Yazarlar Arabidopsis bitkilerin bakımı için Bayan Jennifer M. Lommel ve Tara N. Rognan teşekkür ederim. Bu çalışma, Saint Louis Üniversitesi ve Ulusal Sağlık Enstitüleri Sağlık hibe 1R15GM086846-01 ve 3R15GM086846-01S1 W. Xiao başlangıç ​​fonu tarafından desteklenmiştir.

I. Genetik Crossing 1. Kadın ebeveyn Emasculating DNA dizisi polimorfizm, iki farklı ekotipler, örneğin Columbia-0 (Col-0) ve Ler, kadın ve erkek ebeveynler olarak seçilecektir ile anne ve baba allel ayırmak için. Bitkiler genç ve sağlıklı olmalıdır. Bir büyüteç visor, ya da çıplak gözle, bir diseksiyon mikroskobu kullanılarak dişi ebeveyn hadım. Evre-12 çiçek bulun (Smyth ve ark, 1990) ve makas ile sapçık temel kırpma herhangi bir çiçek veya siliques yukarıda ve aşağıda onları çıkarın . Forseps% 95 etanol forseps üzerinde herhangi bir polen taneleri kaldırmak ve aynı zamanda polen öldürecek bir beher, içine hafifçe ucunun taban batırarak sterilize edin. Yavaşça forseps kullanarak çiçek tomurcukları dışında gözetlemek ve yavaşça dişilik organı çıplak ve zedelemeden, 4 sepals, 4 yaprakları, 6 organlarındaki kaldırmak. Bu işlem sırasında carpels zarar görmesini önlemek için deneyin. 2. Polen donör Toplama ve tozlaşma yürüten Iyi polen donörlerin polen bir çok dökülme olduğu dişilik organı (Smyth ve ark, 1990), 90 ° açıyla uzanan yaprakları ile 14 sahne açık çiçek anthesed. Üssünde çiçek alın ve sapçık hemen üstünde, çiçek açık yayılmasına sebep olacaktır. Anter ile hazırlanan dişilik organı stigma Toz. Tozlaşma sonra, bir mikroskop altında kolayca görülebilir sarı polen, damgalanma ile kaplı olacaktır. 3. Çapraz Etiketleme Bir bitkinin tüm hadım pistils pollinating sonra, biz bir mücevher etiketi ve dişi ve erkek ebeveynler ve güncel bilgi ile çapraz etiket. Toprak bitki yakın bir hissesi yerleştirin, bir dize, kazığa gelişme kök bağlamak için kullanın ve plastik bir çanta ile tozlaşma pistils kapağı. II. Arabidopsis endosperm Dokular izolasyonu 1. Hazırlanması malzemeleri : Biz genellikle bir sıvı nitrojen tankı, sıvı azot, bir diseksiyon mikroskobu ucu ince forseps (Dumont biyoloji tarafından 5 INOX FST, İsviçre), mikroskop cam slaytlar iki yeni çift (endosperm ve embriyo dokuları hasat öncesi gerekli malzemeler hazır 3 &quot;X 1&quot; X 1.0 mm), sorbitol 0.3 M pH 5.7 çözümü ve 5 mM (MES). 2. Toplama siliques 7 - veya 8-gün sonra-tozlaşma (DAP), tohum, orta ve geç torpido sahne embriyogenez hasat ve endosperm ve embriyo için disseke hazırız. Bazen hadım çiçekler çok genç, 9-10 DAP sürebilir. 3. Yalıtımlı endosperm ve embriyo Arabidopsis tohumu küçük olduğundan, endosperm ve embriyo izole etmek için bir diseksiyon mikroskobu kullanın. 8-DAP silique bir mikroskop altında bir koyun, silique sapçık tutmak için forseps, bir çift ve iki carpels sigorta marjı açık silique slayt forseps başka bir çift ucu kullanın. PH 5.7 sorbitol 0.3 M ve 5 mM MES çözümler üzerine bir tohum almak için forseps bir çift kullanın, embriyo slayt micropyle sonunda küçük bir kesim yapmak, endosperm ve tohum kabuğu ayrı endosperm dışarı itmek için kesilmemiş sonuna sıkmak (Kinoshita ve ark, 2004). Embriyo ve endosperm sıvı nitrojen içinde ayrı mikrotüpler içine koyun. 10-15 siliques embriyo ya da endosperm birikir kadar bu devam edin ve sonra tüp -80 ° C derin dondurucuda saklayın. Bazı durumlarda, endosperm, tohum kabuğu, yani ayrılmış olması değil, tek bir gen imprinting analizi için endosperm ve tohum kabuğu karışımı total RNA izole. III. Bisülfit Sıralama 1. Gerekli reaktifleri Genomik DNA, restriksiyon enzimleri, 3 M NaOH (Taze yapılmış), 6,42 M üre / 4 M sodyum bisülfit (190 2 M sodyum metabisülfit, Sigma-Aldrich, S9000, Na2S2O5, Moleküler Ağırlık) hazırlamak için Cetyltrimethyl amonyum bromür (STAB) 10 mM hidrokinon, bir DNA temizleme kiti (Promega Sihirbazı DNA Temizlik Sistemi, Kedi # A7280), TE tampon, 6.3 M NaOH (taze yapılmış), 10 M NH 4 oac, 20 mikrogram / ​​mcL tRNA ve% 100 etanol . 