이 작품은 인간 프론티어 과학 프로그램 보조금 RGP0024에 의해 재정 지원되었다.

1. DNA를 템플릿 준비 및 시험 관내 스크립트 작성에 <ol><li> 관심있는 유전자는 어떤 T7 및 / 또는 예 : 플라스미드 기반 SP6 발기인에 복제됩니다. 관심 RNCs를 얻으려면 폴리펩티드 대상의 C-말단은 SecM FXXXXWIXXXXGIRAGP 7 구속 유도 시퀀스를 추가하여 연장됩니다. 관심 폴리펩티드 조각은 ribosomal 터널 밖으로 압출 성형 것을 보장하기 위해서, 대상 단백질의 C-말단 부분은 12-14 최소한 30 아미노산에 의해 확장되어야한다. 유연한 글리신 - 세린 풍부 링커는 모든 가능한 conformational 제약을 피하기 위해 단백질과 SecM 체포 시퀀스 사이에 도입 할 수 있습니다. </li><li> 시험 관내 전사의 경우, 템플릿 DNA가 ORF의 하류를 절단 제한 효소로 선형되어야합니다. 하나는 아가로 오스 겔 전기 영동에 제한 소화 제품을 실행하여 플라스미드 DNA의 완전한 선형화를 확인해야합니다. </li><li> 선형 혈장id는 추가 시험 관내 전사 반응에 사용됩니다. 템플릿 DNA의 서로 다른 농도는 시험 관내 전사에 필요한 최적의 DNA 농도를 확인하는 테스트를 할 수 있습니다. 일반적으로 Ambion의 MEGAscript 높은 항복 전사 키트 (Ambion / 생활 기술, 그랜드 아일랜드, NY)로, 1 μg 선형 DNA는 40-60 μg의 mRNA를 얻을. 시험 관내 전사에는 제조 업체의 지침 (Ambion / 생활 기술, 그랜드 아일랜드, NY)에 따라 이루어집니다 . </li><li> 시험 관내 전사에 따라 mRNA는 제조 업체의 지침 (Ambion의 MEGAscript 높은 항복 전사 키트, Ambion / 생활 기술, 그랜드 아일랜드, NY)에 따라 염화 리튬의 석출에 의해 정화된다. </li><li> mRNA 무결성이 더욱 아크릴 아미드 또는 아가로 오스의 젤을 사용하여 전기 영동으로 확인됩니다. </li></ol> 2.에서 체외 번역 RTS 100 E. 대장균 HY 키트 (5 프라임, Gaither 이용한 체외 번역에 들어sburg, MD)는 다음 단계를 따르십시오 : <ol><li> 의 체외 번역 반응이 다음과 같은 제조 업체의 지침을 100 μl를 준비합니다. 간단히, nuclease 무료로 물속에 24 μl 아미노산 혼합물 마이너스 메티오닌 (1 ㎜), Ribonuclease 억제제의 10 단위 (Invitrogen / 생활 기술, 그랜드 아일랜드, 뉴욕), 방사성의 20 μCi [35 S] (-메티오닌 MP의 Biomedicals를 추가 솔론, OH), 20 μl 반응 믹스, 24 μl Reconstitution 버퍼와 E의 24 μl 대장균은 Lysate. </li><li> 따뜻한 번역 반응을 미리하는 5 분 동안 30 ° C에서 반응을 품어. 10-15 분 30 ° C에서 번역 반응과 부화 mRNA 1-2 μg을 추가합니다. </li><li> 얼음 번역 반응을 배치하여 반응을 중지합니다. </li></ol> 3. 체외 번역 반응의 초기 폴리펩티드의 분리 <ol><li> RNCs를 분리하려면 번역 반응 20 MM HEPES-코이의 산도 7 5-30%의 자당 기울기 4.5 ML 위에 뛰어넘는 세월을 거슬러 올라가있다0.5, 15 밀리미터 MgCl 2, 100 MM 칼륨 아세테이트, 1 밀리미터 DTT와, 41,000 rpm으로 4 Beckman 보습 바로 앞에 달린 풀베는 날 SW55-티 로터를 사용 ° C를 2 시간 동안 centrifuged. </li><li> 원심 분리 후, 자당 기울기 다른 ribosomal 인구 및 관련 초기 체인을 분리 fractionated입니다. 분리는 ISCO UA-6 흡광 검출기를 사용하여 254 nm의에서 연속 녹화로 ISCO 프로그램 밀도 구배 시스템을 사용하여 모니터링할 수 있습니다. </li><li> 70 년대의 변이를 포함하는 분획 (들) 수집 및 추가 실험 목적에 따라 분석하고 있습니다. </li></ol> 4. 단백질의 관찰 트리스 - tricine SDS 페이지와 Ribosome뿐 이잖아 SecM 확장 단백질은 ribosome 70 년대에 붙어 남아 있는지, 확인하려면 그라데이션 분수를 모으고 다음과 같이 취급되었다 : <ol><li> 각 분획의 단백질은 10 %의 최종 농도로 Trichloroacetic 산 (TCA)를 추가하여 시켰던 4 ° C overni에서 incubated되었다ght. </li><li> 하룻밤 배양 후 시료 펠렛 ~ 15 분 단백질을 위해 14,000 X g에서 centrifuged되었다. 다음 원심 분리의 표면에 뜨는이 제거하고 펠릿는 솔루션이 포함된 아세톤으로 두번 씻었습니다 : 1 ㎜ 트리스-HCL의 산도 7.6 (4시 1분). 