SV NIH K22AI077353 ve Landenberger Vakfı&#39;nın finansman desteği için minnettar olduğunu. KR minnetle NIH (5SC1GM083754) fon kabul eder.

<ol>
	<li>Maniatis, T., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extensive+network+of+coupling+among+gene+expression+machines.">An extensive network of coupling among gene expression machines.</a> Nature. 416, 499-506 (2002).</li><li>Wahle, E., Ruegsegger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3'-End+processing+of+pre-mRNA+in+eukaryotes.">3'-End processing of pre-mRNA in eukaryotes.</a> FEMS microbiology reviews. 23, 277-295 (1999).</li><li>Colgan, D. F., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+regulation+of+mRNA+polyadenylation.">Mechanism and regulation of mRNA polyadenylation.</a> Genes Dev. 11, 2755-2766 (1997).</li><li>Matoulkova, E., Michalova, E., Vojtesek, B., Hrstka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+3'+untranslated+region+in+post-transcriptional+regulation+of+protein+expression+in+mammalian+cells.">The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells.</a> RNA biology. 9, 563-576 (2012).</li><li>Danckwardt, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prothrombin+3'end+formation+signal+reveals+a+unique+architecture+that+is+sensitive+to+thrombophilic+gain-of-function+mutations.">The prothrombin 3'end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations.</a> Blood. 104, 428-435 (2004).</li><li>Moreira, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+upstream+sequence+element+of+the+C2+complement+poly(A)+signal+activates+mRNA+3'+end+formation+by+two+distinct+mechanisms.">The upstream sequence element of the C2 complement poly(A) signal activates mRNA 3' end formation by two distinct mechanisms.</a> Genes Dev. 12, 2522-2534 (1998).</li><li>Hall-Pogar, T., Zhang, H., Tian, B., Lutz, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+polyadenylation+of+cyclooxygenase-2.">Alternative polyadenylation of cyclooxygenase-2.</a> Nucleic Acids Res. 33, 2565-2579 (2005).</li><li>Brackenridge, S., Proudfoot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+a+basal+polyadenylation+factor+by+the+upstream+sequence+element+of+the+human+lamin+B2+polyadenylation+signal.">Recruitment of a basal polyadenylation factor by the upstream sequence element of the human lamin B2 polyadenylation signal.</a> Molecular and Cellular Biology. 20, 2660-2669 (2000).</li><li>Moore, C. L., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+cleavage+and+polyadenylation+of+exogenous+RNA+substrate.">Accurate cleavage and polyadenylation of exogenous RNA substrate.</a> Cell. 41, 845-855 (1985).</li><li>Gilmartin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> mRNA formation and function. , 79-98 (1997).</li><li>Wahle, E., Keller, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA Processing. Vol. II. , 1-34 (1994).</li><li>Chabot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA Processing Vol I. 1, 1-29 (1994).</li><li>Valente, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+mRNA+3'+end+processing+is+distinctively+regulated+by+eIF3f,+CDK11,+and+splice+factor+9G8.">HIV-1 mRNA 3' end processing is distinctively regulated by eIF3f, CDK11, and splice factor 9G8.</a> Mol Cell. 36, 279-289 (2009).</li><li>Dignam, J. D., Lebovitz, R. M., Roeder, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+transcription+initiation+by+RNA+polymerase+II+in+a+soluble+extract+from+isolated+mammalian+nuclei.">Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.</a> Nucleic Acids Res. 11, 1475-1489 (1983).