연구는 건강의 펜실베니아학과 엘리슨 의료, V 및 에메랄드 기초에 의해 지원되었다 여기를 발표했다.

1. 재조합 TC TERT의 표현 <ol><li> TEV cleavable N - 터미널로 합성 TC TERT 플라스미드를 포함하는 (DE3) 로제타 변환 pLysS 세포 여섯 신선한 접시에서 2YT 미디어 6 패 (6 X 2 L은 각 2YT 국물 1 L를 포함 Erlenmeyer의 Flasks들을 당황하게)를 예방 hexahistidine - 태그. </li><li> 37 세포를 성장 ° C 0.5-0.6의 OD 600-220 rpm으로 흔들어 동안. </li><li> 1 ㎜의 IPTG를 추가하여 단​​백질 발현을 유도. </li><li> 30 온도를 감소 ° C, 150 RPM에 떨고 천천히, 그리고 세포가 4-5 H. 위해 단백질을 표현하는 수 </li><li> 4 원심 분리하여 세포를 수확 ° C 30 분 3,500 RPM. </li><li> 25 MM 트리스, 산도 7.5, 10 % 글리세롤, 0.5 M KCl, 15 MM 이미다졸, 5 MM의 β - 메르 캅 토 에탄올과 0.1 MM phenylmethyl sulphonyl 불화 (PMSF)와 benzamidine를 포함하는 버퍼의 20 ML 각 1 패 세포 펠렛을 Resuspend. </li><li> 플래시 고정 천천히 각 드롭 액체 N 2 미치는 영향에 멈추도록 액체 질소로 가득 50 ML 플라스틱 원뿔 튜브에 세포를 흘리지하여 세포 혼합물을 resuspended. -80에서 냉동 세포를 보관 ° C 단백질 정화 준비까지. </li></ol> 2. TC TERT의 정화 <ol><li> 액체 일관성 때까지 실온에서 해동 세포에 도달 후 sonication을 위해 얼음 금속 비커로 이동합니다. </li><li> 30초 권력의 약 51 W에서 일시 중지 30 초 간격으로 2 분 얼음에 세포를 Sonicate. </li><li> 가용성과 불용성 단백질 분수를 별도 조심스럽게 니켈 친화성 크로마 토그래피를위한 뜨는을 제거 20 분 18,000 rpm으로 lysed 세포를 스핀 다운. </li><li> 니켈 - NTA (니켈 - nitrilotriacetic 산성) 25 MM 트리스, 산도 7.5, 10 % 글리세롤, 0.5 M KCl, 15 MM 이미다졸, 5 MM의 β - 메르 캅 토 에탄올과 0.1 MM PMSF 및 benzamidine를 포함하는 버퍼로 열을 평형. </li><li> 수지 위에 뜨는을로드하고를 통해 흐름을 수집합니다. </li><li> 모든 잔류 및 비 특별히 바운드 단백질을 제거하는 25 MM 트리스, 산도 7.5, 10 % 글리세롤, 500 MM KCl, 15 MM 이미다졸, 5 MM의 β - 메르 캅 토 에탄올과 0.1 MM PMSF를 포함하는 버퍼로 컬럼을 씻으십시오. </li><li> Elute TC 15 이미다졸 그라데이션을 사용하여 니켈 NTA 수지의 TERT - 300 MM 및 SDS 페이지 분석을위한 3 ML 분수를 수집합니다. </li><li> 어떤 분수가 TC TERT의 단백질을 포함하고 결정하고 Amicon 30 KDA (Millipore) 필터에 분수 집중 PAGE 분석을 사용합니다. </li><li> 볼륨 더 정화를 위해 미만 10 ML에 TC TERT의 분수를 집중. </li><li> TC TERT은 더욱 양이온 교환 컬럼 (HS poros, 응용 Biosystems) 25 MM 트리스, 산도 7.5, 10 % 글리세롤, 500 MM의 KCl, 1 MM의 DTT를 포함하는 버퍼로 사전 equilibrated을 통해 정화됩니다. </li><li> HS poros 칼럼에있는 단백질 믹스를로드하고 모든 단백질과 핵산의 오염 물질을 제거하기 위해 그것을 씻으십시오. </li><li> 남아있는 오염 물질을 제거하는 KCl 700 MM 500 MM 다음의 KCl을 포함하는 버퍼로 컬럼을 씻으십시오. </li><li> 1 M KCl 버퍼와 HS poros 칼럼에서 TC TERT의 단백질을 Elute. 이 시점에서 단백질 이상 99% 순수해야합니다. </li><li> Superdex - 200 크기 조정 칼럼 (크기 배제 크로마 토그래피 - GE 헬스케어) 위에 단백질을 통과하여 단백질 집계를 제거, 25 MM 트리스, 산도 7.5, 10 % 글리세롤, 500 MM KCl, 1 MM의 TCEP를 포함하는 버퍼로 사전 equilibrated. </li></ol> 3. Tribolium castaneum 비틀 문화 <ol><li> T. castaneum 처음 박사 S. 브라운, 생물 학부, 캔사스 주립 대학에서 선물로 제공 어디 딱정벌레. </li><li> 시작 T. 