Name: targeted in situ mutagenesis of histone genes in budding yeast
Uploaded: 2017-01-26T15:00:00.000+00:00
Duration: 8 min 48 s
Description: Watch this Scientific Journal Video about Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast at JoVE.com

We thank Reine Protacio for helpful comments during the preparation of this manuscript. We express our gratitude to the National Science Foundation (grants nos. 1243680 and 1613754) and the Hendrix College Odyssey Program for funding support.

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The authors declare that they have no competing financial interests.

반수체 S. cerevisiae의 세포에서 4 개의 핵심 히스톤 단백질의 각각에 대한 코드가 구체적으로 돌연변이 유발을위한 두 개의 유전자 중 하나를 타겟팅하려는 연구자에 대한 도전을 나타낼 수있는 두 개의 비 대립 유전자 사이의 서열 상 동성의 높은 수준. 이전에 종종 의존 상기 Delitto PERFETTO, 부위 - 특이 적 유전자 (SSG) 돌연변이 유발, 클로닝없이 PCR 기반 대립 유전자 교환 방법 <sup class...

네 개의 핵심 히스톤 단백질 H2A, H2B, H3 및 H4는 진핵 세포 염색체의 압축, 조직 및 기능에 중심 역할을한다. 이러한 히스톤 각각 두 세트의 궁극적 뉴 클레오 (1)의 형성의 결과로, 히스톤 옥타 자체 주변 DNA의 ~ 147 염기쌍의 감쌈 지시 분자의 스풀을 형성한다. 뉴 클레오 솜은 염색체 전체 유전자 전사의 조절과 euchromatin의 형성 및 이질 같은 염색체 기반 프로세스의 다양한 활성 참가자이며, 같은 과거 수십 년에 걸쳐 집중적 연구의 초점이되어왔다. 이러한 메커니즘은 히스톤 잔류의 번역 후 변형, ATP 의존적 인 뉴 클레오 리모델링 및 ATP 독립적 인 뉴 클레오 재구성을 포함 - 메커니즘의 수는있는 뉴 클레오는 특정 프로세스의 실행을 용이하게 할 수있는 방법으로 조작 할 수있는 기술되어있다조립 / 해체 (2, 3). 신진 효모 사카로 마이 세스 세레 비지는 진핵 생물에서 히스톤 함수의 이해에 특히 강력한 모델 생물이다. 이것은 주로 도메인 eukarya 걸쳐 히스톤 단백질의 진화 적 보전의 높은 유전 생화학 실험 다양 효모의 가공성에 기인 할 수있다 (4)에 접근한다. 효모 역방향 유전자 접근법 널리 염색질 생물학의 다양한 양상의 특정 히스톤 돌연변이의 효과를 연구하기 위해 사용되었다. 실험은 이러한 유형의 비정상적인 세포 (인해 세포에서 플라스미드의 수를 가변으로) 히스톤 단백질의 수준을 초래할 수 자율 플라스미드로부터 발현 같은 돌연변이 히스톤은 그 나라의 게놈 유전자좌로부터 발현 된 세포를 사용하는 것이 바람직하다 크로 엔의 수반 변경최종적으로 결과의 해석을 혼동 할 수 많은 환경. 여기서는 게놈 남은 외래 DNA 서열없이, 원하는 돌연변이 (들)의 발생에 클로닝 단계 및 결과를 필요로하지 않고 그 나라의 게놈 위치에서 히스톤 유전자 표적화 돌연변이 유발을 허용하는 PCR 계 기술을 설명한다. 이 기술은 효모에서 효율적인 상동 재조합 시스템을 활용하고 다른 그룹에 의해 개발 된 다른 유사한 기술과 공통점이 몇 가지 기능이있다 - 가장 특히 Delitto PERFETTO, 사이트 별 게놈 (SSG) 돌연변이 유발 및 복제없이 PCR 기반의 대립 유전자를 대체 방법 5, 6, 7. 그러나,이 설명 된 기술은 특히 히스톤 유전자의 돌연변이 유발에 적합하게 한 측면을 갖는다. 반수체 효모 세포에서, 네 개의 코어 히스톤의 각각은 두 개의 비 a로 인코딩llelic 높은 상 동성 유전자는 예를 들어, 히스톤 H3가 HHT1 HHT2 및 유전자에 의해 암호화되고,이 유전자의 오픈 리딩 프레임 (ORF를는) 시퀀스의 90 % 이상 동일하다. 상 동성이 높은 수준은 구체적으로 돌연변이 유발을위한 두 개의 히스톤를 코딩하는 유전자 중 하나를 목표로 설계된 실험을 복잡하게 할 수 있습니다. 상기 방법들은 상동 재조합을 유도하는 표적 유전자의 ORF 내의 적어도 몇몇 시퀀스의 사용을 필요로하는 반면, 우리는 여기서 설명하는 기술에 대한 (더 적은 서열 상 동성을 공유하는) 히스톤 유전자의 ORF를 플 랭킹 서열을 이용한다 재결합 단계, 따라서 원하는 궤적에 돌연변이를 성공적으로 표적의 가능성을 증가시킨다. 또한, 재조합 드라이브 상동 지역이 더 효율적 대상 상동 재조합에 기여하고, 매우 광범위 할 수 있습니다.

