analysis <ol><li> Bölüm 7.1 olarak RNA-protein kompleksleri çöktürün. % 0.1 SDS ihtiva eden liziz tamponu içinde her bir RNA-protein çökeltisinin yeniden süspanse edin. Bir 55 ° C su banyosu içinde 30 dakika boyunca 2 mg / ml proteinaz K ile çökeltinin protein bileşeni Digest. </li><li> &quot;: Kloroform fenol&quot;, kloroform 2 birimleri ve 2 M NaOAc pH 4 0.1 hacim ve 20 dakika boyunca 4 ° C&#39;de 10,000 x g&#39;de santrifüj 1 hacim ekleyerek Çökeltinin RNA bileşenleri çıkarmak. Izopropanol içinde 1 hacim ve glikojen 0.2 ug / ul ekleyerek -20 ° C &#39;de bir gece boyunca, her numunenin edilen sulu fazdan Çökelti RNA. 4 ° C&#39;de 10,000 x g&#39;de 1 saat boyunca çökelti spin % 70 soğuk etanol ile RNA pelet yıkayın. </li><li> RNAse içermeyen su, küçük bir hacim içine RNA topağı yeniden süspanse edin. Miktarını değerlendirmek ve kalite RNA spektrofotometre kullanılmıştır. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Sukroz dereceleri üzerinde Polizom profil analizi hücreler ya da dokulardan 9,11-14 izole polizom yoğunluğu analiz ederek çeviri başlangıç ​​ölçümünü sağlar. Bu teknik, en iyi yaklaşım (değil benzersiz ise) in vivo çeviri inisiyasyon ölçmek için. Bu, hücre döngüsü boyunca 15 hücrelerinin büyüyen translasyonel durumunu izlemek için ve çeviri başlatma üzerinde, viral enfeksiyonlar, hipoksi 13,16, radyasyon 17 ve kimyasal ilaçlar tera...

Ökaryotik hücreler sürekli bir hızlı adaptif hücre yanıtı gerektiren zararlı fizyolojik ve çevresel stres koşulları bir dizi karşılaşma. Hücre stres yanıtı, anti-hayatta kalma ve pro-sağkalım etkili faktörler arasındaki hassas denge içerir. Bu dengeyi bozarak kanser ve nörodejeneratif hastalıklar gibi insan patolojilerin gelişimine öncülük geri dönülmez sonuçlar doğurabilir. Stres yanıtı ilk adımı sırasında, hücreler mRNA çeviri düzeyinde gen ekspresyonunda değişiklikler koordine kontrolü ile ilgili pro-hayatta kalma yolunu aktive edebilir. ökaryotlarda mRNA çevirisi başlatma faktörleri (EIFS), spesifik RNA bağlayıcı protein (RBDS) ve RNA molekülleri 1 ila koordineli etkileşimleri içeren karmaşık hücresel bir süreçtir. başlatma, uzama ve sonlanma: mRNA çeviri üç ayrı evreye ayrılır. Her üç faz dede tabi olsa da,translasyonel kontrol mekanizmaları, böylece protein sentezi 2&#39;nin oran-sınırlayıcı bir adım teşkil etmektedir çeviri çoğunlukla başlangıç ​​aşaması, hedef merciin düzenleyici mekanizmalar,. Çeviri başlatma i ön başlatma kompleksinin oluşumuna yol açan, üçlü kompleks ve 40S ribozom alt birimine yönelik daha sonraki bağlanma Met eIF2a.GTP.Met-tRNA oluşumu ile başlayan bir çok düzenli bir süreçtir. Bir sonraki adım, eIF4F ve eIF3 gibi çeviri başlatma faktörlerinin aktivitesi içerir mRNA için preinitiation kompleksinin işe olan. Bu şekilde oluşturulan 48S preinitiation kompleksi AUG başlangıç ​​kodonunun tanıyıncaya kadar mRNA&#39;nın 5&#39;-ötelenmemiş bölge tarama başlatmak için bu makine sağlayacak özel yapısal değişikliklere uğrar. Çeviri başlangıç ​​faktörlerin çoğu daha sonra serbest bırakılır ve 60S alt birimleri bir 80S oluşturmak için işe alınırlar ribozom kompleksi çeviri, yetkili birt hangi nokta protein sentezi başlar (Şekil 1). Birden fazla 80S monosome sözde polisomlann (veya poliribozomların) üreten bir zamanda aynı mRNA&#39;nın tercüme edilebilir. Bir mRNA üzerinde polizom yoğunluğu başlatma, sonlandırma ve uzama oranları yansıtır ve böylece, belirli bir transkriptin çevrilebilirlik bir ölçüsüdür. Bununla birlikte, polisom profili esas olarak başlangıç ​​aşamasında mRNA çevirisinde değişiklikleri değerlendirmek için kullanılır. Burada çeviri başlatma inhibitörü gibi bir proteazom önleyicisi kullandık. Bu ilaç kanser hücrelerinin tedavisi translasyon başlatma faktörü eIF2a 3 fosforile HRI adlı stres kinazın aktivasyonu ile karakterize edilen bir stres tepkisi oluşturur. EIF2a fosforilasyonu, memeli hücrelerinde 4 çeviri başlatma inhibisyonuna yol açan önemli olaylardan biridir.

