Name: a cell free assay to study chromatin decondensation at the end of mitosis
Uploaded: 2015-12-19T13:00:00
Description: Watch this Scientific Journal Video about A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis at JoVE.com

AKS 1 &amp; 2가 발달 셀 (31) (3), Magalska에서 다시 인쇄되어 그림에이 작품은 독일 연구 재단과 ERC (WA에 AN377 / 3-2 309528이 CHROMDECON)와 베링거 인 겔 하임 퐁의 박사 원정대에 의해 지원되었다 등의 등. 유사 분열, 305-318, 2014 년 말에 염색질 decondensation에서, RuvB 같은 -ATPase를 기능, 엘스 비어의 종류 권한.

HeLa 세포에서 유사 분열 염색질 클러스터 격리 1. 준비 <ol><li> 세포 배양 솔루션 <ol><li> DMEM에 10 % 소 태아 혈청, 100 유닛 / ㎖ 페니실린, 100 μg의 / ㎖ 스트렙토 마이신 및 2 mM의 글루타민을 추가하여 완전한 둘 베코 변성 이글 배지 (DMEM)를 준비한다. 2.7 밀리미터의 KCl, 137 mM의 염화나트륨, 10 mM의 나 2 2H 2 O HPO 4, 10 N NaOH로 7.4 2 mM의 KH...

<ol>
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제노 계란 추출물 충실히 시험 관내 세포 과정을 재현하는 매우 유용한 도구이다 laevis의,이 시스템은 성공적으로 세포주기 및 세포 분열 이벤트 2, 3,5,6,17의 특성화에 사용 하였다. 때문에 oogenesis 동안 계란에서 압수 핵 구성 요소의 대형 상점, 계란 추출물은 세포 성분의 훌륭한 소스입니다. 포유 동물 조직 세포주 또는 유전자 조작에 RNAi를 같은 다른 방법에 ?...

Xenopus의 laevis의 달걀 추출물은 무 세포 분석의 단순 복잡한 세포 이벤트를 연구하는 강력하고 광범위하게 적용되는 도구입니다. Lohka &amp; Masui 1에 의해 처음으로 설명하기 때문에 그들은 광범위하게 같은 염색질 응축 2, 스핀들 조립 3, 핵 봉투 고장 4뿐만 아니라 nucleocytoplasmic 전송 5 DNA 복제 (6) 등의 유사 분열 과정을 연구하는 데 사용되었다. 핵 봉투 개혁과 핵 기공 복잡한 재 조립 등의 interphasic 핵의 개혁에 필요한 유사 분열의 끝에서 발생하는 이벤트는, 초기 유사 분열 이벤트에 비해 이해 훨씬 덜하지만 유사 Xenopus의 계란 추출물 (7)를 사용하여 공부하실 수 있습니다. 우리는 최근에 유사 분열 (8)의 끝에서 염색질 decondensation을 연구하기 위해 Xenopus의 계란 추출물을 기반으로 분석을 설립, 언더 조사 과정은 내가 기다리고 그자세한 특성의 TS. 후생 동물에서 염색질은 높은 유전 물질의 분리를 충실히 수행하기 위해 유사 분열 진입 집광된다. 염색질은 간기 동안 유전자 발현과 DNA 복제를 위해 액세스 할 수 있는지 확인하기 위하여, 유사 분열의 끝에서 압축 해제 될 필요가있다. 척추 동물에서, 염색질은 최대 유사 분열 다짐 보통 훨씬 ​​낮은 효모 달리, S. 예에서, 단지 두 배 계면 (9)에 비해 유사 분열 동안 더 괴성 오십 배이며 cerevisiae의 10. 척추 염색질 decondensation은 대부분 계란 수정 후 정자의 DNA 구조 조정의 맥락에서 연구되어왔다. 분자 메커니즘은 어떤 nucleoplasmin, 풍부한 난자 단백질, 계란에 저장 히스톤의 H2A와 H2B에 정자 별 프로타민을 교환한다. 이 과정은 또한 Xenopus의 계란 추출물 11, 12를 이용하여 규명 하였다. 그러나, 발현nucleoplasmin의이 정자 별 프로타민을 포함하지 않는 난 모세포 (13)과 유사 분열 염색질로 제한됩니다. 따라서 유사 분열의 끝에서 decondensation을 염색질 8 개의 독립적 인 nucleoplasmin입니다. 체외 decondensation 반응을 위해 우리는 동기화 된 HeLa 세포에서 분리 된 활성화 된 X를 laevis의 달걀과 염색질 클러스터에서 발생 추출물을 사용합니다. 칼슘 이온 운반체와 계란의 치료는 수정시 정자의 항목에 의해 생성 된 난자에 칼슘 방출을 모방. 칼슘 웨이브 제 상간 14로 이행, 감수 분열의 중기 제 체포 세포주기 재개와 달걀을 트리거한다. 따라서, 양식 활성화 계란 준비 계란 추출물은 유사 분열 출구 / 계면 상태를 나타내고 염색질 decondensation, 핵 봉투와 같은 유사 분열 종료에 대한 특정 이벤트를 유도 할 능력이 복잡한 개혁 기공. 유사 분열 채널의 분리를위한클러스터 romatin 우리는 염색체 클러스터가 헬라 세포 유사 분열에 동기화 및 그라데이션 회 원심에 의해 폴리아민 포함하는 버퍼에 격리에서 용해에 의해 방출된다 가세 &amp; 램 믈리 15 일 발표 한 프로토콜의 약간 수정 된 버전을 사용했다.

