Name: isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders
Uploaded: 2016-12-05T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders at JoVE.com

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01 GM33397. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. J.G.G. is American Cancer Society Professor of Developmental Genetics.

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The authors have nothing to disclose.

개구리와 도롱뇽의 손으로 고립 GVS에서 &quot;생활&quot;LBCs의 첫 번째 관찰은 형광 면역 염색 전에 FISH 전에 미국의 생물 학자 윌리엄 Duryee, 위상차 및 DIC 현미경의 도입 전에 (19)에 의해 거의 80 년 전에 만들어졌다. 실제와 방식으로이를 해결하기 위해, 그들의 기본 형태를 파괴하지 않고 분자 기술을 적용, 유리 슬라이드에 LBCs를 연결하는 기술의 개별 유전자 레벨에 필요한 개발에서 염색?...

대부분의 척추 동물은, 유대류와 태반 포유류의 주목할만한 예외로 큰 노른자위의 계란을 생산하고 있습니다. 그들의 때로는 거대한 크기에도 불구하고,이 계란은 여성의 난소에있는 동안 아직 최종 차원에 도달 한 세포이다. 난소 계란 난자라고 각각은 일반적으로 단순히 새싹 소포 또는 GV으로 초기 19 세기부터 알려진 하나의 거대한 핵을 포함하고 있습니다. 일반 실험실 개구리, Xenopus의의 laevis의 및 Xenopus의의 tropicalis의 1 난 모세포는 1.2 mm 및 0.8 mm 각각 (그림 1)의 최대 직경에 도달한다. 직경 0.4 mm (그림 2, 3) -이 두 개구리의 성숙한 난자에서 GVS는 0.3이다. 도롱뇽은 일반적으로 더 큰 난자와 GVS 있습니다. 멕시코 아 홀로 틀, Ambystoma mexicanum의 완전히 성숙 난자는 직경이 2mm 이상이며, GV는 약 0.5 mm이다. 따라서, 이러한 핵 육안 및 캔을 쉽게 볼 수 있습니다전형적인 체세포 핵으로 불가능한 다양한 방법으로 조작 될 수있다. 마찬가지로 현저한은 GV, 19 세기 말에 이미 인식 사실 내 염색체의 거대한 크기이다. Ambystoma 다른 도롱뇽의 개별 염색체의 길이는 최대 1mm (그림 4, 5)이 될 수 있습니다. 100 μm의 이상 최대 길이로, 그들은 대부분의 생물의 일반적인 체세포 염색체를 난쟁이하지만 Xenopus의의 사람들은 작은 상당한 있습니다. 한 쌍의 측면 루프 백 (그림 5) - 난자의 염색체의 중요한 특징은 자신의 가장 특징적인 형태 학적 특징 중 하나에 이르게 자신의 특별한 전사 활동이다. 각 루프는 적극적으로 RNA를 합성 하나 또는 몇 전사 단위로 구성되어 있습니다. 루프는 난자가 피상적 후 이름이 &quot;lampbrush&quot;염색체에지도 퍼지 모양을 염색체 제공등유 램프 굴뚝을 청소하기 이전 시대에 사용 된 브러쉬에 유사. 이 이 논문의 초점은 LBCs 핵 세포 소기관 (핵소체, 히스톤 궤적 기관 및 얼룩을) 연구 고립 GVS의 사용에 있습니다. 두 아니라 다른 기술을 설명한다. 첫번째, 일반적인 기술에서, GVS 간단히 부착 난황을 제거하는 세정 보석의 집게를 이용하여 식염수에서 격리하고, 핵막은 보석의 집게 다시 제거된다. LBCs 핵 세포 소기관을 포함하는 젤라틴 내용은 유리 현미경 슬라이드 나 커버 슬립 상에 침강한다. 이러한 제제는 위상차 또는 DIC 현미경으로 직접 관찰 할 수있다. 또한, 준비는 슬라이드 또는 커버 슬립에 LBCs와 세포 기관을 연결하는 원심 분리 될 수있다. 이러한 준비 후, 주로 면역 및 fluorescen하여 핵산 및 단백질 분자의 상세한 분석을 위해 처리 될 수있다현장 하이브리드 (FISH)에서 t. 3-7 두 번째 기술은 광유에서 GV의 분리를 포함한다. 8 오일 절연 GVS는 많은 시간 동안 전사적으로 활성 상태로 유지 한 핵 내용이 가능한 한 실물되고 싶어 연구를위한 잠재적으로 유용하다. 핵 &quot;SAP&quot;의 굴절률이 LBCs 및 기타 핵 세포 소기관 (그림 3)에 가까운이기 때문에 9, 10는, 현미경 기술은 석유 고립 GVS와 도전이 될 수 있습니다. 마지막으로, 그들의 크기와 조작의 용이성, GVS는 핵 봉투에 대한 연구에 이상적인 재료입니다. 핵 기공 단지는 최초의 수륙 양용 GV 봉투 (11)과 최근 수퍼 해상력의 관찰 전자 현미경 연구에서 설명 된 것과 동일한 재료를 사용하고 있습니다. 12, 13