2. Bisülfit tedavi protokolü Detayları <ol><li> Genomik endosperm veya CTAB yordamı kullanarak embriyonun DNA (Rogers ve Bendich, 1988) kesin. </li><li> Digest 100 ng - 20 mcL 100 mcL toplam hacmi analiz edilecek bölge dışından kesen restriksiyon enzimleri ile genomik DNA 2 mg. MEA organizatörü için, biz XhoI, NdeI ve PstI veya HindIII kullanın. </li><li> DNA beş dakika kaynatarak restriksiyon enzimlerinin denatüre ve ardından buz üzerinde gidermek. </li><li> 15 dakika boyunca 37 1 / 9, 3 M NaOH ve inkübe hacmi (20 mcL sindirilir DNA 2.2 mcL) ° C ekleyin. </li><li> 250 mcL PCR tüp çözüm aktarın. </li><li> 10 ml steril distile su, 7.5 g üre çözülür; yavaş yavaş ekleyin ve 1-2 saat içinde 7.6 g sodyum metabisülfit ısıtma genellikle erime yardımcı olur; pH 5 ile 10 M NaOH taze yapılmış ayarlayın; bir final steril distile su ekleyin 20 ml hacim. Bu 6.24 M üre / 4 M sodyum bisülfit çözüm. </li><li> Sırasıyla 6.24 M üre son konsantrasyonu 5.36 M / 4 M sodyum bisülfit çözüm ve 3.44 M (Paulin ve ark, 1998). Örneğin, 6,42 M üre / denatüre genomik DNA (Xiao ve ark, 2003) Yukarıdaki 22.2 mcL 4 M sodyum bisülfit çözümü 208 mcL ekleyin. </li><li> 0.5 mM (20 mcL sindirim için 12 mcL) son bir konsantrasyonda DNA 10 mM hidrokinon ekleyin. </li><li> 55 ° C de 15 dakika ve 95 ° C &#39;de 30 saniye için 30 kür: PCR makinesi bisülfit tedavi yapılması </li><li> Promega Wizard DNA Temizlik-Up Sistemi kullanılarak bisülfit tedavi DNA Desalt ve protokolü takip (Jacobsen ve ark, 2000). </li><li> TE tuzsuzlaştırma sonra sütun kurtarıldı tam hacmi ölçün ve 37 0.3 M. inkübe son konsantrasyonu 6.3 M NaOH eklemek ° C 15 dakika boyunca. </li><li> 3 M nihai bir konsantrasyon, 2 mcL 20 mikrogram / ​​mcL tRNA ve 3 hacim% 100 etanol ile 10 M NH 4 oac (pH 7.0) ekleyin ve karıştırın. 14.000 rpm&#39;de 15 dakika santrifüj. </li><li> Bir kez,% 70 etanol ile pelet yıkayın kısa bir santrifüj yapmak ve ekstra etanol çıkarın. </li><li> 5-10 dakika ve 25 tekrar süspansiyon için speedvac Kuru pelet - DNA miktarı başlangıç ​​bağlı olarak 100 mcL TE tampon. Sodyum bisülfit ile tedavi edilen DNA PCR analizi için hazırdır. </li></ol> 3. PCR <ol><li> Unmethylated cytosines urasil dönüştürülmüş olduğundan, bu büyük bir parçası, bir şablon olarak bisülfit ile tedavi edilen DNA kullanarak yükseltmek için zordur. Böylece, genellikle bir ürün artık 500 bp daha yükseltmek için primerler dizayn. 4-kb MEA organizatörü dizisi için astar birçok setleri tasarlanmış ve tüm bölgeyi kapsayacak şekilde 14 örtüşen parçaları amplifiye (Xiao ve ark, 2003). </li><li> Üst iplikçik dizisi için ileriye astar, I) tasarımında primerleri ekstra uzun nükleotidler olmadan daha yüksek bir tavlama sıcaklığı için G (guanin) açısından zengin bir bölge seçin; II) değişiklik C (Sitozin) Y ( CG ve CNG bağlamlarda pirimidin) ve T (Timin) kalan C değiştirin. Ters astar, I) tasarımı C-zengin bir bölge seçin; II), CG ve CNG bağlamlarda R (pürin) G değiştirmek ve kalan G A (adenin) değiştirin. </li><li> CG ve CNG bağlamlarda R G değişikliği ve seçim ters astar, I) tasarımı A. kalan G değiştirmek II); alt sırasına iplikli, ileriye astar, I) tasarımı C-zengin bir bölge seçin G-zengin bir bölge; II) Y (pirimidin), CG ve CNG bağlamlarda C değiştirmek ve T. kalan C değiştirmek </li><li> Biz genellikle her PCR (Xiao ve ark, 2003) için bir şablon olarak sodyum bisülfit ile tedavi edilen DNA 1-2 mcL kullanın. PCR ürünü Parçanın doğru boyutu onaylamak için sonra bir ekleme olarak jel saflaştırılmış ve TOPO TA klonlama vektörü pCR2.1 (Invitrogen) klonlanmış olması, jel elektroforezi ile analiz edilmesi gerekiyor. Tek bir koloni aldım ve kültür; plazmid DNA ayıklanır ve sıralama için gönderilir. </li></ol> 4. Sekans analizi Bisülfit sıralama ilkesi unmethylated sitozin PCR ürünü timin olarak yükseltilecek pH5.0 yüksek konsantrasyonda sodyum bisülfit hidrolitik deaminasyon nedeniyle urasil dönüştürülür olacak, 5-metil sitozin sodyum bisülfit tarafından güncellenmiştir izin vermeyecek ve PCR (. Frommer ve ark, 1992. Clark ve ark, 1994) sonra sitozin kalır. Sıralama sonucu elde ettikten sonra, PCR için kullanılan iplikçik belirli bir şablon ile karşılaştırın. Şablonu bir sıralama sonucu bir timin sitozin kalıntı olarak okursa, sitozin metil olmadığını gösterir. Şablonda bir sitozin kalıntı sıralama bir sitozin kalırsa, sitozin metil olduğu anlamına gelir. IV. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2327/2327fig1.jpg" alt="Şekil 1 /" /> Şekil 1.