밀가루의 28.5 g, 말린 베이커의 효모 1.5 g과 컨테이너 백 성인 딱정벌레를 추가하여 castaneum 문화. </li><li> 상온에서와 3~4주에 대한 어두운, 습한 환경에서 저장 충 문화 재생산 및 성장 수 있도록. 습한 환경을 보장하기 위해 충 저장 영역에 물이 들어있는 비커를 놓습니다. </li><li> 로 RNA 분리에 필요한 충 유충을 수확. 나머지 애벌레 같은 문화에서 그들을 유지, 어른 성숙한 수 있습니다. </li><li> 매월, 그들의 식량 공급을 보충하기 위해 신선한 밀가루와 효모를 포함하는 새로운 플라스크에 가장 적극적으로 딱정벌레를 (더 이상 수백 이상) 전송. </li></ol> 4. T. castaneum 총 RNA 분리 <ol><li> 성인 딱정벌레와 밀가루 / 효모 문화를 분리하여 문화에서 수확 20 충 유충 (각 문화가 500-1000 애벌레 사이에 포함되어야합니다). 밀가루하거나 분쇄하기 전에 딱정벌레에 연결된 효모 입자를 제거합니다. </li><li> 벤치 지역, 박격포 및 딱정벌레를 분쇄하기 전에 ribonucleases의 모든 흔적을 제거하는 RNaseZap (앰비온 주식 회사)이 절차에 사용된 유봉을 (물론 장갑을 치료 고려) 소독. </li><li> 절구와 유봉과 T를 사용하여 미세 분말로 액체 N 2에 애벌레를 갈다추출 버퍼가 추가 때까지 가루가 냉동 유지하도록 액체 N 2를 유지하면서 50 ML의 원심 분리기 튜브에 분말 ransfer. </li><li> 초과 액체 N 2 분말에서 증발 수 있습니다. 액체 N 2 증발 후, 25 MM 트리스 - HCL, 5 MM의 β - 메르 캅 토 에탄올, 1 MM EGTA, 0.1 당연 benzamidine 200 MM KCl, 10를 포함하는 추출 버퍼 200 μl의 충 분말 (20 충 유충 당) 균질 %의 글리세롤, 30 분 얼음 RNasin, 산도 7.5과 부화 10 MM 이미다졸 및 20U. </li><li> 4 20 분 동안 15,000 rpm으로 homogenate를 원심 ° C는 뜨는와 플래시를 수집하는 액체 N 2의 샘플을 동결. 필요한 경우, 샘플은 -80 ° C 하룻밤에 저장할 수 있습니다. </li><li> RNeasy 미니 키트 (Qiagen)를 사용하여 총 RNA를 추출합니다. </li></ol> Tribolium의 castaneum의 telomerase 5.에서는 체외의 reconstitution <ol><li> T. 50 μl와 재조합 그의 - TC 태그 TERT 20 μg 믹스 25 MM 트리스 - HCL, 200 MM KCl, 10 % 글리세롤, 5 MM의 β - 메르 캅 토 에탄올, 10 MM 이미다졸, 산도 7.5를 포함하는 바인딩 버퍼에 castaneum 총 RNA. </li><li> 22 두 시간 동안 총 RNA 혼합물 ° T.에 C - TC TERT을 품어 RNA 저하를 방지하기 위해 RNasin 억제제의 존재에 castaneum의 lysate. </li><li> lysate 불순물 및 과도한 RNA를 제거하는 니켈 - NTA 칼럼을 통해 복잡한 정화. </li></ol> 6. Telomerase의 반복 증폭 프로토콜 (트랩)는 assays <ol><li> 니켈 - NTA 정화 T. 4 μg를 섞어 50 μl 신장 버퍼에 castaneum의 telomerase 20 MM를 포함 트리스 - HCL (산도 - 8.3), 7.5 MM MgCl 2, 63 MM KCl, 0.05 % 트윈 20, 1 ㎜ EGTA, 0.01 % BSA, 0.5 MM의 dNTPs, 1 μm의 Tcas - TS DNA 프라이머 (5&#39; - AAGCCGTCGAGCAGAGTC - 3 &#39;), 그리고 60 분 ° C의 재구성 T.하여 Tcas - TS의 DNA 프라이머의 연신율을 허용하는 30 혼합물을 품어 castaneum의 telomerase. </li><li> 반응 혼합물을 페놀 클로로포름의 동일한 볼륨을 추가하여 길쭉한 단일 좌초된 DNA (ssDNA)을 추출합니다. 유제가 균질는 부드럽게 될 때까지 섞은 쉐이크. </li><li> 4 5 분 동안 12,000 rpm으로 샘플을 원심 ° C는 오염 물질의 ssDNA 분리 수 있습니다. </li><li> 1.5 ML eppendorf 튜브에 샘플의 정상 수성 층을 전송, 10 MM NaOAc (산도 5.0)의 최종 농도, MgCl 2 1 ㎜와 66.4 % EtOH 후 -20에서 하룻밤을 촉진하기 위해 샘플을 허용을 추가 ° C. </li><li> 4 삼십분을 위해 15,000 RPM에서 샘플을 스핀 ° C를 펠렛 시켰던 ssDNA 수 있습니다. pipettor를 사용하여 솔루션을 가만히 따르다 다음 예제에서 남은 NaOAc 및 MgCl 2 씻어 샘플을 70 % EtOH 200 μl를 추가합니다. 