펠렛은 더 이상 공기가 건조하고 SDS-PAGE 로딩 버퍼에 해산했습니다. </li><li> 처리 시료가 해결 및 트리스 - tricine SDS-PAGE에 의해 분석되었다. </li><li> 전기 영동 후, 젤류가 수정되었습니다, 진공 젤 다이어를 사용하고 autoradiography를 받게 건조. 어렸을때 polypeptides의 분포는 phosphoimaging을 사용하여 관찰되었다. </li></ol> 5. 대표 결과 여기에서 우리는 안정적으로 ribosome 70에 바인딩 장편 소 감마-B Crystallin의 고립을 설명하는 실험을 제시한다. 그림 1은 소 감마-B Crystallin의 RNCs의 격리에 관련된 단계를 묘사. 감마-B Crystallin의 C-말단는 32 아미노산의 t으로 전체적인 확대되었다ribosomal 터널의 전체 길이 단백질 extrudes 아웃을 보장 해줬, 이것은 또한 폴리펩티드 융합의 C-말단 매우 배치 SecM 끄는 순서를 포함합니다. 체외 번역에 이어 70 년대의 리보솜은 자당의 기울기 원심 분리 (그림 2.1)에 의해 분리되었다. 단백질이 안정적으로 ribosome에 바인딩 상태를 유지하기 위해, 분수를 포함하는 70가 풀링되었다 desalted, 버퍼 교환 및 자당의 기울기 원심 분리의 추가 라운드 (그림 2.2)에 노출. 그림 2에 제시된 결과는 명확하게 그 SecM 효율적으로 감마-B Crystallin의 RNCs의 translational 체포를 유도 수 있습니다 제안합니다. <img alt="그림 1" src="/files/ftp_upload/4027/4027fig1.jpg" /> 1 그림. RNCs의 분리에 관여 단계 도식 설명. 소 감마-B Crystallin의 C-말단는 32 아미노산 서열에 의해 확대되었다. 이 확장C-말단 영역은 또한 SecM 체포 시퀀스를 포함합니다. SecM 순서와 감마-B Crystallin은 pIVEX 2.3 (T7 기준) 플라스미드에 복제되었습니다. 시험 관내 전사의 내용은 템플릿 DNA가 XbaI으로 선형되었다. 선형화된 템플릿은 추가 시험 관내 전사에 사용되었습니다. DNA를 템플릿에서 T7은 T7 프로 모터의 감마-B Crystallin 유전자 위치 하류를 transcribes T7 RNA 중합 효소에 의해 인정되고 있습니다. mRNA는 염화 리튬의 석출 방법을 사용하여 정화되었다. 정화 mRNA 그러면 체외 변환에 사용되었다. 다음과 부화는 번역 반응은 5-30%의 자당 기울기 위에 뛰어넘는 세월을 거슬러 올라가, 2 시간 동안 41,000 RPM, 4 ° C에서 Beckman 보습 바로 앞에 달린 풀베는 날 SW55-티 로터에 centrifuged되었다. <sp클래스 = &quot;pdflinebreak&quot;&gt; <img alt="그림 2" src="/files/ftp_upload/4027/4027fig2.jpg" /> 그림 2. 소 감마-B Crystallin의 분리가 안정적으로 70 년대의 ribosome에 바인딩 RNCs. 1 μg의 mRNA는 100 μl 반응 E.에 혼합되었다 대장균 S30 추출 시스템 20 μCi [35 S]와 프로토콜 절에서 설명한대로-메티오닌과 15 분 동안 30 ° C에서 incubated. 다음과 부화는 번역 반응 20 MM HEPES-코이 산도 7.5, 15 밀리미터 MgCl 2, 100 MM 칼륨 아세테이트, 1 ㎜ DTT에 5-30%의 자당 기울기 위에 뛰어넘는 세월을 거슬러 올라가와 41,000에서 Beckman 보습 바로 앞에 달린 풀베는 날 SW55-티 로터에 centrifuged되었다 RPM, 2 시간 4 ° C에서. 속도 침강에 따라 그라데이션이 언로 드되었으며 ribosome 프로 르가 얻은 것입니다. 데이터 PeakTrak 프로그램 (ISCO 구배 밀도 기울기 분획화 시스템)에 의해 기록되었다. 분수가 수집되었다 단백질은 TCA는 시켰던와 해결 16.5 %의 T에 6퍼센트 C 트리스-T였다ricine 페이지 겔 15. 겔은 건조하고 autoradiography에 대해 접했습니다. 아래 노출이 젤은 태풍 9,410 이미징 스캐너를 사용하여 스캔했습니다. 그림 2.1 전체 길이 감마-B Crystallin은 70 ribosome 분수에 있는지 보여줍니다. 따라서 SecM 끄는 순서는 안정 소 감마-B Crystallin의 RNCs의 절연을 허용합니다. 그림 2.2에서 데이터가 명확하게 고립된 RNCs 실제로 안정을 나타냅니다. 현재 실험에서 자당의 기울기 원심 분리의 첫 라운드 이후 70 년대의 분수는 풀링된으며 자당은 HEPES-코이 산도 7.5, 15 MM 20 밀리미터를 포함하는 용액에 버퍼 교환 뒤에 Amicon 울트라-4 원심 필터 유닛 (Millipore)을 사용하여 삭제되었습니다 MgCl 2, 100 밀리미터 칼륨 아세테이트, 1 밀리미터 DTT. 이 샘플은 더욱 5-30%의 자당 기울기와 fractionated 통해 원심 분리의 추가 라운드를 받게되었다. 각 분획은 취급 그림 2.1에서처럼 분석되었다.