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Embriyo, endosperm ve tohum kabuğu ayırmak için nispeten kolaydır, ancak embriyogenez erken veya orta-torpido aşamada özellikle tohum tohum kabuğu, endosperm ayrı sıkıcı. Tohum kabuğu, sadece bazı genler için çok küçük bir miktar doku katkıda bulunmasından dolayı, örneğin, MEA ve FWA, endosperm, tohum kabuğu ayırmak zorunda değilsiniz. Endosperm ve tohum kat dokularda oluşan bir karışım RNA&#39;lar tecrit anlamına gelir ki, bir genin anne ve baba allel ifade, embriyo ifad...

Sarf Malzemeleri <ul><li> Diseksiyon Mikroskop </li><li> Makas </li><li> İnce İpucu Forseps </li><li> Takı Etiketi </li><li> Bitki Stakes </li><li> String veya Döndür-Kravatlar </li><li> 4 &quot;X 2&quot; X 8 &quot;Polietilen Çanta </li><li> 3 &quot;X 1 X 1,0 mm Mikroskop Slaytlar </li><li> Sıvı Azot </li><li> Sıvı Azot Konteynerler </li><li> Isı Blok </li><li> PCR Tüpleri </li><li> PCR </li><li> Mikrosantrifüj Tüpler </li><li> Mikrosantrifüj </li><li> Jel Elektroforez tesisi </li><li> Arabidopsis thaliana Columbia-0 Tesisleri </li><li> Arabidopsis thaliana Landsberg erecta&#39;nın Tesisleri </li></ul> Reaktifler <ul><li> % 70 Etanol </li><li> % 95 Etanol </li><li> % 100 Etanol </li><li> Sorbitol 0,3 M ve 5 mM MES-pH 5.7 </li><li> Cetyltrimethyl Amonyum Bromür (STAB) </li><li> % 100 Etanol </li><li> Cholorform </li><li> Kısıtlama Enzimler </li><li> 3 M NaOH </li><li> 6.3 m NaOH </li><li> 6,24 M Üre / 4 M sodyum bisülfit </li><li> Steril distile H 2 O </li><li> 10 mM hidrokinon </li><li> Sihirbazı DNA Temizlik Sistemi (Promega) </li><li> 10 M NH 4 oac </li><li> 20 mikrogram / mcL tRNA </li><li> TE tampon </li><li> TOPO TA Klonlama Kiti (Invitrogen) </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

