이 작업은 FAPESP 보조금 (15/06477-9)에 의해 지원되었다.

1. 효모 총 추출물 준비
<ol><li>TAP태그9에 융합된 접합인자 중 하나를 발현하는 효모 세포를 성장시키는 데 적합한 아미노산 또는 핵염기와 함께 L YNB-glu 미디어(효모 질소 기준 2%m/v 포도당으로 보충됨), 이 경우 아데닌(20 μg/mL), 류신(30 μg/mL), 트립토판(30 μg/mL), OD600 = 1.0까지.</li>
	<li>1L 배양으로부터 100mL 원심분리기 병 3개에서 17,000x g에서 4°C에서 10분 동안 원심분리하여 세포를 수집하고 10mL 의 차가운 멸균물로 두 번 세척한다.</li>
	<li>수집된 효모 세포를 콜드 버퍼 A의 세포 부피 1/10에서 재연하여(10mmolL-1 HEPES pH = 7.9, 1.5mmol L-1 MgCl2,50mmolL-1 KCl, 5% v/v 글리세롤, 0.5mmolL-1 DTT, EDTA-프리 프로제스트 억제제).</li>
	<li>액체 질소에 셀 현탁액의 작은 방울을 동결. 작은 방울은 재중단 된 세포 용액을 파이프팅하고 50 μL을 액체 질소에 직접 떨어뜨려 얻을 수 있습니다. 주의: 액체 질소는 피부 또는 눈과 접촉하여 부상을 입을 수 있습니다. 적절한 안전 장비를 사용하여 처리하십시오. 참고: 프로토콜은 여기에서 일시 중지할 수 있습니다. 셀 서스펜션의 냉동 방울은 -80 °C에 저장 될 수있다.</li>
	<li>20Hz/s에서 6사이클을 통해 볼밀 장치에서 3분 동안 효모 세포용광을 준비합니다. 참고: 각 주기 후에, 녹지 않도록 액체 질소에 얼어붙은 세포를 품고 있는 용기를 담그세요. 프로토콜은 여기에서 일시 중지할 수 있습니다. 냉동 추출물은 -80°C에 보관할 수 있습니다. 효모 접합 추출물은 균질화제를 사용하여 구엽세포10을용해하거나 박격포 및유봉(11,12)을사용하여 제조할 수 있다.</li>
	<li>실내 온도에서 물에 들어 있는 튜브를 물에 넣고 가끔 흔들어 서 추출물을 녹입니다.</li>
	<li>원심분리기는 4°C에서 1h에 45,000 x g에서 추출합니다.</li>
	<li>BCA방법(13)에의해 지워진 상체의 단백질 함량을 정량화한다.</li>
	<li>추출물의 알리쿼트를 준비하고 액체 질소에 빠르게 고정하고 -80 °C에 보관하십시오. 참고: 프로토콜은 여기에서 일시 중지할 수 있습니다.</li>
</ol>2. 글리세롤 그라데이션 준비
<ol><li>버퍼 A에 두 개의 글리세롤 솔루션을 준비, 하나는 포함 10% v/v 와 다른 포함 30% v/v 글리세롤.</li>
	<li>30% v/v 글리세롤 용액에 교차 연결 제글루타랄데히드를 0.1% v/v에 넣고 혼합하여 균질화합니다. 주의: 적절한 안전 장비를 사용하여 연기 후드 내부의 글루타랄데히드를 처리하십시오.</li>
	<li>12mL 원심분리기 튜브(14 x 89mm) 하단에 차가운 10% v/v 글리세롤 용액 6mL를 추가합니다.</li>
	<li>튜브 하단에 그라데이션 마스터 장치가 제공된 긴 바늘에 부착된 주사기로 글루타랄데히드로 보충된 감기 30% v/v 글리세롤 용액6mL을 10% v/v 글리세롤 용액 아래에 추가합니다. 참고: 또는, 10% v/v 글리세롤 용액은 30% v/v 글리세롤/글루타랄데히드 용액의 상단에 조심스럽게 파이프화될 수 있다.</li>
	<li>그라데이션 마스터 장치를 사용하여 연속 밀도 그라데이션을 생성합니다. 참고: 그라데이션 마스터 장치에서 튜브는 매개 변수(시간/각도/속도)를 결정하기 위한 제조업체의 권장 사항에 따라 적절한 랙에 배치되고 잠시 회전됩니다. 이 작품에서 우리는 사용 : 2:25 분 / 81.5 ° / 11 rpm.</li>
	<li>셀 추출물을 첨가하기 직전에 7% v/v 글리세롤/버퍼 A 쿠션의 200 μL을 조심스럽게 상단에 넣습니다. 참고: 쿠션의 글리세롤 농도는 선형 그라데이션을 만드는 데 사용되는 농축 글리세롤 용액보다 낮어야 합니다.</li>
</ol>3. 원심분리추출
<ol><li>쿠션으로 12mL 선형 글리세롤 그라데이션 10%-30% 글루타랄데히드의 상단에 총 단백질 약 2 mg을 적재합니다.</li>
	<li>미리 냉각된 스윙 버킷 로터에 튜브를 놓습니다. 참고: 초원심분리 전에 혼합하지 않도록 튜브를 부드럽게 처리합니다.</li>
	<li>원심분리기는 4°C에서 16h의 194,000 x g에서 원심분리기.</li>
	<li>Aliquot각 12 mL 튜브24 500 μL 분획적응 된 EconoSystem을 사용 하거나 신중 하 게 파이펫팅에 의해 분수. 참고: 적응된 EconoSystem은 연동 펌프, UV 검출기 및 튜브 천포링 장치에 연결된 분수 수집기로 구성됩니다. 침전 프로파일은 280 nm에서 흡광도의 측정에 의해 모니터링될 수 있다.</li>
	<li>연동 펌프를 통해 40% v/v 글리세롤 용액을 사용하여 튜브에서 분획 수집기로 글리세롤10%-30%의 그라데이션을 밀어넣습니다. 참고: 프로토콜은 여기에서 일시 중지할 수 있습니다. 사용 전까지 분획을 -80°C에 보관합니다.</li>
	<li>점 얼룩 또는 슬롯 블롯 장치에 니트로 셀룰로오스 멤브레인에 직접 각 분획의 50 μL을 로드합니다. CBP에 대한 항체를 이용한 면역블롯(1:6,000, TAP 태그의 CBP 부분을 검출함) 및 호스레디시어 페록시다제(1:15,000)와 결합한 항래빗 IgG를 이차 항체로 검출한다.</li>
</ol>