다시 펠렛 샘플에 동일한 속도와 온도에서 5 분 동안 샘플을 원심 분리기. </li><li> pipettor를 사용하여 EtOH 솔루션을 가만히 따르다하여 eppendorf 튜브 세 분 상온에서 열 부화함으로써 에탄올의 남은 흔적을 제거합니다. 예제에서 에탄올 선물 것은 PCR의 증폭 단계에 방해가됩니다. </li><li> 10 MM 트리스 - HCL (산도 8), 50 MM KCl, 2 MM MgCl 2, 100 μm의 dNTPs를 (dATP, 포함하는 PCR 반응 버퍼의 50 μl의 ssDNA 펠렛을 (telomerase 길쭉한 제품과 Tcas - TS의 프라이머를 포함) Resuspend dGTP 및 dTTP), 10 μm의 [32 P] dCTP, 1 μm의 Tcas - CX 프라이머 (5&#39; - GTGTGACCTGACCTGACC - 3 &#39;)와 1.25 U DNA 형성 촉매의 DNA 중합 효소. </li><li> PCR들은 12 % polyacrylamide 젤에서 볼 수 있도록 telomerase의 연신율 제품 (94 ° 30 초 동안 C, 50 ° 30 초 동안 C, 72 ° C에 대한 일분의 29 사이클)을 증폭. </li><li> 트리스 - Borate - EDTA (에틸렌 다이아 민 테트라 초산) (TBE)이 44.5 MM 트리스, 44.5 MM 붕산, 1 MM EDTA (에틸렌 다이아 민 테트라 초산), 서른 분 산도 8 전에 PCR 샘플을 실행하기를 포함하는 버퍼를 실행과 함께 12 % 젤을 미리 실행합니다. 사전 실행 및 PCR 반응으로 젤로드 후, 섭씨 또는 얼음에 4 명이에서 1.5-2 시간 동안 185 볼트에서 젤을 실행합니다. </li><li> 젤 건조하고 활동을 시각화하기 위해 형광체 이미징 플레이트를 사용합니다. </li></ol> 7. 대표 결과 : 크기 배제 크로마 토그래피 후 정화 TC TERT의 예제는 그림 2에 표시됩니다. 정제 후 농축 단백질은 12% SDS - polyacrylamide 젤에서 실행되며 순수 이상 99% (그림 2A)로 표시됩니다. 크기는 제외 (S200) 크로마토 그램은 또한 순수와 정화 단백질 샘플에서 집계 부족을 설명하기 (그림 2B) 제공됩니다. 최종 생산량은 달라질 수 있지만 완료된 모든 정화 단계 후 5 MG 단백질의 수율은 보통 얻어진다. 재구성 T.에 대한 대표적인 함정 분석 이전 미첼 외 의해 보고된로 castaneum의 telomerase는 그림 3에 표시됩니다. 12. telomerase 제품의 단계 현명한 사다리는하지만, 레인 1에서 볼 수있다그들은 차선이 각각 3 RNase 취급 telomerase 및 TERT 활성 사이트에 돌연변이 (D251A) 샘플 모두에 결석하고 있습니다. <img src="/files/ftp_upload/2799/2799fig1.jpg" alt="그림 1"/> 그림 1. 재조합 TC TERT의 reconstitution에 관련된 단계 플로우 차트. 첫째, 재조합 TC TERT는 E. 언덕에 표시됩니다 대장균. 단백질은 다음 니켈, 양이온 교환 및 크기 배제 크로마 토그래피에 의해 정화됩니다. T. castaneum 딱정벌레는 집안에 교양하고 그들의 애벌레는 총 RNA를 분리하는 데 사용됩니다. 정화 TC TERT와 총 RNA는 다음 reconstitute telomerase의 RNP 단지를 혼합하고 있으며, 후속 트랩 분석이 RNP 복합을 확인하는 데 사용되는 것은 활성 telomerase입니다. <img src="/files/ftp_upload/2799/2799fig2.jpg" alt="그림 2"/> 그림 2. TC TERT 단백질의 크기 제외 크로마토 그램 및 SDS 페이지 분석. 크기 배제 크로마 토그래피 후 TC TERT 단백질의 (A) 12% SDS - polyacrylamide 젤. 그것이 높은 isoelectric 포인트 (PI - 9.8)를 가지고 있기 때문에 TC TERT 단백질 (70 KDA)는 SDS 페이지 젤에 신속하게 마이 그 레이션합니다. TC TERT의 단백질 (B) 사이즈 제외 크로마토 그램 (Superdex S200). <img src="/files/ftp_upload/2799/2799fig3.jpg" alt="그림 3"/> 그림 3. 체외에의 트랩 assays는 Tribolium castaneum의 telomerase가 미첼 외에서 맞게 재구성. 12. 레인 1 : 야생 타입 TC TERT와 총 RNA는 벌레의 유충으로부터 격리. 레인 2 : 야생 유형 TC TERT와 RNase는 애벌레의 총 RNA와 세포 추출물을 처리. 레인 3 : 활성화된 사이트의 돌연변이 TC TERT (D251A)와 벌레의 유충의 총 애벌레의 RNA.