Arabidopsis thaliana epigenetik mekanizmalar çalışmak için mükemmel bir model organizmadır. Nedenlerinden biri DNA methyltransferases kaybı fonksiyon-boş mutant böylece bir genom DNA metilasyon kaybı, büyüme ve gelişme nasıl etkilediğini incelemek için bir sistem sağlayarak, yaşayabilir. Basma maternal ve paternal allellerin diferansiyel ifadesi anlamına gelir ve hem de memeli hayvan ve bitkilerin üreme gelişmesinde önemli bir rol oynar. DNA metilasyon baskılı bir gen anne veya baba allel ifade ya da susturulmuş olup olmadığını belirlemek için kritik öneme sahiptir. Çiçekli bitkiler, bir çift üreme döllenme olayı var: tek bir sperm hücresi yumurta hücresi embriyo ve endosperm yol vermek için merkezi bir hücre ile ikinci bir sperm sigortalar oluşturmak için döller. Endospermi imprinting, bitkilerde meydana gelen bir dokudur. MEDEA, bir SET etki Polycomb grup gen, FWA, çiçekli düzenleyen bir transkripsiyon faktörü, endosperm ve kendi ifade baskılı gösterilen ilk iki gen, DNA metilasyon ve demetilasyon tarafından kontrol edilir bitkiler. Bir gen ve endosperm metilasyon paterni imprinting durumunu belirlemek amacıyla, ilk endosperm izole etmek gerekir. Tohum Arabidopsis ufak olduğundan, Arabidopsis endosperm izole ve metilasyon incelemek için zorlu kalır. Bu video protokolde, tohumları endosperm doku izole etmek için, genetik bir çapraz yapmak ve bisülfit sıralama metilasyon durumunu belirlemek için nasıl rapor.

Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Basma, bitki ve memeli üreme bir olgudur. DNA metilasyon, imprinting mekanizmaları önemli bir rol oynar. Endosperm Yalıtımlı ve baskılı genlerinin metilasyon durumunun belirlenmesinde<em&gt; Arabidopsis</em&gt; Zor olabilir. Bu protokol, endosperm izole ve metilasyon bisülfit sıralama tarafından nasıl belirleneceği açıklanmaktadır.

Baskılı Genler DNA metilasyon Belirlenmesi Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

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Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

17.3K Görüntüleme.  Saint Louis University. Basma, bitki ve memeli üreme bir olgudur. DNA metilasyon, imprinting mekanizmaları önemli bir rol oynar. Endosperm Yalıtımlı ve baskılı genlerinin metilasyon durumunun belirlenmesinde Arabidopsis Zor olabilir. Bu protokol, endosperm izole ve metilasyon bisülfit sıralama tarafından nasıl belirleneceği açıklanmaktadır.

Video: Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA Metilasyonu: bisülfit modifikasyonu ve Analiz

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Biyoloji

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: Sayısal ve Yapısal Değişmeler Algılama Tüm 24 İnsan Kromozomlar Aynı zamanda Roman OctoChrome BALIK Test kullanma

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

Genetiği keşfedin A Tohum Kabuğunun Yatak Deneyi<em&gt; In Vitro</em&gt; Nasıl Endosperminde Denetimler Çimlenme içinde<em&gt; Arabidopsis thaliana</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro Metilasyona Tahlil Protein Arginin metilasyon Okuyacak

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

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

Baz Çifti Çözünürlük DNA metilasyon Değerlendirme Temsil bisülfit sıralamayı İndirimli pekiştirilmiş

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

Gen spesifik ifadesi ile DNA Methylation değişiklikler astrositik transkripsiyonel Aktivite ilişkilendirilmesi<em&gt; KCNJ10</em&gt; (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

DNA Metilasyon Analizi nesil Dizilimine tarafından hedef

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

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

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

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

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Arabidopsis Protein-DNA Etkileşimleri Tespiti için Kromatin Immunoprecipitation Deneyi<em&gt; İn Vivo</em

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

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Kristalin Selüloz Birikimin Yüksek Çözünürlüklü Niceleme<em&gt; Arabidopsis</em&gt; Kökler Doku spesifik Hücre Duvarı Değişiklikler Monitör

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...