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			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
			<td height="21" style="height:21px;width:279px;">Cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">HeLa cervical cancer cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Schneider Drosophila embryonic cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CRL-1963</td>
			<td></td>
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		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Culture medium and Supplements</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Schneider&#8217;s Drosophila Medium</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>SO146-500ml</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM</td>
			<td>Life technologies</td>
			<td>11995-073</td>
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			<td>Fisher Scientist Scientist</td>
			<td>SH30396-03</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">penicillin/streptomycin</td>
			<td>Life technologies</td>
			<td style="width:119px;">15140122</td>
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		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
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			<td>Fisher Scientist</td>
			<td>BP220-212</td>
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			<td height="21" style="height:21px;width:279px;">Glycerol</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>49767</td>
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			<td height="21" style="height:21px;width:279px;">Blue Bromophenol</td>
			<td>Fisher Scientist</td>
			<td>B3925</td>
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			<td style="width:311px;"></td>
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			<td></td>
			<td height="21" style="height:21px;width:279px;">Lysis buffer</td>
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			<td height="21" style="height:21px;width:279px;">Tris Hydrochloride</td>
			<td>Fisher Scientist</td>
			<td>BP153-500</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:279px;">MgCl2 </td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NaCl</td>
			<td>Tekniscience</td>
			<td>3624-05</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DTT</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D 9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Nonidet P40 (Igepal CA-630 )</td>
			<td>MJS Biolynx</td>
			<td>19628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">SDS</td>
			<td>Tekniscience</td>
			<td>4095-02</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:279px;">RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor)</td>
			<td>Life technologies</td>
			<td>10777-019</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Antiproteases (complete, mini, EDTA free)</td>
			<td>Roche</td>
			<td>11,836,170,001</td>
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		<tr height="21">
			<td style="width:311px;"></td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteinase K</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM2542</td>
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		<tr height="21">
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			<td>Fisher Scientist</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Chloroforme</td>
			<td style="width:311px;">Fisher Scientist</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycogen</td>
			<td>Life technologies</td>
			<td>10814-010</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isopropanol</td>
			<td style="width:311px;">Acros organics</td>
			<td>327270010</td>
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			<td height="21" style="height:21px;width:279px;">anti-FMRP antibody</td>
			<td colspan="2" style="width:429px;">Fournier et al., Cancer Cell International, 2010</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-Ribosomal Protein L28 antibody</td>
			<td style="width:311px;">Santa Cruz Biotechnology, Inc.</td>
			<td style="width:119px;">SC-50362</td>
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		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Others</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteasome inhibitor : Bortezomib</td>
			<td style="width:311px;">LC Laboratories</td>
			<td style="width:119px;">B-1408</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DEPC (Diethylpyrocarbonate)</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D5758-25ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">RNaseZAP Solution</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM9780</td>
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		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">T25 cell culture flask</td>
			<td style="width:311px;">Corning</td>
			<td style="width:119px;">430639</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">1cc U100 Insulin Syringe 28 G1/2</td>
			<td>Fisher Scientist</td>
			<td>148291B</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tube ultra-centrifugation, PA, 12ml</td>
			<td>Fisher Scientist</td>
			<td>FSSP9763205</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco Model 160 gradient former</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Sorvall OTD Combi</td>
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Thermo Scientific Sorvall Rotor TH-641</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;">54295</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Automated Density Fractionation System</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;">67-9000-177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco UA-6 UV-vis detector</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ultracentrifuge C 5415</td>
			<td style="width:311px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Optical Microscope</td>
			<td style="width:311px;">Olympus CK2</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of translation initiation during stress conditions by polysome profiling

