CJN laboratuarında Araştırma NSERC, Kanada Kanser Derneği Araştırma Enstitüsü (CCSRI) ve Kanada Meme Kanseri Vakfı (BC-Yukon Şubesi) faaliyet hibe tarafından desteklenmektedir.

NOT: Bu prosedür genel iş akışı, Şekil 2&#39;de belirtilmiştir. Büyüme inhibe edici Doz 1. belirlenmesi <ol><li> Maya, gece boyunca kültürün hazırlanması NOT: Bu protokolde kullanılan maya-özü-pepton-dekstroz büyüme ortamı (YEPD) standart tarifi 13 olduğunu. <ol><li> BY4741 (mata his3 Δ1 leu2Δ0 met15Δ0 ura3Δ0) bir YEPD agar plakası üzerine hücreler üzerinden çizik ve 30 ° C de ya da görünür koloniler oluşana kadar 48 saat boyunca inkübe edilir. </li><li> Tek bir koloni ile steril bir kültür kabı içinde YEPD sıvı ortam 5 ml aşılayarak bir gecelik kültürler olarak hazırlanır. Sürekli dönme veya çalkalama ile 30 ° C&#39;de gece boyunca inkübe edin. Kültür tipik sabah (~ 2 × 10 8 hücre / ml) doygunluk ulaşacak. </li></ol></li><li> Katı ağar ortamının hazırlanması çeşitli kimyasal maddeyi ihtiva eden <ol><li> Birkaç stokları hazırlayınkimyasal, uygun bir çözücü içinde, 100x arzu edilen nihai konsantrasyonda test edilecek. Not: etkili konsantrasyonları kimyasal bağlıdır ve tecrübi olarak tespit edilmelidir. Yüksek mM düşük mcM gelen, yani; Başlangıçta geniş bir nihai konsantrasyon aralığını test etmek bu nedenle tavsiye edilir. </li><li> Kimyasal her bir konsantrasyonu için, bir kaç steril kültür tüpüne kısım erimiş YEPD agar 3 ml test edilecek. 55 ° C su banyosunda Yeri katılaşan tutmak için. </li><li> Test edilecek olan kimyasal her bir konsantrasyonu için, bir 12-çukurlu plaka içindeki çift havuzdan her bir çifti içine erimiş ortam, girdap karıştırmak için 2-3 saniye ve pipet 1 mi 100x bileşik 30 ul ekle. Yalnızca kontrol bir araç dahil emin olun. Plakalar oda sıcaklığında bir gece boyunca bekleyin. Herhangi bir ekstra medya atın. </li></ol></li><li> Forma kaplama hücreler <ol><li> Aşama 1 &#39;de olduğu gibi, gece boyunca büyütüldü doymuş bir maya kültürünün 2 ul ile YEPD sıvı ortam 10 ml inoküle.1.2. </li><li> Yuva başına seyreltilmiş maya kültürü 25 ul steril cam boncuklar veya çubuk ile düzgün biçimde yayılmış ve plaka, bir alev altında kurumasına izin plaka. 48 saat süre ile 30 ° C &#39;de plaka inkübe edin. </li><li> Plakalar üzerinde maya büyüme (koloniler hem toplam sayısını ve boyutunu) değerlendirir. Ekran gerçekleştirmek için daha fazla% 10 -15% gelişimini çoğunlukla inhibe etmemektedir bileşiğin bir alt öldürücü konsantrasyonunu belirleyin. </li></ol></li></ol> 2. Sistematik Kimyasal genetik Ekran <ol><li> Kaynak Plate silinmesi mutant-dizi Bakım (DMA) ve Hazırlık <ol><li> Gliserol stoklarından silme mutant diziler inşa ilgili ayrıntılı bilgi için Baryshnikova ark. 14 bakınız. Iptal mutantlarının plaka başına 384 koloni bir yoğunluğa dizilmiş edildikten sonra, DMA, 4 ° C&#39;de birkaç ay boyunca saklanabilir. Her birine büyüyen koloniler önlemek için G418 gerekli 200 ug / ml ihtiva eden YEPD agarı üzerinde çoğaltmaDiğer. NOT: Birden seri tekrarlamalı yavaş büyüyen suşlar kaybını önlemek ve