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저자는 이해 상충이 없습니다.

단백질 단백질 및 리보핵산 단백질 상호 작용은 교차 연결제를 사용하여 안정화될 수 있다. 결과 복합체는 글리세롤 그라데이션에 대한 초원심분리를 견딜 수 있는 안정성이 있는 것이 중요합니다. 또한 버퍼 조건은 상호 작용을 허용해야하지만 비특정 바인딩을 피할 수 있을 만큼 엄격해야 합니다. 여기에 표시된 실험에서, 우리는 이미 시험관 내 접합 반응(15)에대해 확립 된 ?...

접합은 조정된 방식으로 다양한 요인의 바인딩 및 방출이 필요한 매우 역동적인 프로세스입니다. 이러한 접합 요인은 RNA 결합 단백질, ATPases, 헬리케이스, 단백질 키나아제 및 인산염, 유비퀴틴 리개세, 그 외1,2,3을포함한다. 그리고 분자 재배열이 일어날 수 있도록, 이러한 요인 중 일부는 매우 일시적인 이 RNP 중간 복합체의 격리 및 식별을 매우 어렵게 만듭니다.
여기서, 그라픽스 법4,5는 B액트 복합체 6과 효모 접합인자 Cwc24의 상호작용을 안정화하고, 유비퀴틴 리구아제 Prp19가 19(NTC)의 결합 또는 방출에 어떤 역할을 하는지 여부를 결정하기 위해B액트 복합체6을 사용하였다. 장소. 글리세롤 그라데이션을 따라 크로스링커의 농도가 증가하는 거시분자를 노출시키는 장점은 복합간 교차링크4,5,따라서 골재형성을 피한다는 것이다.
이 방법은 단백질 면역 침전 및 풀다운 분석에 대한 보완으로 사용되었으며, 이는 큰 복합체의 분리를 허용함에도 불구하고 큰 동적복합체7,8내에서 과도 상호 작용을 유지하는 데 신뢰할 수 없을 수 있다. 글리세롤 그라데이션에서 고정 시약을 사용하면 이러한 요인의 결합을 안정화시켜 접합 서브콤플렉스와 특정 단백질의 상호 작용을 확인할 수 있습니다. 선택된 크로스링커는 화학적으로 돌이킬 수 없기 때문에, 회수된 분획에 존재하는 단백질은 그라데이션 원심분리 후 점블롯에 의해 분석되었다.