</li><li>Aurup, H., Williams, D. M., Eckstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2'-Fluoro-+and+2'-amino-2'-deoxynucleoside+5'-triphosphates+as+substrates+for+T7+RNA+polymerase.">2'-Fluoro- and 2'-amino-2'-deoxynucleoside 5'-triphosphates as substrates for T7 RNA polymerase.</a> Biochemistry. 31, 9636-9641 (1992).</li><li>Keller, W., Bienroth, S., Lang, K. M., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+and+polyadenylation+factor+CPF+specifically+interacts+with+the+pre-mRNA+3'+processing+signal+AAUAAA.">Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3' processing signal AAUAAA.</a> EMBO J. 10, 4241-4249 (1991).</li><li>Takagaki, Y., Ryner, L. C., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+factors+are+required+for+3'-end+cleavage+of+pre-mRNAs.">Four factors are required for 3'-end cleavage of pre-mRNAs.</a> Genes Dev. 3, 1711-1724 (1989).</li><li>Takagaki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multisubunit+factor,+CstF,+is+required+for+polyadenylation+of+mammalian+pre-mRNAs.">A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs.</a> Genes Dev. 4, 2112-2120 (1990).</li><li>Ruegsegger, U., Blank, D., Keller, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pre-mRNA+cleavage+factor+Im+is+related+to+spliceosomal+SR+proteins+and+can+be+reconstituted+in+vitro+from+recombinant+subunits.">Human pre-mRNA cleavage factor Im is related to spliceosomal SR proteins and can be reconstituted in vitro from recombinant subunits.</a> Mol Cell. 1, 243-253 (1998).</li><li>Vries, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pre-mRNA+cleavage+factor+II(m)+contains+homologs+of+yeast+proteins+and+bridges+two+other+cleavage+factors.">Human pre-mRNA cleavage factor II(m) contains homologs of yeast proteins and bridges two other cleavage factors.</a> EMBO J. 19, 5895-5904 (2000).</li><li>Gilmartin, G. M., Nevins, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ordered+pathway+of+assembly+of+components+required+for+polyadenylation+site+recognition+and+processing.">An ordered pathway of assembly of components required for polyadenylation site recognition and processing.</a> Genes Dev. 3, 2180-2190 (1989).</li><li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+the+human+pre-mRNA+3'+processing+complex.">Molecular architecture of the human pre-mRNA 3' processing complex.</a> Mol Cell. 33, 365-376 (2009).</li><li>Bentley, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rules+of+engagement:+co-transcriptional+recruitment+of+pre-mRNA+processing+factors.">Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors.</a> Curr Opin Cell Biol. 17, 251-256 (2005).</li><li>Tian, B., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+cleavage+and+polyadenylation:+the+long+and+short+of+it.">Alternative cleavage and polyadenylation: the long and short of it.</a> Trends Biochem Sci. 38, 312-320 (2013).</li><li>Ryner, L. C., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+accurate+and+efficient+mRNA+3'+end+cleavage+and+polyadenylation+of+a+simian+virus+40+early+pre-RNA+in+vitro.">Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro.</a> Molecular and Cellular Biology. 7, 495-503 (1987).</li><li>Hirose, Y., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatine+phosphate,+not+ATP,+is+required+for+3'+end+cleavage+of+mammalian+pre-mRNA+in+vitro.">Creatine phosphate, not ATP, is required for 3' end cleavage of mammalian pre-mRNA in vitro.</a> J Biol Chem. 272, 29636-29642 (1997).</li><li>Ryan, K., Khleborodova, A., Pan, J., Ryan, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+activators+of+pre-mRNA+3'+cleavage.">Small molecule activators of pre-mRNA 3' cleavage.</a> RNA. 15, 483-492 (2009).</li></ol>

