우리는 RNAP와 Mfd 단백질과 표현 벡터를 제공하는 세스 다라 특히 감사합니다. 우리는 또한 기술 지원을 GreB와 댄 장를 제공 세르게이 Borukhov 감사합니다. 이 작품은 건강 (MOD)의 국립 연구소에서 교부금에 의해 및 마리 - 조시와 록펠러 대학 (RTP)에서 헨리 Kravis 원정대에 의해 지원되었다.

이 방법에 보고된 연구에 사용된 <a href="http://www.sciencemag.org/cgi/content-nw/full/327/5965/590/F3">Pomerantz와 오도넬, 과학 327, 590-592 (2010)</a> . 1. 중단 RNAP의 연신율 복잡한 조립은 다음과 같이 수행되었습니다 E. 500 NM 최종 농도 콜리 RNAP 사람은 (20 MM 트리스 - CL (산도 7.5), 8 MM MgCl 2, 0.5 MM EDTA (에틸렌 다이아 민 테트라 초산), 5 버퍼 100 μl의 T7A1 모터를 포함하는 biotinylated 3.6 이하 DNA 템플릿 5 NM 최종 농도로 혼합했다 70 σ 37 10 분 MM DTT, 10 % 글리세롤) ° C. APU 40 μm의 GTP, ATP와 CTP의 각 100 μm의는 37 추가 10 분 ° C.에 추가되었습니다 2. streptavidin 비즈에 중단 RNAP의 신장 복합 고정은 다음과 같이 수행되었습니다 streptavidin 자기 코팅 구슬 (Invitrogen) 200 μl는 방 온도에서 10 분 이상 반응 추가되었습니다. 3. 고정 중단 RNAP의 신장 복합 정제는 다음과 같이 수행되었습니다 구슬이 (위에서) 버퍼의 0.9 ML 포함하는 0.75 M NaCl, 200 μg / ML의 헤파린, 20 μg / ML 다음 ssDNA (뜨는은 자기 분리하여 각 세척 단계 후 삭제되었습니다.) 5 번 씻어되었습니다 구슬은 버퍼 A.의 0.9 ML로 2 번 씻어되었습니다 4. :에 정면 RNAP 다음과 같이 수행되었다 하류 상대 복제 포크의 형성 구슬이 (위에서) 뉴잉글랜드 Biolabs 버퍼 4 삽 I (뉴잉글랜드 Biolabs) 10 단위 100 μl에 resuspended되었습니다 것은 37 10 분 추가되었습니다 ° C. 구슬은 버퍼의 0.9 ML 3 번 씻어 후 빠른 T4 결합 반응 버퍼 (뉴잉글랜드 Biolabs) 50 μl에 resuspended되었습니다. 빠른 T4 ligase (뉴잉글랜드 Biolabs) 2 μl는 방 온도에서 10 분 사전 annealed 포크드 DNA 6 nm의 최종 농도 (RP10, RP22, RP25)와 함께 추가되었습니다. 구슬은 버퍼 A.의 0.9 ML 3 번 씻어되었습니다 5. 복제 분기점에서 최고의 스트랜드 합성의 replisome와 개시의 총회는 다음과 같이 수행되었습니다 DnaB (hexamer 등)의 448 pmol는 버퍼의 비즈 150 μl와 incubated되었습니다 (20 MM 트리스 - CL (산도 7.5), 8 MM MgCl 2, 0.5 MM EDTA (에틸렌 다이아 민 테트라 초산), 5 MM DTT, 10 % 글리세롤) 30 s에 대한 23 ° C. 투표소 III의 4.9 pmol * (폴 III HE 마이너스 β), β 15 pmol, 2 MM ATP, 60 μm의 dGTP 및 dATP 각각 200 μl의 볼륨에 추가 37에서 추가로 5 분 ° C.