| In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
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Author:
Date:
2017-09-20
[Abstract] The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their ‘correct’ strands, as well as quality control mechanisms that evict polymerases when they associate with an ‘incorrect’ strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.
[摘要] 真核生物复制品是重复DNA的多蛋白复合物。 复制品被雕刻成连续的前导链合成与不连续的滞后链合成，主要通过DNA聚合酶ε和δ以及解旋酶，聚合酶α-引发酶，DNA滑动夹，夹带载体和许多其它蛋白质进行。 我们以前已经建立了聚合酶ε和δ靶向其“正确”链的机制，以及在与“不正确”链相关联时驱赶聚合酶的质量控制机制。 在这里，我们提供了使用纯蛋白质在体外差异测定前导和滞后链复制的实用指南。
Using pure proteins from Saccharomyces cerevisiae, our lab was the first to reconstitute a functional eukaryotic DNA replisome, a ~2 MDa complex that includes the 11-subunit CMG helicase (complex of Cdc45, Mcm2-7, GINS heterotetramer), the 4-subunit DNA polymerase (Pol) ε, the 4-subunit Pol α-primase, the PCNA (Proliferating Cell Nuclear Antigen) clamp homotrimer ring shaped processivity factor that ...
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| Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings
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Author:
Date:
2017-08-05
[Abstract] The plant endomembrane system plays vital roles for synthesis, modification and secretion of proteins and lipids. From the classic view, only mRNAs encoding secreted proteins could be targeted to the endoplasmic reticulum (ER) for translation via a co-translational translocation manner, however, recently this model has been challenged by accumulative evidence that lots of cytosolic mRNAs could also associate with ER, and that some categories of small RNAs are enriched on ER. These results suggested unrevealed functions of ER beyond our current knowledge. The large scale identification of RNAs and proteins on microsome is crucial to demonstrating the ER function and the studies will be boosted by next generation sequencing technology. This protocol provides a technical workflow to isolate ...
[摘要] 植物内膜系统对蛋白质和脂质的合成，修饰和分泌起着至关重要的作用。 从经典观点来看，只有编码分泌蛋白质的mRNA才能通过协同翻译方式靶向内质网（ER）进行翻译，然而最近，这一模型已经被大量的细胞溶质mRNA也可能与 ER，并且一些类别的小RNA在ER上富集。 这些结果表明ER的功能超出了目前的知识。 在微粒体上大规模鉴定RNA和蛋白质对于显示ER功能至关重要，研究将由下一代测序技术提升。 该协议提供了从植物组织中分离细胞质，微粒体，游离多聚体（FP）和膜结合多聚体（MBP）的技术工作流程。 分离的级分适用于mRNA，小RNA和蛋白质的基因组广谱分析。
【背景】植物内膜系统对于细胞壁形成，脂质生物合成，蛋白质合成，修饰，折叠和贩运非常重要。根据共翻译易位模型，分泌蛋白N末端的信号肽由细胞溶质多核糖体合成，然后由ER上的信号识别粒子识别，其余蛋白质部分随后在ER上合成。根据该模型，只有编码分泌蛋白的mRNA可以被带到ER进行翻译（Peter和Johnson，1994）。然而，从哺乳动物和植物细胞ER（Lerner等人，2003; de ...
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| Protocol for Enrichment of the Membrane Proteome of Mature Tomato Pollen
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Author:
Date:
2017-06-05
[Abstract] We established and elaborated on a method to enrich the membrane proteome of mature pollen from economically relevant crop using the example of Solanum lycopersicum (tomato). To isolate the pollen protein fraction enriched in membrane proteins, a high salt concentration (750 mM of sodium chloride) was used. The membrane protein-enriched fraction was then subjected to shotgun proteomics for identification of proteins, followed by in silico analysis to annotate and classify the detected proteins.
[摘要] 我们建立并阐述了利用番茄茄子（番茄）的例子丰富经济相关作物的成熟花粉膜蛋白质组的方法。为了分离富含膜蛋白质的花粉蛋白质级分，使用高盐浓度（750mM氯化钠）。然后将富含膜蛋白的级分进行霰弹枪蛋白质组学鉴定蛋白质，然后进行计算机分析，以对所检测的蛋白质进行注释和分类。
背景 由于蛋白质和溶质在不同细胞区室之间的适当分布或将新合成的蛋白质插入膜中很大程度上取决于膜蛋白质，所以膜蛋白质组是维持细胞和细胞器内稳态的核心（Paul等人 2013，2014和2016a）。考虑到膜蛋白的重要性，这些对于花粉功能和发育也是至关重要的（Paul等人，2016b）。许多全球花粉蛋白质组学研究已经在过去进行（Chaturvedi等人，2013和2016）;然而，很少讨论关于蛋白质的胞内分布和膜蛋白质组学在花粉中的组成的信息（Pertl et al。，2009）。一个原因可能是膜蛋白的低丰度和溶解度。在这里，我们描述了一种方法，用于分离和分析富含成熟花粉的膜蛋白的蛋白质级分，这是为番茄建立的（图1）。
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