Yazarlar ifşa hiçbir şey yok.

HeLa hücre nükleer özütlerinde veya bu ekstrelerden parçalandı bölme faktörleri ile gerçekleştirilen in vitro pre-mRNA 3 &#39;bölünme reaksiyonu, çekirdek bölünmesi faktörler ve ana komplekslerinin 16-21 tanımlanmasını sağladı. Bu faktörlerle ilişkili daha birçok proteinlerin son zamanlarda 22 tespit edilmiştir ve in vitro reaksiyon bu proteinlerin reaksiyonu nasıl katkıda ışık tutacak devam edebilir. In vivo reaksiyon cotranscriptional ...

En olgun ökaryotik mesajı RNA&#39;lar (mRNAlarının) biyosentezi gibi kapatma, yapıştırma ve poliadenilasyonun gibi çeşitli transkripsiyon sonrası değişiklikler gerektirir. Bu değişiklikler genel olarak doğru işlem 1 yürütülmesini sağlamak ve güçlü bir mRNA&#39;nın stabilitesini arttırmak kuple edilir. 3 &#39;, memeli ön mRNA uç oluşumu 5&#39;e adenilat tortularının ilave edildi ve ardından yeni oluşan RNA endonükleolitik yarılmasıyla üretilir&#39; poly (A) polimeraz (PAP) 2-4 ürünü ayrıldı. Memelilerde, bölünme özel ön-RNA dizileri üzerinde toplanır, yaklaşık 800 kDa, ve, bir çok-bileşenli protein kompleksi ile gerçekleştirilir. Poli (A) sinyal sekansı, yüksek oranda korunmuş bir kanonik hexanucleotide sekansı AAUAAA, yaklaşık 10-30 nt alt bölünme sitesi yönlendirir. Bu site spesifik yarılma ve poliadenilasyon özgüllük faktörü (CPSF) ve 73 kD alt ünitesi tarafından tanınanCSPF endonükleaz aktivitesi içerir. Bölünme uyarım faktörü (CSTF) alt poli (A) Alanı daha dejenere GU-ya da U-zengin sekansı element bağlar. Ayrıca memeli bölünme faktörü I (CFIm) ve memeli bölünme faktörü II (CFII) bölünmesi için gerekli olan. CFIm gen bir dizi için tanımlandığı gibidir ve önemli fizyolojik işlemleri 5-8 dahil olmak gibi olmuştur üst dizisi elemanları (kullanım) spesifik UGUA (N) sitelerine bağlanır. İn vitro olarak, RNA işleme tepkimelerinin genel olarak radyo-etiketli RNA alt tabakaların 9-12 kullanımı ile analiz edilmiştir. Bunlar, bakteriyofaj promotör T7 ya da SP6 ikinci run-off transkripsiyonu ile sentezlenebilir. Daha önce karakterize edilmemiş bir poliadenilasyon sitesi incelenirken, önemli alt dizileri cDNA içinde mevcut olmayabilir gibi, RNA alt tabakanın üretilmesi için, genomik DNA yerine cDNA kullanmak gereklidir. En az 150 nt upstrea dahil tasarım yüzeylerm ve 50 nt aşağısında olgun mRNA üzerinde yarılma sitesi / ucundan. Alt-tabaka bölünme oluşumu daha hızlı göç eder; Diğer parçalar spesifik olmayan nükleaz hareketiyle oluşturulmuş olabilir, çünkü ancak, reaksiyonun özgüllüğü doğru işlem sinyal dizileri üzerinde bağımlılığı ile doğrulanmalıdır. Bu nedenle, AAUAAA dizisi (örneğin, AAGAAA) bir nokta mutasyonu ile alt-tabakalar RNA bölünme reaksiyonu için bir negatif kontrol olarak hizmet etmektedir. Radyo işaretli RNA küçük bir miktar bölünme reaksiyonları için kullanıldığı göz önüne alındığında, çoğu nükleer özütlerinde de yüksek bolca mevcut RNazlar sorunlu olabilir ve özü hazırlanması için başlangıç ​​malzemesinin seçimi sınırlayabilir. HeLa hücreleri, endojen RNases düşük seviyelerini ihtiva edebilmekte ve bu yüzden, bu deneylerde iyi bir performans göstermektedir. In vivo ve in vitro RNA substratların endonükleolitik klivaj hemen poli (A) ekleme, Per izlemektedirs bölünmüş ara tespit edilebilir miktarlarda mevcut değildir. Bu nedenle, belirli bir RNA dizisini ya da bir bölünme reaksiyonunda yer alan proteinleri ya da çalışma için, deneyler meydana gelen poliadenilasyon önlemek koşullarda yapılır. Bir parçalanma reaksiyonunu zarar vermeden poliadenilasyona durdurmak böylece hiçbir poliadenilasyonunun üzerinde bölünme bağımlılığı, ya da tam tersi vardır. Bu nedenle, ATP tek bir nükleotid poli (A) bölgesinde dahil edilebilir ve sadece bir RNA klivaj tespit edilebilir, böylece 3 &#39;hidroksil grubuna sahip olmayan bir zincir sonlandırma analogu ile değiştirilir. Karmaşıklığı ve tahlil bu tür yüksek derecede belirli özellikler göz önüne alındığında, in vitro mRNA öncülerinin ayırma / Poli (A) makine ile endonükleolitik bölünme incelemek için ayrıntılı bir video protokol açıklar. Biz, yetkili nükleer özleri hazırlamak radyoetiketlenmiş RNA alt tabakalar oluşturmak, parçalanma reaksiyonunu gerçekleştirmek ve sonucu analiz etme ve yorumlama nasıl tarifing ürünleri. Şekil 1, bir bölme deneyi için kullanılacak olan HIV-1 ön-mRNA öğeleri içerisinde 3 &#39;ucunda kodlama için alt-tabaka RNA&#39;ların bir örneğini göstermektedir. HIV RNA genomunun 3 &#39;ucu, örneğin poli (A) sitesi, G+ U zengin bölge ve bütün viral mRNA transkriptlerinin 13 etkin bir şekilde olgunlaşması için gerekli olan USE elemanı olarak çok önemli bir düzenleyici sekansların oluşmaktadır . Bu örnekte giriş RNA substrat 338 nt ve bölünme 237 üzerine nt olması beklenir. Poliadenilasyon oluşmasına izin verilir ise, ürün bir yayma 237 ve 437 nt arasında anlamlı olacaktır.