를 incubated되었습니다 복제가와 함께 10 μg SSB와 24 μm의 dATP와 dTTP 각 추가시 시작되었다 [α - 32 P] dTTP와 [α - 32 P] dCTP (특정 활동, 3,000-5,000 CPM / pmol) 250 마지막 볼륨 μL. 반응 500 MM EDTA (에틸렌 다이아 민 테트라 초산) 12 μl를 추가시 10 분 이후에 종료되었습니다. 6. 정화 및 라디오 레이블이 붙은 DNA 제품의 농도는 다음과 같이 수행되었습니다 반응이 구슬에서 DNA를 제거하기 위해 종료 후에 구슬이 (위에서) 삶은되었습니다. 비즈에 연결된 나머지 잔여 DNA는 50 30 분 10 UL의 볼륨에 proteinase K와 구슬을 치료 ° C.를하여 제거되었습니다 구슬 그런 다음 삶은되었으며 뜨는은 자기 분리에 의해 제거되었습니다. DNA를 포함하는 결합 뜨는은 Qiagen PCR 정리 키트를 사용하여 정화되었다. 라디오 표시 DNA 제품은 다음 알칼리성 아가로 오스 겔에서 분석되었다 정화. 정면 RNAP 신장 복합은 위와 같이 수행되었다 중단 단, σ 70 부재에 * 복제가 생략되었습니다. 대표 결과 : 함께 replisome의 충돌 정면 RNAP 보통 2.5 킬로바이트와 길이 3.6 킬로바이트 두 제품에 발생합니다. 2.5 이하 제품은 중단 RNAP와 RNAP와 충돌시 연기 replisome에서 결과를 분기점에서 DNA의 길이를 나타냅니다. 3.6 이하 제품은 불완전한 RNAP에 의해 모터의 숙박 또는 일부 replisome 정면 RNAP의 읽기를 통해서에서 전체 길이 DNA와 결과를 나타냅니다. 좋은 또는 정확한 결과는 약 50 % 전체 길이 DNA 생산 것을 보여줍니다. 그러나, 경우에 RNAP에 의해 모터의 적은 인가 발생하고 전체 길이의 DNA보다 높은 비율이 replisomes의 큰 인구 이후 관찰된 것은 정면에 RNAP가 발생하지 않습니다. 단 전체 길이 DNA (3.6 KB)에서 중단 RNAP 결과의 부재에 복제. <img src="/files/ftp_upload/1919/1919fig1.jpg" alt="그림 1" /> 그림. 1 replisome는 정면에서 RNAP에 의해 장애가 있습니다. (A) 실험 설정은. E. 안부나는 중단 연신율 복잡한는 이전에 설명한대로 T7A1 발기인 (9)를 포함하는 biotinylated DNA를 조립했다 RNAP. 간단히 중단 연신율 복합은 더욱 10 분 APU, GTP, ATP와 CTP의 또한 다음 10 분 biotinylated DNA와 RNAP의 holoenzyme을 잠복기로 모터 20 BPS 하류를 형성했다. Streptavidin 비즈는 다음 비즈가 0.75M NaCl, 200 MG / ML 헤파린, 100 nm의 단일 스트랜드 불안정 RNAP - DNA의 단지 및 초과 NTPs를 제거하는 DNA를 포함하는 버퍼의 0.9 ML, 5 번 각각 씻어 줄 추가 10 분 추가되었습니다 . DNA는 다음 SAPI와 소화, 세탁 및 사전 annealed 포크드 DNA에 출혈도 잡았습니다. 구슬은 replisome의 조립하기 전에 다시 한번 씻어되었습니다. 발기인은 분기점에서 2.5 킬로바이트에 위치하고 분기점으로 직접 전송하는 방향입니다. (B) DnaB는 다음 replisome 조립했을 추가되었습니다 및 복제는 존재하거나 시작 (레인 2) 또는 부재 (레인 1)되었습니다 정면 RNAP.