<table><tbody><tr><td>Anti-Calmodulin Binding Protein Epitope</td><td>Millipore</td><td>07-482</td><td></td></tr><tr><td>ECL anti-Rabbit IgG</td><td>GE Healthcare</td><td>NA934</td><td></td></tr><tr><td>EconoSystem</td><td>Bio-Rad</td><td>1-800-424-6723</td><td>Parts of the EconoSystem used: peristaltic pump, the UV detector and the fraction collector</td></tr><tr><td>EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Fraction Recovery System</td><td>Beckman Coulter</td><td>270-331580</td><td>Tube-perforating device that was connected to the parts of the EconoSystem</td></tr><tr><td>Gradient Master Model 107ip</td><td>Biocomp</td><td>107-201M</td><td></td></tr><tr><td>Mixer Mill MM 200</td><td>Retsch</td><td>207460001</td><td>Ball Mill device</td></tr><tr><td>Rotor F12-6x500Lex</td><td>Thermo Scientific</td><td>096-062375</td><td></td></tr><tr><td>Sorvall RC 6 Plus Centrifuge</td><td>Thermo Scientific</td><td>36-101-0816</td><td></td></tr><tr><td>Swinging Bucket Rotor P40ST</td><td>Hitachi</td><td></td><td></td></tr><tr><td>Ultracentrifuge CP 80 NX</td><td>Hitachi</td><td>901069</td><td></td></tr><tr><td>Ultra-Clear Centrifuge Tubes (14 x 89 mm)</td><td>Beckman Coulter</td><td>344059</td><td></td></tr></tbody></table>

utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes

pre-mRNA 접합은 복잡한 성분의 조립, RNA 처리 및 방출 중에 화려한 하위 복합체의 많은 분자 재배열을 포함하는 매우 역동적인 과정입니다. 글리세롤 그라데이션 원심분리는 기능적 및 구조적 연구를 위해 단백질 또는 RNP(RiboNucleoProtein) 복합체의 분리를 위해 사용되어 왔다. 여기서는 단일 입자 저온 전자 현미경 검사법을 위한 거대 분자 복합체를 정화하고 안정화하기 위해 처음 개발된 Grafix(그라픽스(Gradient Fixation)의 활용을 설명하며, 이는 스플렉션 컴플렉스에 일시적으로 결합되는 접합 인자 간의 상호 작용을 식별합니다. 이 방법은 시료의 원심분리를 복합체안정화를 위한 고정 시약의 농도증가로 기초한다. 글리세롤 그라데이션에 적재된 효모 총 추출물의 원심분리 후, 회수된 분획은 점블롯에 의해 분석되어 개별 접합 인자의 존재의 현물 및 의결을 위해.

Cwc24-TAP의 퇴적 프로파일을 분석하고 Grafix 방법이 서브콤플렉스를 접합하는 데 결합을 안정화시키는 데 효과적인지 여부를 결정하기 위해, 우리는 글리세롤 그라데이션에 원심분리를 통해 Cwc24-TAP를 표현하는 세포의 총 효모 추출물을 분리하여 교차 연결제로 글루타랄데히드의 존재 또는 부재에 있다. 24μL 분획의 샘플은 TAP 태그의 CBP 부분에 대한 항체를 가진 슬롯 블롯에 의해 분석되었다. 결과?...

Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

여기서는 크로스링커가 있는 글리세롤 그라데이션 원심분리인 그라픽스(Gradient Fixation)의 활용을 설명하여 쌍향적인 복합체에 일시적으로 결합하는 접합 요인 간의 상호 작용을 식별합니다.

사카로미세스 세레비시아스 스플리케소터 서브콤플렉스의 과도성 행위자 검출을 위한 그라픽스의 활용

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

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Research

JoVE Journal

Biochemistry

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

3.5K 조회수.  University of Sao Paulo. 여기서는 크로스링커가 있는 글리세롤 그라데이션 원심분리인 그라픽스(Gradient Fixation)의 활용을 설명하여 쌍향적인 복합체에 일시적으로 결합하는 접합 요인 간의 상호 작용을 식별합니다.

비디오: Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

단일 분자 형광에서 제자리 교 잡 (smFISH) 신진 효 모 무성 한 성장 및 감수 분열 분석

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

연구

유전학

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

생물학

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

ACT1-CUP1 분석은 신진 효모에서 스플라이세오솜 돌연변이체의 기질 특이적 민감도를 결정합니다.

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

사카로마이세스 세레비시아에서 근접 결찰 및 정량적 PCR을 통한 상동 재조합 중간체의 검출

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

구슬에 갈렉틴-3 - U1 snRNP 복합체에 의한 접합 활동의 보완

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

생화학

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

화학 유전자 상호 작용의 신속한 식별<em&gt; 사카로 미세스 세 레비 시아</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

성장 기반의 결정 및 단백질 분해에 대한 유전 요구 사항의 생화학 적 확인에<em&gt; 사카로 미세스 세 레비 시아</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

진핵 세포에서 번역 단지의 신속한 생체 내 고정 및 격리

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

선택적 리보솜 프로파일링을 통한 공동 번역 상호 작용 네트워크의 글로벌 식별

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...

사카로미세스 세레비시아스 스플리케소터 서브콤플렉스의 과도성 행위자 검출을 위한 그라픽스의 활용

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