baskılayıcı mutantlar görünümünü en aza indirmek için kaçınılmalıdır. Bu haploidlerin gibi koleksiyonları muhafaza eğer özellikle ilgilidir. </li><li> Robotik sistem arraying bir mikrobik kullanarak tüm çoğaltma kaplama adımları uygulayın. Alternatif olarak, manuel çivileme araçlarını kullanarak koloniler dizilmiş manipüle. NOT: maya silme toplama çeşitli ticari kaynaklardan haploidlerin ve diploid olarak kullanılabilir ve tipik olarak 96 oyuklu plakalar içinde gliserol stoklan halinde sevk edilir. </li></ol></li><li> Silme mutant dizi Yoğuşmalı <ol><li> G418, 200 ug / ml ihtiva eden YEPD agar ortamı 250 ml hazırlayın. G418 etkili konsantrasyon değişebilir ve her bir çok deneysel olarak test edilmelidir. </li><li> Plaka başına G418 200 ug / ml ihtiva eden YEPD agar 50 ml dökülerek beş YEPD agar hazırlayın. Dizi yoğuşmalı önce bu bir gün yapın ve plakalar oda sıcaklığında soğumaya bırakınerature gecede düz bir hatta yüzey üzerinde. NOT: Dört tabak 1.536-gerilme yoğunluğu toplanması için gerekli olacak, beşinci plaka ekstra olarak dökülür. </li><li> Plaka başına 384 koloni bir yoğunlukta mutant silme koleksiyonu içeren 16 Petri kapları çıkarın ve oda sıcaklığında (~ 1 saat) gelmek için izin verir. </li><li> Hassas bir görev silme kullanarak her plakanın kapak üzerinde oluşturulmuş olan herhangi bir yoğunlaşma silin. Su damlacıkları neden olabilir Aksi takdirde dizi ve dizilmiş mutantlar çapraz kontaminasyon biriken ediliyor. </li><li> Mikrobiyal dizi iğneleme robotu kullanarak, plaka başına 1.536 koloniler (4 Petri kapları) plaka başına 384 koloniler (16 Petri kapları) bir yoğunluktan DMA yoğunlaşmak. Plaka 1 pimleri 1. koloni yoğunlaştırılmış dizinin 1 sütun 1 satır böylece bu gerçekleştirin, plakanın 2 1. koloni 1 sütun 2, 2 ve sütun 1 satır plakanın 3 1. kolonisi satır, ve plakanın 4 1. koloni 2 satır içinSütun 2. </li><li> Gecede 30 ° C&#39;de konsolide dizi inkübe. </li><li> Kaynak plakalardan kolonilerin üniforma aktarımını sağlamak için dizi inceleyin. DMA doğru (Şekil 4A) yoğunlaşmış olan sağlamak için bir rehber olarak hizmet bilerek diziye dahil birçok boş pozisyonları, olacak. Not: Bu test bileşiği ihtiva eden ortam üzerinde çoğaltma kaplama için kaynak plakaları olacaktır. Tek bir kaynak plakası birden pinnings için kullanılabilir. Ayrıca birçok robotlar her zaman transfer ediliyor maya benzer numaraları sağlayacak bir mahsup özelliği, sahip olacaktır. </li></ol></li><li> Kopya plaka silme mutant dizisi <ol><li> Kontrol, 700 ml (yalnızca araç) ve deney ortam (kimyasal ihtiva eden) 700 ml hazırlayın. Bu üç nüsha olarak tahlil (koşul başına 12 plakaları, artı iki ekstra) gerçekleştirmek için yeterli. </li><li> Test kimyasal veya plaka başına denetimi içeren YEPD agar 50 ml dökün. Plakalar t izin vero düz bile yüzeyde gece boyunca oda sıcaklığında soğutun. </li><li> Çoğaltma plakasına robot kullanarak, deneysel belirlenen konsantrasyonda kimyasal içeren plakalar üç set yanı sıra araç denetimi içeren plakalar üç set üzerine DMA aşılamak. 