<ol>
	<li>Rudolph, C. J., Dhillon, P., Moore, T., Lloyd, 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=Avoiding+and+resolving+conflicts+between+DNA+replication+and+transcription.">Avoiding and resolving conflicts between DNA replication and transcription.</a> DNA Repair (Amst). 6, 981-98 (2007).</li><li>Vilette, D., Ehrlich, S. D., Michel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-induced+deletions+in+plasmid+vectors:+M13+DNA+replication+as+a+source+of+instability.">Transcription-induced deletions in plasmid vectors: M13 DNA replication as a source of instability.</a> Mol Gen Genet. 252, 398-398 (1996).</li><li>Trautinger, B. W., Jaktaji, R. P., Rusakova, E., Lloyd, 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=RNA+polymerase+modulators+and+DNA+repair+activities+resolve+conflicts+between+DNA+replication+and+transcription.">RNA polymerase modulators and DNA repair activities resolve conflicts between DNA replication and transcription.</a> Mol Cell. 19, 247-247 (2005).</li><li>McGlynn, P., Lloyd, 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=Modulation+of+RNA+Polymerase+by+(p)ppGpp+Reveals+a+RecG-Dependent+Mechanism+for+Replication+Fork+Progression.">Modulation of RNA Polymerase by (p)ppGpp Reveals a RecG-Dependent Mechanism for Replication Fork Progression.</a> Cell. 101, 35-35 (2000).</li><li>McGlynn, P., Guy, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+forks+blocked+by+protein-DNA+complexes+have+limited+stability+in+vitro.">Replication forks blocked by protein-DNA complexes have limited stability in vitro.</a> J Mol Biol. 381, 249-249 (2008).</li><li>Labib, K., Hodgson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+barriers:+pausing+for+a+break+or+stalling+for+time.">Replication fork barriers: pausing for a break or stalling for time.</a> EMBO Rep. 8, 346-346 (2007).</li><li>Bidnenko, V., Ehrlich, S. D., Michel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+collapse+at+replication+terminator+sequences.">Replication fork collapse at replication terminator sequences.</a> Embo J. 21, 3898-3898 (2002).</li><li>Pomerantz, R. T., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replisome+mechanics:+insights+into+a+twin+DNA+polymerase+machine.">Replisome mechanics: insights into a twin DNA polymerase machine.</a> Trends Microbiol. 15, 156-156 (2007).</li><li>Pomerantz, R. T., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+replisome+uses+mRNA+as+a+primer+after+colliding+with+RNA+polymerase.">The replisome uses mRNA as a primer after colliding with RNA polymerase.</a> Nature. 456, 762-762 (2008).</li><li>Heller, R. C., Marians, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+reactivation+downstream+of+a+blocked+nascent+leading+strand.">Replication fork reactivation downstream of a blocked nascent leading strand.</a> Nature. 439, 557-557 (2006).</li><li>Yuzhakov, A., Turner, J., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+reactivation+downstream+of+a+blocked+nascent+leading+strand.">Replication fork reactivation downstream of a blocked nascent leading strand.</a> Cell. 86, 557-557 (1996).</li><li>Hinkle, D. C., Ring, J., Chamberlin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replisome+assembly+reveals+the+basis+for+asymmetric+function+in+leading+and+lagging+strand+replication.">Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication.</a> J Mol Biol. 70, 197-197 (1972).</li><li>Kaplan, D. L., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+binding+of+Escherichia+coli+RNA+polymerase+to+DNA.+V.+T7+RNA+chain+initiation+by+enzyme-DNA+complexes.">Studies of the binding of Escherichia coli RNA polymerase to DNA. V. T7 RNA chain initiation by enzyme-DNA complexes.</a> Mol Cell. 15, 453-453 (2004).</li><li>Park, J. S., Marr, M. T., Roberts, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twin+DNA+pumps+of+a+hexameric+helicase+provide+power+to+simultaneously+melt+two+duplexes.">Twin DNA pumps of a hexameric helicase provide power to simultaneously melt two duplexes.</a> Cell. 109, 757-757 (2002).</li><li>Hanawalt, P. C., Spivak, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=coli+Transcription+repair+coupling+factor+(Mfd+protein)+rescues+arrested+complexes+by+promoting+forward+translocation.">coli Transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation.</a> Nat Rev Mol Cell Biol. 9, 958-958 (2008).</li><li>Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+DNA+repair:+two+decades+of+progress+and+surprises.">Transcription-coupled DNA repair: two decades of progress and surprises.</a> Science. 266, 1957-1957 (1994).</li><li>Selby, C. P., Sancar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+repair+and+human+disease.">Transcription-coupled repair and human disease.</a> J Biol Chem. 270, 4882-4882 (1995).</li><li>Liu, B., Alberts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+transcription-repair+coupling+factor.+I.+Structural+domains+and+binding+properties.+Head-on+collision+between+a+DNA+replication+apparatus+and+RNA+polymerase+transcription+complex.">Structure and function of transcription-repair coupling factor. I. Structural domains and binding properties. Head-on collision between a DNA replication apparatus and RNA polymerase transcription complex.</a> Science. 267, 1131-1131 (1995).</li><li>George, D. L., Witkin, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+excision+repair+in+an+mfd+mutant+of+Escherichia+coli+B/r.">Slow excision repair in an mfd mutant of Escherichia coli B/r.</a> Mol Gen Genet. 133, 283-283 (1974).</li></ol>