<ol>
	<li>Gillis, A. J., Schuller, A. P., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Tribolium+castaneum+telomerase+catalytic+subunit+TERT.">Structure of the Tribolium castaneum telomerase catalytic subunit TERT.</a> Nature. 455, 633-637 (2008).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+specific+telomere+terminal+transferase+activity+in+Tetrahymena+extracts.">Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.</a> Cell. 43, 405-413 (1985).</li><li>Harley, C. B., Futcher, A. B., Greider, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+shorten+during+ageing+of+human+fibroblasts.">Telomeres shorten during ageing of human fibroblasts.</a> Nature. 345, 458-460 (1990).</li><li>Hayflick, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Limited+in+vitro+Lifetime+of+Human+Diploid+Cell+Strains.">The Limited in vitro Lifetime of Human Diploid Cell Strains.</a> Exp Cell Res. 37, 614-636 (1965).</li><li>Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres:+no+end+in+sight.">Telomeres: no end in sight.</a> Cell. 77, 621-623 (1994).</li><li>Kim, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+association+of+human+telomerase+activity+with+immortal+cells+and+cancer.">Specific association of human telomerase activity with immortal cells and cancer.</a> Science. 266, 2011-2015 (1994).</li><li>Bodnar, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+life-span+by+introduction+of+telomerase+into+normal+human+cells.">Extension of life-span by introduction of telomerase into normal human cells.</a> Science. 279, 349-352 (1998).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telomere+terminal+transferase+of+Tetrahymena+is+a+ribonucleoprotein+enzyme+with+two+kinds+of+primer+specificity.">The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity.</a> Cell. 51, 887-898 (1987).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+telomeric+sequence+in+the+RNA+of+Tetrahymena+telomerase+required+for+telomere+repeat+synthesis.">A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis.</a> Nature. 337, 331-337 (1989).</li><li>Jacobs, S. A., Podell, E. R., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+essential+N-terminal+domain+of+telomerase+reverse+transcriptase.">Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase.</a> Nat Struct Mol Biol. 13, 218-225 (2006).</li><li>Rouda, S., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+RNA-binding+domain+of+telomerase:+implications+for+RNA+recognition+and+binding.">Structure of the RNA-binding domain of telomerase: implications for RNA recognition and binding.</a> Structure. 15, 1403-1412 (2007).</li><li>Mitchell, M., Gillis, A., Futahashi, M., Fujiwara, H., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+telomerase+catalytic+subunit+TERT+binding+to+RNA+template+and+telomeric+DNA.">Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA.</a> Nat Struct Mol Biol. 17, 513-518 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기서 제시하는 방법은 T.의 촉매 subunit의 대량 생산을 허용 구조적 및 생화 학적 연구에 대한 가용성, 적극적인 형태로 castaneum의 telomerase의 TERT. TC TERT 통해 표현의 방법은 위에서 언급한 이들의 온도 또는 셀 밀도에 미묘한 변화에 민감하고 극적으로 단백질 표현의 수준에 영향을 줄 수 있습니다. 특히, 우리는 광학 밀도가 600 나노미터에서 0.5에 도달하기 전에 단백질 발현을...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 로제타 (DE3) plysS 셀 </td><td> Novagen </td><td> 70,956 </td></tr><tr><td> 2YT 맛을 </td><td> Teknova </td><td> Y0215 </td></tr><tr><td> IPTG </td><td> 골드 생명 공학 </td><td> I2481C </td></tr><tr><td> MISONIX Sonicator 3000 </td><td> Qsonica, LLC. </td><td></td></tr><tr><td> 악타 정수기 FPLC </td><td> GE 생명 과학 </td><td></td></tr><tr><td> NI - NTA의 Superflow 수지 </td><td> Qiagen </td><td> 30,410 </td></tr><tr><td> Amicon 울트라 - 15 원심 필터 장치 </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> 포장 POROS 50 HS 강한 양이온 교환 </td><td> 응용 Biosystems </td><td> 1-3359-06 </td></tr><tr><td> 포장 POROS 50 HQ 강한 양이온 교환 </td><td> 응용 Biosystems </td><td> 1-2559-06 </td></tr><tr><td> Superdex 200 3백분의 10 사이즈 - 제외 칼럼 </td><td> GE 생명 과학 </td><td> 17-5175-01 </td></tr><tr><td> 페놀 : 클로로포름 : 10mM 트리스, 산도 8, 1mM EDTA (에틸렌 다이아 민 테트라 초산)과 Isoamyl 25:24:1 </td><td> 시그마 </td><td> P3803 - 100mL </td></tr><tr><td> RNaseZap </td><td> 앰비온 </td><td> AM9780 </td></tr><tr><td> 재조합 Rnasin의 Ribonuclease 억제제 </td><td> Promega </td><td> N251B </td></tr><tr><td> RNeasy 미니 키트 </td><td> Qiagen </td><td> 74,104 </td></tr><tr><td> 의 DNA oligonucleotides </td><td> 통합 DNA 기술 </td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

