这项工作得到了维罗纳大学在联合项目2014的框架内的支持 (在葡萄藤中的 ATL 基因家族的特性及其对霜霉病霜霉病的抗性)。

<ol>
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Rep. 6, 38260 (2016).</li><li>Vitulo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+deep+survey+of+alternative+splicing+in+grape+reveals+changes+in+the+splicing+machinery+related+to+tissue,+stress+condition+and+genotype.">A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype.</a> BMC Plant Biol. 14 (1), 99 (2014).</li><li>Overbeek, R., Fonstein, M., D'Souza, M., Pusch, G. D., Maltsev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+gene+clusters+to+infer+functional+coupling.">The use of gene clusters to infer functional coupling.</a> Proc. Natl. Acad. Sci. USA. 96 (6), 2896-2901 (1999).</li><li>Dalquen, D. 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作者没有什么可透露的。

在基因组时代, 许多植物物种被深深地刻画了。这些信息是初步的功能研究, 并提供一个框架, 进一步调查不同成员在一个家庭中的作用。在这方面, 还需要一个命名系统, 允许唯一地识别家庭中的每个成员, 避免在不同的研究组独立于不同的基因分配姓名时可能出现的冗余和混乱。
经过深思熟虑的考虑, 葡萄科学界同意命名的葡萄基因在一个家庭的基础上与拟南芥基因的...

在过去的十年中, 对葡萄基因组学进行了大量的研究。葡萄是一种公认的经济相关作物, 它已成为研究果实发育和木本植物对生物和非生物学胁迫反应的模型。在此上下文中,葡萄亚种cv. PN40024 基因组在 2007年1及其更新版本2中的发布导致了 "学" 数据的快速积累和大量高通量的研究。根据所公布的序列数据, 对某一特定基因家族 (通常由分享保守主题的蛋白质、结构和/或功能相似性和进化关系) 的综合分析, 现在可以进行, 以揭示其分子功能, 进化和基因表达谱。这些分析有助于理解基因家族如何控制整个基因组的生理过程。
植物生命周期的许多方面是由泛素介导的关键蛋白降解, 这需要一个 fine-tuned 的营业额, 以确保正常的细胞过程。泛素介导的降解过程的重要组成部分是 E3 泛素酶, 这是负责系统的灵活性, 由于具体目标的招聘3。因此, 这些酶代表一个巨大的基因家族, 约 1400 E3 连接编码基因预测在拟南芥基因组4, 每 E3 泛素连接作用于特定靶蛋白泛。尽管基质特异泛在植物细胞调控中的重要性, 但很少有人知道如何调节泛通路和目标蛋白只在少数情况下被识别。对这种特异性和调节机制的破译首先依赖于系统的不同组成部分的识别和定性, 特别是 E3 酶。在泛素酶中, ATL 亚科的特征是: 在a. 南芥中, 显示一个 RING-H2 手指域5,6, 其中一些在防御和荷尔蒙应答中扮演了一个角色7。
定义新基因家族成员的第一个关键步骤是对家庭特征的精确定义, 如一致主题、关键域和蛋白质序列特征。事实上, 基于爆炸分析的所有基因家族成员的可靠检索需要一些强制性的序列特性, 特别是蛋白质的功能/活动, 作为蛋白质的签名。这可以通过先前对其他植物物种的同一基因家族的描述来促进, 或者通过分析不同植物物种中属于同一家族的不同基因认定来分离共同的序列。然后, 家庭成员可以根据国际财团为某种特定植物所定的共同规则, 单独命名。例如, 在小道消息, 这种程序受到了葡萄基因注释 (sNCGGa) 的超级命名委员会的建议, 建立了一个系统系统树的构建, 包括五亚种和a. 南芥基因家族成员允许基于核苷酸序列的基因注释8。
染色体定位的家庭成员和基因重复调查允许突出的存在, 全基因组或串联复制基因。这种信息似乎有助于解开假定的基因功能, 因为它可能显示功能冗余或揭示不同的情况,即, non-functionalization, 新官能化, 或 sub-functionalization9。新的和 sub-functionalization 都是重要的事件, 创造了基因的新奇, 为植物适应变化的环境提供了新型的细胞组件10。特别是, 在葡萄基因组的进化过程中, 祖先基因的重复和新基因的产生是非常频繁的, 而新形成的基因则是来自葡萄的近端和串联重叠, 更有可能产生新函数11。
破译基因家族功能的另一个关键因素是组剖面。因此, 可以利用公共数据库访问大量的组数据, 从而利用 large-scale在 "硅片" 表达式分析中为基因家族成员分配假定函数。的确, 特定植物器官中某些基因的奇特表达或对某些压力的反应, 可以给出一些关于在定义条件下相应蛋白质的假定作用的提示, 并支持关于可能的假设sub-functionalization 重复的基因来应对不同的挑战。为此, 重要的是要考虑几个数据集: 这些可以已经可用的基因表达矩阵, 如葡萄器官的全基因组组图谱和发育阶段12, 或者可以通过以下方式生成ad hoc检索特定植物物种的组数据集的定义应力。此外, 一个简单的方法使用两个矩阵, 一个具有成对相似性的数据和另一个具有两两表达系数, 可以用来评价序列相似性和表达模式之间的关系在一个基因家族。
这项工作的目的是提供一个全球性的方法, 定义基因结构, 保守的蛋白质主题, 染色体位置, 基因重复, 和表达模式, 以及预测蛋白质本地化和磷酸化的网站, 以达到植物基因家族的详尽描述。这种综合的方法在这里适用于描述的 ATL E3 泛素连接家族的葡萄。根据 ATL 亚科成员在调控关键细胞过程中的新作用7, 这项工作可以很好地帮助识别功能性研究的强候选者, 并最终解开控制这一重要作物对其环境的适应。