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+a+peptide+tagging+system+in+degradation+of+proteins+synthesized+from+damaged+messenger+RNA.">Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA.</a> Science. 271, 990-993 (1996).</li><li>Evans, M. S., Ugrinov, K. G., Frese, M. A., Clark, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+stalled+ribosome+nascent+chain+complexes+produced+in+vivo+or+in+vitro.">Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro.</a> Nat. Methods. 2, 757-762 (2005).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

시험 관내 전사와 번역에 사용된 부품의 재현성 결과, 품질과 집중이 중요하십시오. 우리는 시험 관내 전사 및 번역 추출물에서 시판 사용하고 신중하게 처리한다면 그들은 효과적이고 재현성 결과를 제공합니다. mRNA의 품질이 번역에 영향을 미칠 수 있으므로, 그것은 체외의 번역을 위해 사용하기 전에 mRNA의 무결성을 테스트하기 위해 가장 중요합니다. 체외...

<table border="1"><tbody><tr><td> 시약 / 키트의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> MEGAscript T7 높은 항복 전사 키트 </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> RTS 100 E.coli HY 키트 </td><td> 5 프라임 </td><td> 2,401,100 </td></tr><tr><td> Ribonuclease 억제제 </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> 트랜스 [35 S]-라벨 </td><td> MP의 Biomedicals </td><td> 0,151,006 </td></tr><tr><td> Amicon 울트라-4 원심 필터 유닛 </td><td> Millipore </td><td> UFC801008 </td></tr><tr><td> 저장 형광체 autoradiography </td><td> GE 헬스케어 </td><td> 태풍 9,410 가변 모드 영상기 </td></tr><tr><td> 밀도 그라데이션 분획화 시스템 </td><td> Teledyne Isco 주식 회사 </td><td> ISCO 프로그램 밀도 기울기 시스템 </td></tr><tr><td> 자당 그라데이션 Centrifugation </td><td> Beckman 보습 바로 앞에 달린 풀베는 날 </td><td> Optima L-90 K 예비의 Ultracentrifuge </td></tr></tbody></table>