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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Slide-A-Lyzer Dialysis Cassettes Kit</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:119px;">66372</td>
			<td>MWCO 7000 0.5ml-3ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15ml Dounce Tissue grinder set</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9938-1SET</td>
			<td>Different sizes available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Expand High Fidelity PCR Kit</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">11 732 650 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10mM dNTP Mix</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:119px;">Y02256</td>
			<td>10mM each nucleotide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MaxiScript SP6/T7 Kit</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM1322</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">m7G(5&#39;)ppp(5&#39;) G RNA Cap</td>
			<td style="width:141px;">New England Biolabs</td>
			<td style="width:119px;">S1404S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Century Marker Template Plus</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM7782</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easytides UTP [alpha-32p]-250uCi</td>
			<td style="width:141px;">Perkin Elmer</td>
			<td style="width:119px;">BLU507H250UC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel loading buffer II</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">8546G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DEPC treated water</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM9906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x TAE</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP13354</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x TBE</td>
			<td style="width:141px;">Ameresco Life Sciences</td>
			<td style="width:119px;">0658-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x PBS</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP399-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP169-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Persulfate</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP150-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Acetate</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">A637-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40% 19:1 Acrylamide:Bis-acrylamide</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">10 901 393 001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100% Absolute Ethanol 200 Proof</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">61509-0040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acid Phenol-Chloroform</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">9720</td>
			<td>For RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintilation Fluid</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">SX18-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase Inhibitor</td>
			<td style="width:141px;">Promega</td>
			<td style="width:119px;">N261B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly (Vinyl alcohol) PVA</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine Phosphate</td>
			<td style="width:141px;">Calbiochem</td>
			<td style="width:119px;">2380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100mM dATP</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP2560-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">23042-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP1700-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adjustable Sequencing Unit</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351881-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Binder Clips</td>
			<td style="width:141px;">Office Depot</td>
			<td style="width:119px;">838-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx42cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352543</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx22cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35252-7</td>
			<td>1SET</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20cmx42cm Aluminum Cooling Plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352667</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx22cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35230-6</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx42cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352314-1</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35195-4</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">16-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351962</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351970</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Repel Coating</td>
			<td style="width:141px;">C.B.S. Scientific</td>
			<td style="width:119px;">SGR-0401</td>
			<td>or SGR-0101 for individual bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Loading Tips</td>
			<td style="width:141px;">Rainin</td>
			<td style="width:119px;">GT-10-4</td>
			<td>0.1-10uL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sequencing PowerPac HV</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">PowerPac HV</td>
			<td>5000V/500mA/400W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Dryer Model 583</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">Model 583</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrotech Vacuum Pump for gel dryer</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glogos II Glow-in-the-dark Markers</td>
			<td style="width:141px;">Agilent</td>
			<td style="width:119px;">420201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 8x10</td>
			<td style="width:141px;">Midsci</td>
			<td style="width:119px;">BX810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 14x17</td>
			<td style="width:141px;">Phenix</td>
			<td style="width:119px;">F-BX1417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autoradiography Cassette</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">FBCA 1417</td>
			<td>8x10 size available</td>
		</tr>
	</tbody>
</table>

analysis of rna processing reactions using cell free systems: 3' end cleavage of pre-mrna substrates in vitro