24-48 saat süre ile, oda sıcaklığında inkübe edin. </li></ol></li></ol> 3. Görüntüleme Tabaklar ve Veri Analizi <ol><li> Inkübatör plakaları çıkarın ve oda sıcaklığında (~ 1 saat) önce görüntüleme gelmek için izin verir. Bu plakaları görüntüleme sırasında yoğunlaşmayı engeller. </li><li> Kapağı çıkarmadan ve bir düz yatak tarayıcı yüzü aşağı koyarak 24 ve 48 saat sonra görüntü plakaları. En az 300 dpi çözünürlükte görüntüler yakalayın. Alternatif olarak, görüntü yakalamak için bir dijital kamera kullanın. Bunu yaparken ise, yer plakaları karanlık bir arka plan üzerinde yukarı yüz ve görüntü kapakları kaldırın. NOT: Plakaların dosya formatı ve konumlandırma kullanılan miktar programına bağlıdır. </li&gt; <li> Ölçümlerini ve balony 15, SGAtools 16, ve ScreenMill 17 dahil olmak üzere birçok açık kaynak programları, birini kullanarak dizi koloni boyutları karşılaştırma yapın. </li></ol> Ekran 4. Doğrulama NOT: Bu aşamada çeşitli maya silmeleri mutantlar ilgi kimyasal olarak aşırı duyarlı atar. Bu kimyasal-genetik etkileşimler önümüzdeki iki şekilde valide edilmelidir. İlk duyarlı suşların kimliği PCR ile teyit edilmelidir. İkincisi, kimyasal duyarlılık bağımsız attı gerekir. Aşağıda belirtilen gerilme aşırı duyarlılık, maya biyoloji yaygın bir teknik doğrulamak için tespit tahliller yapmak için kısa bir protokoldür. <ol><li> Streak dışarı G418 200 ug / ml içeren YEPD agara maya silme koleksiyonundan (hipersensitif) mutantlar istenen. Koloniler oluşana kadar 30 ° C&#39;de inkübe edilir. Primerler göre diagnostik PCR ile soyun genotip doğrulamaÜreticinin protokolü. </li><li> Her bir suş için YEPD sıvının 5 ml inoküle ve dönme ya da çalkalama ile 30 ° C&#39;de gece boyunca inkübe edilir. </li><li> OD 600 ölçün ve steril dH 2 O. 600 = 1 OD her gerginlik sulandırmak Bunlar şimdi normalize maya kültürleri vardır. </li><li> 96 oyuklu bir plaka de A1 normalize yabani tip maya (BY4741), kültür, 100 ul koyun. Kuyular B1-H1 kadar yedi ek suşların 100 ul koyun. </li><li> A6-H6 ile kuyular A2-H2 steril dH 2 O 90 ul dağıtmak için bir çok kanallı pipet kullanın. Son olarak, normalleştirilmiş maya kültürlerinin 1:10 seyreltme serisi hazırlanır. Seri bir çok kanallı pipet ile sütun 2 sütun 1, 10 ul pipetleme başlayın ve altı seyreltme yapılana kadar 96 oyuklu plaka boyunca devam (yani, 10 0-10 -5). </li><li> Bir çok kanallı kullanarak bir tablo içinde seyreltilmiş maya kültürleri 2-5 ul aktarınKimyasal ve araç kontrolünü içeren YEPD katı ortama pipet. 24-48 saat için uygun sıcaklıkta inkübe edin. </li><li> Plakaları kontrol edin ve (BY4741 kontrole göre) kimyasal select mutantların duyarlılığını karşılaştırmak. Görüntü 24 de plakaları ve adım 3.2 olarak 48 saat. Not: ilgili gen ontolojilerinde veya fonksiyonlar ile bir kaç hiperduyarlı suşunun ekran geçerliliği güvenilir olmaktadır birlikte, genin vahşi tip bir kopyasını içeren bir plazmid ile aşırı duyarlı bir silme soyunun transformasyonu resmi bir gen kurmak için gerekli olan Çevrede silinmesi (ve gizli ikinci sitesi mutasyonlar) ilaç hassasiyeti neden olur. </li></ol>