telomerase의 촉매 subunit을 분리하기 위해 노력 TERT은 구조적 연구를위한 충분한 수량에 10 년 이상 제한된 성공을 만나왔다. 여기, 우리는 재조합 Tribolium castaneum TERT (TC TERT)의 분리 및 활성 T.의 reconstitution에 대한 방법을 제시 체외에서 castaneum의 telomerase의 ribonucleoprotein (RNP) 복잡한. Telomerase는 엔드 - 투 - 엔드의 융합과 파괴로부터 그들을 보호하기 위해 제공 선형 염색체 2의 3 &#39;끝에 telomeres라는 짧은 DNA의 반복을, 추가 전문 역방향 transcriptase 1입니다. DNA 복제에 따라, 짧은 세그먼트는 염색체 3의 끝에와 telomerase없이 손실, 세포는 결국 자신의 헤이 플릭 한계 4에 도달하기 전까지 나누어 진행합니다. 또한, telomerase는 성인에서 가장 체세포 5 휴면이지만, 그것이 세포 영생에게 7 증진 암세포 6 활성화됩니다. 효소의 촉매 subunit와 템플릿 TERT가 telomeres 8,9를 합성하는 데 사용하는 포함하는 필수적인 RNA 구성 요소 (터)를 구성되어 단백질 subunit (TERT) : 최소한의 telomerase 효소는 두 핵심 구성 요소로 구성되어 있습니다. 이전 2008 개별 telomerase 도메인에 대해서만 구조는 10,11을 해결했다. 이 분야에 주요 돌파구는 활성 12 결정 구조의 결정, T.의 촉매 subunit에서 온 castaneum의 telomerase, TC TERT 1. 여기서는 구조적 및 생화 학적 연구에 대한 적극적인, 용해 TC의 TERT, 그리고 telomerase 활동 assays에 대한 체외에서 telomerase의 RNP 복합 reconstitution의 대량 생산을위한 방법을 제시한다. 사용된 실험 방법의 개요는 그림 1에 표시됩니다.