<table>
	<tbody>
		<tr>
			<td>Personal computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Local Alignment Search Tool (BLAST)</td>
			<td></td>
			<td></td>
			<td>https://blast.ncbi.nlm.nih.gov/Blast.cgi</td>
		</tr>
		<tr>
			<td>Molecular Evolutionary Genetics Analysis (MEGA)</td>
			<td></td>
			<td></td>
			<td>http://www.megasoftware.net/</td>
		</tr>
		<tr>
			<td>Motif-based sequence analysis tools (MEME)</td>
			<td></td>
			<td></td>
			<td>http://meme-suite.org/</td>
		</tr>
		<tr>
			<td>Geneious</td>
			<td>Biomatters Limited</td>
			<td></td>
			<td>http://www.geneious.com/</td>
		</tr>
		<tr>
			<td>ProtParam Tool</td>
			<td></td>
			<td></td>
			<td>http://web.expasy.org/protparam/</td>
		</tr>
		<tr>
			<td>ngLOC</td>
			<td></td>
			<td></td>
			<td>http://genome.unmc.edu/ngLOC/index.html</td>
		</tr>
		<tr>
			<td>TargetP v1.1 Server</td>
			<td></td>
			<td></td>
			<td>http://www.cbs.dtu.dk/services/TargetP/</td>
		</tr>
		<tr>
			<td>Protein Prowler</td>
			<td></td>
			<td></td>
			<td>http://bioinf.scmb.uq.edu.au:8080/pprowler_webapp_1-2/</td>
		</tr>
		<tr>
			<td>MUsite</td>
			<td></td>
			<td></td>
			<td>http://musite.sourceforge.net/</td>
		</tr>
		<tr>
			<td>Pfam</td>
			<td></td>
			<td></td>
			<td>http://pfam.xfam.org/</td>
		</tr>
		<tr>
			<td>TMHMM Server v. 2.0</td>
			<td></td>
			<td></td>
			<td>http://www.cbs.dtu.dk/services/TMHMM/</td>
		</tr>
		<tr>
			<td>ProtScale</td>
			<td></td>
			<td></td>
			<td>http://web.expasy.org/protscale/</td>
		</tr>
		<tr>
			<td>Grape Genome Database (CRIBI)</td>
			<td></td>
			<td></td>
			<td>http://genomes.cribi.unipd.it/grape/</td>
		</tr>
		<tr>
			<td>PhenoGram</td>
			<td></td>
			<td></td>
			<td>http://visualization.ritchielab.psu.edu/phenograms/plot</td>
		</tr>
		<tr>
			<td>MCScanX</td>
			<td></td>
			<td></td>
			<td>http://chibba.pgml.uga.edu/mcscan2/</td>
		</tr>
		<tr>
			<td>Interactive Tree Of Life (iTOL)</td>
			<td></td>
			<td></td>
			<td>http://itol.embl.de/</td>
		</tr>
		<tr>
			<td>UniProt</td>
			<td></td>
			<td></td>
			<td>http://www.uniprot.org/</td>
		</tr>
		<tr>
			<td>Phylogeny.fr</td>
			<td></td>
			<td></td>
			<td>http://www.phylogeny.fr/index.cgi</td>
		</tr>
		<tr>
			<td>MUSCLE</td>
			<td></td>
			<td></td>
			<td>http://www.ebi.ac.uk/Tools/msa/muscle/</td>
		</tr>
		<tr>
			<td>Gblocks Server</td>
			<td></td>
			<td></td>
			<td>http://molevol.cmima.csic.es/castresana/Gblocks_server.html</td>
		</tr>
		<tr>
			<td>Vitis vinifera cv. Corvina gene expression Atlas datamatrix</td>
			<td></td>
			<td></td>
			<td>https://www.researchgate.net/publication/273383414_54sample_datamatrix_geneIDs_Fasoli2012</td>
		</tr>
		<tr>
			<td>Multi Experiment Viewer (MeV)</td>
			<td></td>
			<td></td>
			<td>http://mev.tm4.org/#/welcome</td>
		</tr>
		<tr>
			<td>Sequence Read Archive (SRA)</td>
			<td></td>
			<td></td>
			<td>https://www.ncbi.nlm.nih.gov/sra</td>
		</tr>
		<tr>
			<td>R</td>
			<td></td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>EMBOSS Needle (EMBL-EBI)</td>
			<td></td>
			<td></td>
			<td>http://www.ebi.ac.uk/Tools/psa/emboss_needle/</td>
		</tr>
	</tbody>
</table>