<table><tbody><tr><td>1 kb DNA Ladder (DNA standards)</td><td>New England BioLabs</td><td>N3232L</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Sigma</td><td>A5093-100G</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma</td><td>B0394-500G</td><td></td></tr><tr><td>dNTP mix (10 mM each)</td><td>ThermoFisher Scientific</td><td>R0192</td><td></td></tr><tr><td>EDTA solution (0.5 M, pH 8.0)</td><td>AmericanBio</td><td>AB00502-01000</td><td></td></tr><tr><td>Ethanol (200 Proof)</td><td>Fisher Scientific</td><td>16-100-824</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td><td>Sigma</td><td>E4884-500G</td><td></td></tr><tr><td>Lithium acetate dihydrate</td><td>Sigma</td><td>L6883-250G</td><td></td></tr><tr><td>MyCycler Thermal Cycler</td><td>BioRad</td><td>170-9703</td><td></td></tr><tr><td>Poly(ethylene glycol) (PEG)</td><td>Sigma</td><td>P3640-1KG</td><td></td></tr><tr><td>PrimeSTAR HS DNA Polymerase (high fidelity DNA polymerase)&amp;nbsp; and 5x buffer</td><td>Fisher Scientific</td><td>50-443-960</td><td></td></tr><tr><td>Salmon sperm DNA solution</td><td>ThermoFisher Scientific</td><td>15632-011</td><td></td></tr><tr><td>Sigma 7-9 (Tris base, powder form)</td><td>Sigma</td><td>T1378-1KG</td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>Sigma</td><td>236500-500G</td><td></td></tr><tr><td>Supra Sieve GPG Agarose (low metling temperature agarose)</td><td>AmericanBio</td><td>AB00985-00100</td><td></td></tr><tr><td>Taq Polymerase and 10x Buffer</td><td>New England BioLabs</td><td>M0273X</td><td></td></tr><tr><td>Toothpicks</td><td>Fisher Scientific</td><td>S67859</td><td></td></tr><tr><td>Tris-HCl (1 M, pH 8.0)</td><td>AmericanBio</td><td>AB14043-01000</td><td></td></tr><tr><td>a-D(+)-Glucose</td><td>Fisher Scientific</td><td>AC170080025</td><td>for yeast media</td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>DF0140-01-0</td><td>for yeast media</td></tr><tr><td>Peptone</td><td>Fisher Scientific</td><td>DF0118-07-2</td><td>for YPD medium</td></tr><tr><td>Yeast Extract</td><td>Fisher Scientific</td><td>DF0127-17-9</td><td>for YPD medium</td></tr><tr><td>4-aminobenzoic acid</td><td>Sigma</td><td>A9878-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Adenine</td><td>Sigma</td><td>A8626-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Glycine hydrochloride</td><td>Sigma</td><td>G2879-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Alanine</td><td>Sigma</td><td>A7627-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Arginine monohydrochloride</td><td>Sigma</td><td>A5131-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Asparagine monohydrate</td><td>Sigma</td><td>A8381-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Aspartic acid sodium salt monohydrate</td><td>Sigma</td><td>A6683-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Cysteine hydrochloride monohydrate</td><td>Sigma</td><td>C7880-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Glutamic acid hydrochloride</td><td>Sigma</td><td>G2128-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G3126-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Histidine monohydrochloride monohydrate</td><td>Sigma</td><td>H8125-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Isoleucine</td><td>Sigma</td><td>I2752-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Leucine</td><td>Sigma</td><td>L8000-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Lysine monohydrochloride</td><td>Sigma</td><td>L5626-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Methionine</td><td>Sigma</td><td>M9625-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Phenylalanine</td><td>Sigma</td><td>P2126-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Proline</td><td>Sigma</td><td>P0380-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Serine</td><td>Sigma</td><td>S4500-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Threonine</td><td>Sigma</td><td>T8625-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Tryptophan</td><td>Sigma</td><td>T0254-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Tyrosine</td><td>Sigma</td><td>T3754-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Valine</td><td>Sigma</td><td>V0500-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>myo-Inositol</td><td>Sigma</td><td>I5125-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Ammonium Sulfate</td><td>Fisher Scientific</td><td>A702-500</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Yeast Nitrogen Base</td><td>Fisher Scientific</td><td>DF0919-07-3</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>5-Fluoroorotic acid (5-FOA)</td><td>AmericanBio</td><td>AB04067-00005</td><td>for&amp;nbsp; 5-FOA medium</td></tr></tbody></table>