using secm arrest sequence as a tool to isolate ribosome bound polypeptides

광범위한 연구는 세포에 개고 단백질이 공동 translational 절차 1-5는 것을 제안 충분한 증거를 제공했습니다. 다만, 폴리펩티드 사슬는 기능적인 양식을 달성하기위한 공동 translational 접는 동안 다음과 정확한 경로는 여전히 수수께끼입니다. 이 과정을 이해하고 공동 translational 접힘 중간체의 정확한 형태를 결정하기 위해서는, 그들의 자세한 구조 분석을 허용하도록 미리 정해진 크기의 초기 체인을 들고 RNCs의 절연을 허용 기법을 개발하는 것이 필수적입니다. SecM (분비 모니터)는 170 아미노산 E.입니다 secM-secA 오페론 6 하류 SecA (분비 운전) ATPase의 표현을 규제 대장균 단백질. Nakatogawa와 이토 원래 SecM 단백질의 C-말단 지역에서 17 아미노산 긴 시퀀스 (150-FSTPVWISQA QGIRA G P-166)에 충분하고 필요한 것을 발견따라서 peptidyl-glycyl-tRNA가 안정적으로 7-9 P 사이트 ribosomal에 바인딩 생산, Gly165에서 SecM 신장의 연기자 거든. 더 중요한 건, 그것이 17 아미노산 긴 시퀀스 따라서 미리 결정된 사이즈 7의 초기 체인을 들고 RNCs의 생산을 허용하는 거의 모든 전신 및 / 또는 잘린 단백질의 C-말단에 융합 수있는 사실을 발견했습니다. 따라서 대상 단백질에 융합하거나 삽입할 때 SecM 끄는 순서는 폴리펩티드 사슬의 신장의 체포를 생산하고 E.에서 생체내에 모두 안정적인 RNCs를 생성 대장균 세포와 세포없는 시스템에서 체외 인치 자당의 기울기 원심 분리는 더욱 RNCs을 분리하기 위해 사용된다. 격리된 RNCs는 공동 translational 접힘 중간체의 구조 및 기능적 특징을 분석하는 데 사용할 수 있습니다. 최근에는이 기술이 성공적으로 여러 개의 ribosome 바운드 초기 체인 10,11의 구조에 대한 통찰력을 얻기 위해 사용되었습니다. 다음은 소 감마-B Crystallin의 고립을 설명하는 것은 SecM에 융합 및 체외 번역 시스템에서 발생 RNCs.

Watch this Scientific Journal Video about Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides at JoVE.com

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

우리는 지금 여기 정기적으로 안정적으로 바운드 ribosome 초기 체인 단지 (RNCs)를 분리하는 데 사용되는 기술을 설명합니다. 이 기술은 17 아미노산 긴 SecM &quot;체포 시퀀스는&quot;prokaryotic에서 번역 신장을 중지할 수있는 발견의 활용 (<em&gt; E. 대장균</em&gt; 거의 모든 단백질 (또는 C-말단에 융합)에 삽입) 시스템.

Ribosome 바운드 Polypeptides을 파악하는 도구로 SecM 체포 시퀀스를 사용하여

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

using-secm-arrest-sequence-as-tool-to-isolate-ribosome-bound

Research

JoVE Journal

Biology

12.3K 조회수.  Cleveland State University. 우리는 지금 여기 정기적으로 안정적으로 바운드 ribosome 초기 체인 단지 (RNCs)를 분리하는 데 사용되는 기술을 설명합니다. 이 기술은 17 아미노산 긴 SecM "체포 시퀀스는"prokaryotic에서 번역 신장을 중지할 수있는 발견의 활용 ( E. 대장균 거의 모든 단백질 (또는 C-말단에 융합)에 삽입) 시스템.

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

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Microfabricated 포스트 - 배열 감지기 (mPADs) : 기계적 힘을 분리하는 접근

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

연구

생물학

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

도구로 상호 빛과 전자 현미경 (클레멘 타인)는 Microinjected 분자와 진핵 세포 하위 세포 목표를 시각화하는 방법


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

호스트 식물 휘발성 물질에 대한 선별 도구로 Electroantennographic Bioassay

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

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

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

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

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

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

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus의 번역 손상을 식별 할 수있는 모델로 laevis의

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

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

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

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

포유 동물 세포에서 초기 DNA에 바인딩 단백질의 시험 BrdU의 칩 슬롯 서양 기술을 사용하여

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

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

개별 세포 유형의 전사 프로파일 링을위한 도구로 레이저를 이용한 이외에 Microdissection (LAM)

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

마그네틱 활성화 전략을 분리 하 고 정화 활 액 액체 파생 된 중간 엽 줄기 세포는 토끼 모델에서 정렬 셀

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

마우스에서 대뇌 골후혈구를 분리하는 고출력 방법

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...