<table><tbody><tr><td>Paraformaldehyde (reagent grade, crystalline)</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>Despite warnings in many protocols, a concentrated solution can be stored indefinitely at RT</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma-Aldrich</td><td>A5040-100G</td><td>Sometimes referred to as MS-222</td></tr><tr><td>Ethicon RB-1 1/2 circle taper point 3-0 sutures</td><td>VWR&amp;nbsp;</td><td>95057-000</td><td></td></tr><tr><td>Paraplast (paraffin wax)</td><td>Sigma-Aldrich</td><td>P3558-1KG</td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Phenylenediamine</td><td>Sigma-Aldrich</td><td>P6001</td><td></td></tr><tr><td>Gelatin</td><td>Grocery Store</td><td></td><td>Commercial Knox gelatin works fine</td></tr><tr><td>ProLong Gold antifade mountant</td><td>ThermoFisher Scientific</td><td>P10144</td><td></td></tr><tr><td>Gold Seal cover glass&amp;nbsp; 22 x 22 mm #1 1/2 (0.16 - 0.19 mm thick)</td><td>Electron Microscopy Sciences</td><td>63786-01</td><td>These coverslips are the recommended thickness for superresolution microscopy</td></tr><tr><td>Dumont forceps #5</td><td>Electron Microscopy Sciences</td><td>72700-D</td><td>http://www.emsdiasum.com/microscopy/products/tweezers/dumont_positive_action.aspx</td></tr><tr><td>Paraffin oil (light)&amp;nbsp;</td><td>EMD Chemicals</td><td>PX0047-1</td><td>For isolating GVs in oil</td></tr><tr><td>Adhesive &lt;em&gt;in situ&lt;/em&gt; PCR and hybridization chambers (25 &amp;micro;L)</td><td>BioRad</td><td>Frame-Seal Slide Chambers #SLF0201</td><td>http://www.bio-rad.com/en-us/sku/slf0201-frame-seal-slide-chambers</td></tr><tr><td>Silicone isolators</td><td>Grace-Biolabs</td><td>select from catalog link</td><td>http://www.gracebio.com/life-science-products/microfluidics/silicone-isolators.html</td></tr><tr><td>Coplin jars and staining dishes</td><td>Electron Microscopy Sciences</td><td>select from catalog link</td><td>http://www.emsdiasum.com/microscopy/products/histology/staining.aspx</td></tr></tbody></table>

isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders

우리는 개구리와 도롱뇽의 난자 또는 unlaid 계란에서 발견 된 거대한 전사적으로 활성 lampbrush 염색체 (LBCs)를 공부하는 방법을 설명합니다. 개인 LBCs의 최대 길이는 1mm 될 수 있으며 직경 0.5 mm로, 거대한 핵 자체를 상주합니다. 염색체의 크기가 커서 빛의 광학 현미경으로 활성 유전자 비할 관측을 허용하지만, 동시에 특별한 기술은 핵을 단리 핵막을 제거하고, 현미경 슬라이드 상 염색체 확산 필요하다. 또한 새싹 소포 (GV)라는 난자의 핵은 핵 굴지의 부분 겔을 허용하고 LBCs의 섬세한 구조를 보존하는 매체에 격리됩니다. 이 단계는 보석의 겸자를 사용하여 해부 현미경을 수동으로 수행된다. 다음으로, 핵 봉투는 보석의 집게 다시 수동으로 제거됩니다. 핵 내용은 신속하게 분산 외장 물감 매체로 전송굴지 젤 노변 장치 및 손상되지 않은 LBCs는 현미경 슬라이드에 정착 할 수 있습니다. 미세한 세부 사항은 브라운 운동에 의해 가려하고 있지만이 시점에서 LBCs 및 기타 핵 세포 소기관은, 위상차 또는 미분 간섭 대비 현미경으로 볼 수 있습니다. 고해상도 현미경 관찰 또는 분자 분석을 위해, 전체 제조는 슬라이드에 단단히 섬세한 LBCs를 연결하는 원심 분리된다. 파라 포름 알데히드에 대한 간단한 고정 후 면역 염색으로 또는 현장 하이브리드에 따른다. LBCs는 전사적으로 활성 상태에있는 자신의 거대한 크기는 촛점 또는 초해 현미경을 사용하여 개별 유전자 수준의 분자량 분석을 허용한다.

거대한 lampbrush 염색체를 검사하려면 하나는 개구리 나 도롱뇽에서 난 모세포를 분리하는 것으로 시작한다. 도 1은 개구리 제노의 난소 제거 후 완충 식염수 성숙한 난자들의 그룹을 나타낸다. 이러한 난자는 실온에서 일하기 좋은 상태로 남아 있습니다. 핵 (또는 새싹 소포는) 다음 중 하나 식염수 (그림 2) 또는 기름에, 보석상의 집게와 난?...

Watch this Scientific Journal Video about Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders at JoVE.com

Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

우리는 개구리와 도롱뇽의 난자 생활에서 거대한 전사적으로 활성 lampbrush 염색체 분리에 대한 간단한 기술을 제시한다. 우리는 위상차 또는 미분 간섭 대비하는 방법과 현장 하이브리드 또는 면역 염색에서 형광을 위해 그들을 해결하는 방법에 의해 &quot;살아&quot;이러한 염색체를 관찰하는 방법에 대해 설명합니다.

개구리와 도롱뇽의 생활 난 모세포에서 거대한 Lampbrush 염색체의 분리

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and ...

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DNA and Chromosome Structure

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12.9K 조회수.  Carnegie Institution for Science.  우리는 개구리와 도롱뇽의 난자 생활에서 거대한 전사적으로 활성 lampbrush 염색체 분리에 대한 간단한 기술을 제시한다. 우리는 위상차 또는 미분 간섭 대비하는 방법과 현장 하이브리드 또는 면역 염색에서 형광을 위해 그들을 해결하는 방법에 의해 "살아"이러한 염색체를 관찰하는 방법에 대해 설명합니다. 

비디오: Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

조작 및<em&gt; 체외</em의&gt; 성숙<em&gt; 아프리카 발톱 개구리 laevis의</em세포질 내 정자 주입에 이어&gt; 난 모세포, 배아 발달을 연구하는

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

연구

발생학

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Microemulsions 셀 무료 Xenopus 달걀 추출 물에서 세포 주기 진동의 재구성

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

생화학

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

동시 사전 및 사후 신경 Electrophysiological 녹화에서<em&gt; Xenopus</em&gt; 신경 근육 공동 문화

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

신경과학

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

의 수정 Xenopus의 oocytes 호스트 전송 방법을 사용하여

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

생물학

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

라이브 셀 이미징 및 배아 상피 세포의 정량 분석 Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgf&#223; family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgf&#223; receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgf&#223; precursor proteins. RNA encoding Tgf&#223; precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of&#160;gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgf&#223; cleavage products under physiologic conditions.

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

When Transforming Growth Factor &#223; family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.

제노푸스 라레비스 배아의 블라스코클로로 분비된 성장 인자 변형 분석 가족 분열 제품

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

막 단백질의 Conformational 역학 검사 현장에서 사이트 이동 형광 라벨링과

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

생산<em&gt; Xenopus의 tropicalis</em&gt; 달걀은 미세 소관 관련 RNA를 식별하기 위해 추출

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

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 ...

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

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

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

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 ...

개구리와 도롱뇽의 생활 난 모세포에서 거대한 Lampbrush 염색체의 분리

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