높은 소금과 비즈로 바운드 중지 신장 컴플렉스를 세척하는 것은 어떤 불안이나 비 특별히 바운드 RNAP 분자를 제거하기 위해 중요합니다. RNAP는 프라이머 - 템플릿 타입 DNA 구조에 대해 매우 높은 친화력을했다. 따라서, 그것은 뇌관 - 템플릿에서 복제를 수행하기 위해 이러한 불특정 RNAP - DNA의 단지를 제거하는 것이 필요합니다. Qiagen PCR 정화 키트를 사용하여 라디오 레이블이 붙은 DNA 제품을 집중하면 겔에서 관찰할 수있을만큼 충분히 높은 방사성 신호를 얻기 위해서도 중요합니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dynabeads M-270</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>653.05</td>
<td>&nbsp;</td>
<tr>
<td>SapI</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>R0569S</td>
<td>&nbsp;</td>
<tr>
<td>T4 Quick Ligase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M2200S</td>
<td>&nbsp;</td>
<tr>
<td>PCR Purification Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

direct restart of a replication fork stalled by a head-on rna polymerase

생체내 연구에서 복제 퍽스가 전사 단지 정면에 함께 인한 일로 체포하는 것이 좋습니다. 그러나, replisome 및 RNA 효소 (RNAP)의 운명은 다음과 같은 머리를에 충돌 알 수 있습니다. 여기, 우리가 찾을 수있는 E. 와 충돌시 대장균 replisome 노점을 정면에서 전사 복잡 대신 무너지고의 복제 포크는 매우 안정적인 남아 결국 DNA의 RNAP를 생기죠 후 신장을 다시 시작합니다. 우리는 전사 - 수리 커플링 계수, Mfd이 RNAP의 변위를 촉진하여 충돌을 다음과 포크를 직접 다시 시작을 촉진 것을 또한 확인하십시오. 이러한 결과는 복제 장치의 본질적인 안정성과 RNAP 블록 과거 복제를 추진하고있는 전사 결합 수리 경로에 대한 소설 역할을 보여줍니다.

Watch this Scientific Journal Video about Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase at JoVE.com

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

replisome의 운명과 충돌을 따라 정면에서 RNA 효소 (RNAP) 알 수 있습니다. 우리는 DNA에서 RNAP를 생기죠 후 신장을 replisome 정면에 RNAP와 충돌시 포장 마차지만, 이력서 것을 발견했습니다. Mfd은 충돌 후 RNAP의 변위를 촉진하여 복제 다시 시작을 추진하고 있습니다.

정면 RNA 효소에 의해 지연 복제 포크의 직접 다시 시작

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA ...

direct-restart-replication-fork-stalled-head-on-rna

Research

JoVE Journal

Biology

13.5K 조회수.  Rockefeller University. replisome의 운명과 충돌을 따라 정면에서 RNA 효소 (RNAP) 알 수 있습니다. 우리는 DNA에서 RNAP를 생기죠 후 신장을 replisome 정면에 RNAP와 충돌시 포장 마차지만, 이력서 것을 발견했습니다. Mfd은 충돌 후 RNAP의 변위를 촉진하여 복제 다시 시작을 추진하고 있습니다.

비디오: Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

293T 세포의 과도 Transfection하여 복제 - 결함이 레트로 바이러스의 생산

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

연구

생물학

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

형광 현미경과 함께 머릿속으로 단일 분자의 DNA 복제

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

효소 복제 DNA의 직접 관찰 어세을 스트레칭 단일 분자 DNA를 사용하여

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

머릿속 RNA 현지화에서 Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

사출 및 Electroporation에 의해 Zebrafish Otocyst으로 MIF Morpholinos 직접 배달 내이 (内耳)의 개발 미치는 영향

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

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 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

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

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; 현장에서</em전체 마운트 태아 및 세포 조직학에서&gt; 하이브리드는 해양 Elasmobranchs을위한 적응

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

마우스 췌장에서 RNA 격리 : 리보 뉴 클레아 풍부한 조직

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

내부 양성 대조군과 양적 재조합 효소 중합 효소 증폭 분석 개발

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.