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The authors declare they have no competing financial interests.

Kimyasal genetik etkileşim profilleri oluşturmak için bir yaklaşım burada özetlenen. yöntem basittir: bir kimyasal maddenin varlığı ve yokluğunda geniş gen silinmesi koleksiyonlarında, her bir suş koloni boyutları karşılaştırarak, bir kimyasal hakaret tolere gerekli genler tespit edilir. Hassas soyların ortaya çıkan listesinin Açıklaması zenginleştirme analizi, daha sonra bir etki kimyasal moduna ilişkin bilgi temin etmek üzere kullanılabilir. Bu protokol, maya tomurcuklanma için optimize e...

genetik araçlar ve model organizma Saccharomyces cerevisiae mevcut kaynakların topluca genler ağları biyolojik sistemlerin gerekliliklerini yerine getirmek olarak işlev nasıl yeni anlayış sağlamak büyük ölçekli fonksiyonel genomik çalışmalar sağlamıştır. Bu araçların taşı maya 1,2 bütün açık okuma çerçeveleri zorunlu olmayan gen silme komple bir set işbirlikçi kurulmasıdır. Çarpıcı bir gözlem, standart laboratuvar koşulları altında haploidlerin olarak yetiştirilen maya genlerinin ~% 20 canlılığı için gerekli oldu. Bu alternatif, biyolojik yolların kullanılması ile genomik düzensizliklerin karşı tampon bir hücrenin yeteneğini ortaya koymaktadır. Genetik ayrı ayrı yaşayabilir mutantlar, ancak birlikte öldürücü, bağlı veya yakınsak paralel biyolojik yollar sinyal ve biyolojik işlevi açıklayan genetik etkileşim ağları oluştururlar. Koşullu te gelişmesiyle birlikteEsas teşkil eden genlerin mperature duyarlıdır ve hypomorphic allelleri teknolojisi esansiyel olmayan genlerin 3,4 çalışması ile sınırlı değildir. Bu kavram, benzer hücresel proseslerle genlerin 5 bir araya gelip nasıl çalıştığını gösteren bir tarafsız genetik etkileşimin ilk üreten bir genomik ölçekte uygulanmıştır. Genetik ağların Kimyasal tedirginlikler mimik gen delesyonlar (Şekil 1) 6. Aşırı duyarlılık için, silme soylarının yüksek yoğunluklu dizisine doğru büyüme önleyici bileşikler sorgulama kimyasal stresi tolere için gerekli olan genlerin bir listesini, yani, kimyasal bir genetik etkileşim profili tanımlar. Genetik etkileşimleri gibi kimyasal kütüphanelerin büyük ölçekli ekranlar aksiyon küme bir araya 7 benzer bir modu ile bileşiklerin göstermiştir. Bu nedenle, bir bileşiğin kimyasal genetik etkileşim profili oluşturarak, etki mekanizmaları lar ile karşılaştırarak tahmin edilebilirge-ölçekli sentetik genetik ve kimyasal genetik etkileşim 8,9 veri setlerini. Bileşiklerin puanları, sorguya büyük ölçekli kimyasal-genetik ekranlar, barkod rekabet analizleri ile yapılmıştır. Bu yaklaşımda, silme soylarının havuzlanmış toplama kimyasal içeren, küçük bir ortam hacmi içinde birkaç nesil için topluca yetiştirilir. Her bir kromozom anormalliği mutantı, tek bir genetik barkod barındırır için, silme soylarının havuz içinde tek tek mutantlann canlılığı / büyüme mikrodizi veya yüksek sekanslama 10 tarafından izlenir. Bir biyolojik olarak aktif bileşiği içeren bir katı agar üzerinde büyütülmüş, fiziksel olarak dizilmiş mutantların kolonilerini izleyerek uygunluk çıkarım da kimyasal bir genetik etkileşimleri 11,12 tanımlamasında çok etkili bir yöntemdir. Bu yaklaşım, rekabete dayalı tarama için maliyet-etkin bir alternatif sunuyor ve kimyasallar küçük kütüphaneler tahlil için uygundur.Burada özetlenen S. kimyasal genetik etkileşimin bir listesini üretmek için bir basit bir yöntemdir biyoloji manipülasyonlar veya altyapı moleküler dayanmaz cerevisiae. Bu sadece bir maya silme koleksiyonu, bir robot veya manuel çivileme aparatı ve serbestçe kullanılabilir görüntü analizi yazılımı gerektirir.

<table><tbody><tr><td>Yeast Extract</td><td>BioBasic</td><td>G0961</td><td>For YEPD liquid/solid media add to 1% final concentration (w/v)</td></tr><tr><td>Tyrptone Powder</td><td>BD Biosciences</td><td>211820</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v)</td></tr><tr><td>Dextrose</td><td>Anachemia</td><td>31096-380</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v) &amp;#8212; do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td></tr><tr><td>Agar A</td><td>Bio Basic</td><td>FB0010</td><td>For YEPD solid media add to 2% final concentration (w/v)</td></tr><tr><td>G418</td><td>A.G. Scientific Inc.</td><td>G-1033</td><td>Prepare 1,000x stock at 200&amp;nbsp;mg/ml in dH&lt;sub&gt;2&lt;/sub&gt;O and filter sterilize.&amp;nbsp;</td></tr><tr><td>12-well plate</td><td>Greiner Bio One</td><td>655180</td><td></td></tr><tr><td>5 ml culture tubes</td><td>Evergreen Scientific</td><td>222-2376-080</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>ROTOR HDA</td><td>Singer Instruments&amp;nbsp;</td><td>ROT-001</td><td>high-throughput microbial array pinning robot</td></tr><tr><td>PLUSPLATE&amp;copy; Petri Dish</td><td>Singer Instruments&amp;nbsp;</td><td>PLU-001</td><td>Box of 200 dishes</td></tr><tr><td>384 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-384</td><td>Box of 1,000 pads</td></tr><tr><td>1536 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-1536</td><td>Box of 1,000 pads</td></tr><tr><td colspan="4">Alternative Pinning Tools:</td></tr><tr><td>Fully Automated Robtic Systems</td><td>S&amp;amp;P robotics</td><td>http://www.sprobotics.com</td><td>Several automated colony handling robitic and imagining systems available.</td></tr><tr><td>Manual Pinning Tools</td><td>V&amp;amp;P Scientific</td><td>http://www.vp-scientific.com</td><td>Handheld replication tools and accessories.&amp;nbsp;</td></tr></tbody></table>