<table>
	<tbody>
		<tr>
			<td>spermine tetrahydrochloride</td>
			<td>Fluka analytical</td>
			<td>85610-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>spermidine trihydrochloride</td>
			<td>Sigma&#160;</td>
			<td>S2501-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>high-purity digitonin</td>
			<td>Millipore</td>
			<td>300410-1GM</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Applichem</td>
			<td>A0999,0100</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>thymidine</td>
			<td>Calbiochem</td>
			<td>6060</td>
			<td></td>
		</tr>
		<tr>
			<td>nocodazole</td>
			<td>Calbiochem</td>
			<td>487928</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>37 % formaldehyde solution</td>
			<td>Roth</td>
			<td>7398-1</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>trypan blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>1,4-dithiothreitol (DTT)</td>
			<td>Roth</td>
			<td>6908.2</td>
			<td></td>
		</tr>
		<tr>
			<td>AEBSF hydrochloride</td>
			<td>Applichem</td>
			<td>A1421,0001</td>
			<td></td>
		</tr>
		<tr>
			<td>pepstatin</td>
			<td>Roth</td>
			<td>2936.1/2/3</td>
			<td></td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Roth</td>
			<td>CN334</td>
			<td></td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Roth</td>
			<td>A162.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (colloidal silica particles solution)</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm&#178; tissue culture flasks</td>
			<td>Greiner Bio-one</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>heat-inactivated fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer (40 mL tissue grinder)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Assistent</td>
			<td>441/1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Oak Ridge Centrifuge Tubes, polycarbonate (50 ml)</td>
			<td>Nalgene</td>
			<td>3118-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell strainer, nylon</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>cytochalasin B</td>
			<td>Applichem</td>
			<td>A7657,0010</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Roth</td>
			<td>8682.3</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>L-cystein</td>
			<td>Merck</td>
			<td>1,028,381,000</td>
			<td></td>
		</tr>
		<tr>
			<td>hCG available as Ovogest</td>
			<td>MSD</td>
			<td>1431593</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG available as Intergonan</td>
			<td>MSD</td>
			<td>1431015</td>
			<td></td>
		</tr>
		<tr>
			<td>A23187 (mixed calcium-magnesium-salt)</td>
			<td>Enzo</td>
			<td>ALX-450-002-M010</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>syringe needles (1.20 x 40 mm, 18 G x 1 1/2&#34;)</td>
			<td>Braun</td>
			<td>4665120</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Serva</td>
			<td>10920.03</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP, 2 Na x 3 H20</td>
			<td>Roth</td>
			<td>K056.1/2/3/4</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine phosphat disodium salt</td>
			<td>Calbiochem</td>
			<td>2380</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine phosphokinase</td>
			<td>Sigma</td>
			<td>C3755-35KU</td>
			<td></td>
		</tr>
		<tr>
			<td>DMAP</td>
			<td>Sigma</td>
			<td>D2629-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI&#160;</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma</td>
			<td>P-6148</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>centrifugation tubes for TLA 100 (7 x 10 mm, 5/16 x 13/16 in.)</td>
			<td>Beckman Coulter</td>
			<td>343775</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Cell-Saver&#34; (tips with wide opening, 1000 &#181;L)</td>
			<td>Biozym</td>
			<td>729065</td>
			<td></td>
		</tr>
		<tr>
			<td>50 % glutaraldehyde solution, grade I</td>
			<td>Sigma alderich</td>
			<td>G7651-10 mL</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>0.1 % (w/v) poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>flat-bottom tubes (6 mL, 16.0/55 mm)</td>
			<td>Greiner Bio-one</td>
			<td>145211</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>tubes for TLA120 (11 x 34 mm, 7/16 x 1 3/8 in.)</td>
			<td>Beckman Coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Cell-Saver&#34; (tips with wide opening, 200 &#181;L)</td>
			<td>Biozym</td>
			<td>729055</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm coverslips</td>
			<td>Thermo Scientific</td>
			<td>0784 #1</td>
			<td></td>
		</tr>
	</tbody>
</table>

a cell free assay to study chromatin decondensation at the end of mitosis

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

decondensation 반응의 시간 의존성 그림 1은 decondensation 분석의 전형적인 시간 과정을 보여줍니다. 반응 개시 가시 염색체 클러스터 decondenses 번의 둥글고 부드러운 핵 내로 병합. 계란 추출물 자당으로 대체 될 때 염색체 클러스터 decondensation 활동 계란 추출물에 존재 함을 시사하는 축합 남아 버퍼. 염색질 decondensation는 에너지 의존하는 과정이...

Watch this Scientific Journal Video about A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis at JoVE.com

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

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

셀 무료 분석은 유사 분열의 끝에서 염색질 Decondensation을 공부하기

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

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

A Chromatin Assay for Human Brain Tissue

인간 두뇌 조직에 대한 염색질 어세이

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Operant Learning of Drosophila at the Torque Meter

토크 미터에서 Drosophila의 Operant 학습

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

휴대 전화의 Macromolecular 교통 분석 Planta에 Biolistic 포격을 활용

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

염색질 상호 작용을 매핑 및 전사 규정을 이해하는 데 이점 엔드 태그 장면 (ChIA-PET)과 염색질의 상호 작용 분석

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

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

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

In vitro Methylation Assay to Study Protein Arginine Methylation

In vitro Methylation Assay to Study Protein Arginine Methylation

체외 메틸화 분석에서 단백질 아르기닌 메틸화를 공부합니다

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

정량 면역 형광 분석은 중심체에서 단백질 수준의 변화를 측정하기 위해

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

애기 장대 단백질 DNA 상호 작용의 식별을위한 염색질 면역 침전 분석<em&gt; 생체</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...