comprehensive workflow for the genome-wide identification and expression meta-analysis of the atl e3 ubiquitin ligase gene family in grapevine

家庭中基因的分类和命名可以显著地有助于描述编码蛋白质的多样性, 以及基于几个特征的家庭功能的预测, 如序列主题的存在或特定修饰修改的网站和家庭成员在不同条件下的表达谱。这项工作描述了一个关于基因家族特性的详细的协议。在这里, 这个过程被应用到在 Levadura 中的拟南芥 Tóxicos的描述 (ATL) E3 泛素连接家族的葡萄。这些方法包括家庭成员的全基因组鉴定、基因定位、结构和复制的特征、保守蛋白的分析、蛋白质定位和磷酸化的预测以及基因表达谱在不同的数据集的整个家族。这种程序可以扩展到根据实验目的进一步分析, 可适用于任何基因组的任何植物物种, 为其提供基因数据, 并提供有价值的信息, 以确定感兴趣的候选人为功能性研究, 提供对植物适应环境的分子机制的洞察。

VIT_05s0077g01970 基因, 被确定为最相似的a. 南芥 ATL2 (At3g16720) 通过 BLASTp 搜索, 被用作探针, 以调查的 ATL 家族成员在葡萄基因组 (五亚种cv 皮诺 PN40024)。PSI-爆炸分析在几个周期之后汇合, 揭示了属于葡萄树 ATL 基因家族 (图 1A) 的假定基因列表。在分析中确定的所有条目的肌肉对齐方式的视觉检查 (图 1B) 中, 对每个候选?...

Watch this Scientific Journal Video about Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine at JoVE.com

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

本文介绍了一个基因家族的鉴定和描述的程序, 适用于在 Levadura 的家庭的葡萄Tóxicos 的拟南芥(ATL) E3 泛素酶。

葡萄全基因组鉴定与表达的综合工作流 E3 泛素连接基因家族的 Meta 分析

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

comprehensive-workflow-for-genome-wide-identification-expression-meta

Research

JoVE Journal

Biology

10.4K 次观看.  Università degli Studi di Verona. 本文介绍了一个基因家族的鉴定和描述的程序, 适用于在 Levadura 的家庭的葡萄Tóxicos 的拟南芥(ATL) E3 泛素酶。

视频: Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...