Watch this Scientific Journal Video about In vitro Reconstitution of the Active T. castaneum Telomerase at JoVE.com

In vitro Reconstitution of the Active T. castaneum Telomerase

telomerase의 촉매 subunit을 분리하기 위해 노력 TERT은 구조적 연구를위한 충분한 수량에 10 년 이상 제한된 성공을 만나왔다. 여기, 우리는 재조합 Tribolium castaneum TERT의 절연 (대한 방법을 제시<em&gt; TC</em&gt; TERT)와 활성의 reconstitution<em&gt; T. castaneum</em&gt; telomerase의 ribonucleoprotein (RNP) 복잡한<em&gt; 체외에서</em&gt;.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

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

in-vitro-reconstitution-of-the-active-t-castaneum-telomerase

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JoVE Journal

Biology

In vitro Reconstitution of the Active T. castaneum Telomerase

11.6K 조회수.  University of Pennsylvania. telomerase의 촉매 subunit을 분리하기 위해 노력 TERT은 구조적 연구를위한 충분한 수량에 10 년 이상 제한된 성공을 만나왔다. 여기, 우리는 재조합 Tribolium castaneum TERT의 절연 (대한 방법을 제시 TC TERT)와 활성의 reconstitution T. castaneum telomerase의 ribonucleoprotein (RNP) 복잡한 체외에서.

비디오: In vitro Reconstitution of the Active T. castaneum Telomerase

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

연구

생물학

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

이온화 방사선 다음 γH2AX의 공간적 분포 평가

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

사용 Schwann - astrocyte 상호 작용 분석 체외에서 Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

텔로미어의 길이와 텔로 머라 제 활성을 세포 노화의 음과 양

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

천연 단백질에서 아밀로이드의 빠른 생성<em&gt; 체외</em

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

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

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

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

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

사전 mRNA의 기판의 3 &#39;끝 분열 : 휴대폰 무료 시스템을 사용하여 RNA 가공 반응 분석<em&gt; 시험관</em

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

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

β-catenin의 저하의 재구성<em&gt; Xenopus의</em&gt; 계란 추출

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

반수체 제브라 피쉬 배아의 생산에 의해<em&gt; 체외</em&gt; 시비

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

식물과 녹조류의 빛 수확 단지의 체외의 재구성에

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.