rapid identification of chemical genetic interactions in saccharomyces cerevisiae

Biyolojik olarak aktif kimyasal etki şeklinin belirlenmesi, akademik, ilaç geniş bir ilgi ve endüstriyel bilim göstermektedir. Saccharomyces cerevisiae ya da çiçek mayası bir model ökaryot olduğu ~ 6.000 gen silme mutantları ve hypomorphic elzem genin tam bir koleksiyon mutantlar ticari olarak temin edilebilir. Mutantlar bu koleksiyonları sistematik bir kimyasal tolere gerekli kimyasal gen etkileşimler, örneğin genleri tespit için de kullanılabilir. Bu bilgiler, sırayla, bileşiğin etkime olası moduna bildirir. Burada maya tomurcuklanma kimyasal genetik etkileşimin hızlı tespit edilmesi için bir protokol açıklar. Bu eylem, iyi tanımlanmış bir mekanizmaya sahiptir, kemoterapötik madde, 5-flüorourasil (5-FU), kullanılarak yöntemi göstermektedir. Sonuçlarımız, nükleer TRAMP eksozom ve DNA onarımı enzimler önceki m ile tutarlıdır 5-FU mevcudiyetinde çoğalma için gerekli olan RNA olduğunu göstermektedirtabanlı barkod kimyasal genetik yaklaşımlar ve 5-FU olumsuz hem RNA ve DNA metabolizmasını etkilediğini bilgiyi icroarray. Bu, yüksek verimli taramalara gerekli doğrulama protokolleri de tarif edilmektedir.

Bu yaklaşımın bir doğrulama olarak, yukarıda tarif edilen protokol izlenerek, kemoterapötik madde, 5-florourasil (5-FU) temsil eden bir kimyasal genetik etkileşimin ekran gerçekleştirilir. 5-FU, timidilat sintaz gibi DNA ve RNA metabolizmasını 18 bozmak için bilinmektedir. 5-FU kimyasal genetik etkileşimler de çalışılmış ve heterozigot ve homozigot silme koleksiyonları 8,19 ikisini de kullanarak maya barkod mikroarray teknikleri ile incelenmiştir. Burada içindeki mutant koloni...

Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

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.

Kimyasal Genetik Etkileşimleri Hızlı Kimlik<em&gt; Saccharomyces cerevisiae</em

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

rapid-identification-chemical-genetic-interactions-saccharomyces

Research

JoVE Journal

Biology

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

10.3K Görüntüleme.  University of Victoria. 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.

Video: Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

ChEC-seq ile Protein-DNA Etkileşimlerinin Genom Boyunca Haritalandırılması<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Genetik

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.

Fisyon mayası Schizosaccharomyces pombe derin sıralama yoluyla, kendiliğinden bastırıcı ekran

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 Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

Genom-Geniş Karşılıklı Hemizygosity Analizi ile Saccharomyces Maya Türleri Arasındaki Termotolerans Farklarının Genetik Haritalama

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

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. cerevisiae, kantitatif proteomik protein kompleksleri tanımlanması

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

Biyoloji

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.

Saccharomyces cerevisiae'de Proximity Ligation ve Kantitatif PCR ile Homolog Rekombinasyon Ara Ürünlerinin Saptanması

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 ...

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

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

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

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Saccharomyces cerevisiae Spliceosome Subcomplexes Geçici Interactors Tespiti için Grafix kullanımı

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

Biyokimya

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Mikroarray Analizi için Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

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.

Büyüme-tabanlı Belirlenmesi ve Protein Parçalanabilirliklerinin Genetik Talepler biyokimyasal Onay<em&gt; Saccharomyces cerevisiae</em

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

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Şizosakkaromices pombe'de Multicopy Suppressorların Tanımlanması için Genetik Ekran

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

Kimyasal Genetik Etkileşimleri Hızlı Kimlik

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ChEC-seq ile Protein-DNA Etkileşimlerinin Genom Boyunca Haritalandırılması