targeted in situ mutagenesis of histone genes in budding yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

우리는 시츄 돌연변이 전략 표적의 대표 예로서 글루탐산 (돌연변이 H3-R53E)에 아르기닌에서 위치 53에서의 치환을 보유하는 히스톤 H3 돌연변이 단백질을 표현한 hht2 대립 형질의 발생을 설명한다. 우리 HHT2 전체 ORF가 URA3 유전자 (프로토콜의 단계 1 참고)로 대체 된 균주를 생성. 이 균주 yAAD156 ...

Watch this Scientific Journal Video about Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast at JoVE.com

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

대상<em&gt; 현장에서</em신진 효모에서&gt; 돌연변이 유발 히스톤의 유전자

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

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Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

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15.2K 조회수.  Hendrix College. A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented. 

비디오: Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

히스톤 - miniSOG에서 사용 Optogenetic 무작위 돌연변이 유발<em&gt; C. 엘레</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

연구

유전학

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

히스톤 수정 Saccharomyces cerevisiae 에서 chromatin Immunoprecipitation (칩)

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

분열 효 모 섬으로 불안정 프로테아좀 돌연변이 선택할 임의의 Mutagenesis 유전자 타겟팅

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

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 ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

효 모 신경 Proteinopathy 모델에서 특성화 히스톤 포스트 번역 상 수정 변경

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

분열 효 모 Schizosaccharomyces pombe 에 깊은 시퀀싱 지원, 자발적인 억압 화면

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

인간의 DNA 수리 단백질의 유전자 연구는 모델 시스템으로 효모를 사용하여

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

생물학

효모 세포에서 locus-specific Chromatin 분리

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

저자 스포트라이트: Advancing Chromatin Research and Overcoming Limitations with a High-Enrichment Locus-Specific Chromatin Isolation Protocol (고농축 유전자좌 특이적 크로마틴 분리 프로토콜로 크로마틴 연구 발전 및 한계 극복) 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae (맥주효모균의 단일 복제 유전자 자리, 염색질 정제)

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

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이 비디오의 챕터

히스톤 - miniSOG에서 사용 Optogenetic 무작위 돌연변이 유발

히스톤 수정 Saccharomyces cerevisiae 에서 chromatin Immunoprecipitation (칩)

분열 효 모 섬으로 불안정 프로테아좀 돌연변이 선택할 임의의 Mutagenesis 유전자 타겟팅

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

효 모 신경 Proteinopathy 모델에서 특성화 히스톤 포스트 번역 상 수정 변경

분열 효 모 Schizosaccharomyces pombe 에 깊은 시퀀싱 지원, 자발적인 억압 화면

인간의 DNA 수리 단백질의 유전자 연구는 모델 시스템으로 효모를 사용하여

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae (맥주효모균의 단일 복제 유전자 자리, 염색질 정제)

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae (맥주효모균의 단일 복제 유전자 자리, 염색질 정제)

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae (맥주효모균의 단일 복제 유전자 자리, 염색질 정제)