MRNA çevirisi hassas kontrolü, özellikle fizyolojik ve patolojik strese yanıt olarak, ökaryotik hücre homeostazı için temeldir. Bu programın değişiklikler kanser gelişimi arasında ayırt edici bir özelliği, ya da nörodejeneratif hastalıklarda görüldüğü gibi, prematüre hücre ölümüne hasarlı hücrelerin büyümesine yol açabilir. Translasyonel kontrol için moleküler temel ilişkin bilinenlerin çoğu bir yoğunluk gradyan ayırma sistemi kullanılarak Polizom analizinden elde edilmiştir. Bu teknik, bir doğrusal sakroz gradyanı üzerinde sitoplazmik özler ultrasantrifüj dayanır. Sıkma işlemi tamamlandıktan sonra, sistem, farklı çeviri tekabül eden santrifüj bölgelerinin ayırma ve böylece bir miktar Polizom profili ile sonuçlanan popülasyonları ribozom sağlar. Polisom profilde değişiklikler stres çeşitli tepki olarak meydana gelen çeviri başlatma değişiklikler veya kusurların göstergesidir. Bu teknik aynı zamanda inci değerlendirmek sağlar:e çeviri başlatma belirli proteinlerin rolü ve spesifik mRNA translasyon aktivitesini ölçmek için. Burada, normal ya da stres ya da büyüme koşulları altında, ökaryotik hücrelerin ve dokuların başlatılması için değerlendirmek amacıyla Polizom profilleri gerçekleştirmek için protokol açıklar.

Daha önce belirtildiği gibi, polisom profil stres şartları altında için başlatma değişikliklerin analizi. 1. Daha önce çeviri başlatma komplekslerinin sıralı bir düzeneği içeren bir çok aşamalı bir süreçtir tarif edildiği gibi çeviri başlatma basitleştirilmiş bir görünüşüdür sağlar. Normal büyüme şartları altında, çeviri başlatma kompleksleri olan algılama Polizom profili tarafından bir aktif için başlatma (; İşlenmemiş Şekil 2)</...

Watch this Scientific Journal Video about Analysis of Translation Initiation During Stress Conditions by Polysome Profiling at JoVE.com

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Burada, stres koşullarında tepki olarak ökaryotik hücrelerin mRNA çevirisi başlatılmasında değişiklikleri analiz etmek için bir yöntem açıklanmaktadır. Bu yöntem olmayan çeviri ribozom ribozomlan çeviri sakaroz Yokuşta hız ayrılık dayanmaktadır.

Polizom profilleme Stres Durumlardaki Çeviri başlatılması Analizi

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

analysis-translation-initiation-during-stress-conditions-polysome

Research

JoVE Journal

Biology

18.2K Görüntüleme.  Laval University.  Burada, stres koşullarında tepki olarak ökaryotik hücrelerin mRNA çevirisi başlatılmasında değişiklikleri analiz etmek için bir yöntem açıklanmaktadır. Bu yöntem olmayan çeviri ribozom ribozomlan çeviri sakaroz Yokuşta hız ayrılık dayanmaktadır. 

Video: Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Stres Cevaplarının sırasında Bitki Hormonları İzlenmesi

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Biyoloji

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Antikor Profil Lusiferaz Immunoprecipitation Sistemleri (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Zebra balığı Embriyolar Oksidatif Stres Analizi

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Kantitatif RT-PCR ile Bireysel İnsan Embriyonik Kök Hücreleri profilleme

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Bir genom Ölçeği Polizom Fraksiyonu ve Memeli Translatomes Analizi

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polizom Profil Yöntemi ile memeli hücrelerine selektif mRNA dönüşümüyle Değerlendirilmesi

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Bitki Polizom Profiling için bir Kolay Yöntem

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP Mikroskopisi ile Gap Birleşimine Bağlı MiRNA Transferinin Analizi

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In Vitro E3 Ubiquitin Ligase fonksiyonunun analizi

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...