Memeli mRNA&#39;ların 3 &#39;ucu RNA polimeraz II (RNPII) ile transkripsiyonun sonlandırılması ile ani oluşmuş değildir. Bunun yerine, RNPII olgun RNA&#39;lar ötesinde ön-mRNA sentezler ve endonükleaz aktivitesi olan bir süreç belirli bir site gereklidir. Öncü RNA bölünmesi normalde CA dinükleotidleri sonra konsensüs polyA bölgesi (AAUAAA) aşağı nt 10-30 oluşur. Yarılma kompleks, yaklaşık olarak 800 kDa&#39;lık çok faktörlü bir protein kompleksi, ikinci proteinler, bu özel nükleaz aktivitesi gerçekleştirmek. Belirli RNA sekansları, yukarı akış ve poliA site alt bölünme kompleksinin işe kontrol eder. Hemen bölünmesinden sonra, pre-mRNA stabil olgun RNA mesajları üretmek için poli A polimerazı (PAP) ile poliadenile edilmiştir. Bir RNA transkriptinin 3 &#39;ucunun radyo-etiketli RNA Processing özel alt-tabakalar ile hücre nükleer özler kullanılarak incelenebilir. Özetle, uzun bir 32 &lt;/ Sup&gt; P-etiketli Parçalanmamış öncü RNA, in vitro olarak nükleer ekstreler ile inkübe edilir, ve bölünme jel elektroforez ve otoradyografi ile değerlendirilir. Uygun bölünme oluştuğunda, 5 &#39;kısa bir ürün olarak algılanır ve ölçülür ayrıldı. Burada, bir örnek olarak, kullanılarak ayrıntılı olarak HIV-1 mRNA&#39;ların 3 &#39;ucu işleme bölünme tahlili tarif eder.

RNA poli (A) HIV-1 siteleri (Şekil 2) bir bölünme tahlili temsil edici sonuçları. Bu jelin üstünde yavaş göç eden bir bant Parçalanmamış RNA alt tabakanın, gözlemleyebiliriz. Belirli ürün, daha hızlı parçalanan beklenen boyutta jelde çalışan en yoğun kısa fragman bandı, ve poliA sekansı (mutPolyA) RNA, bir nokta mutasyon ihtiva mutpoly (A) kontrolü yarılma tahlilinden özellikle yoktur alt-tabaka. Giriş substratın degradasyon ürünleri, bazen gözlemlenebilir. Yarılma ak...

Watch this Scientific Journal Video about Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro at JoVE.com

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polimeraz II olgun mRNA&#39;nın 3 &#39;ucunun ötesine uzanan bir ön-madde RNA sentezler. Olgun RNA uç yarılma kompleksinin endonükleaz etkinliği ile, RNA dizileri tarafından dikte bir bölgede, cotranscriptionally oluşturulur. Burada, detay in vitro bölünme reaksiyonları çalışma yöntemi biz.

Pre-mRNA Substratlarm 3 &#39;End Bölünme: Hücre Ücretsiz Sistemlerini Kullanma RNA İşleme Reaksiyonları Analizi<em&gt; In vitro</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

analysis-rna-processing-reactions-using-cell-free-systems-3-end

Research

JoVE Journal

Biology

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

12.7K Görüntüleme.  The Scripps Research Institute.  RNA polimeraz II olgun mRNA'nın 3 'ucunun ötesine uzanan bir ön-madde RNA sentezler. Olgun RNA uç yarılma kompleksinin endonükleaz etkinliği ile, RNA dizileri tarafından dikte bir bölgede, cotranscriptionally oluşturulur. Burada, detay in vitro bölünme reaksiyonları çalışma yöntemi biz. 

Video: Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video Biyoinformatik İnsan Embriyonik Kök Hücre Colony Büyüme Analizi

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Biyoloji

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

MG53-aracılı Hücre Zarı Tamiri Görselleştirme In vivo Ve In vitro Sistemleri

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

10 nanogram Total RNA tek okuyun ve Eşleştirilmiş Sona mRNA-Seq Illumina Kütüphaneler

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

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 ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Hücre Özgül Analizi<em&gt; Arabidopsis</em&gt; Floresans Aktif Hücre Ayırma kullanma Leaves

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / Cas Sistemleri Endonükleazlar için Substrat Üretimi

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

İfadesi için HeLa Tabanlı Hücre Özgür İfade Sistemleri<em&gt; Plasmodium</em&gt; Rhoptry Proteinler

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

Hücre Testi Mitoz Sonu Kromatin dekondenzasyon Eğitim için

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA bağlama proteinlerinin tespiti ile<em&gt; İn Vitro</emAdiposit Kültür&gt; RNA pull-down

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

3' End Sequencing Library Preparation with A-seq2

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3&#39;UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3&#39; end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3&#39; end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3&#39; ends in a wide range of conditions, from 106 or fewer cells.

3&#039; End Sequencing Library Preparation with A-seq2

This protocol describes a method for mapping pre-mRNA 3&#39; end processing sites.