| Determination of VPS34/PIK3C3 Activity in vitro Utilising 32P-γATP
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Author:
Date:
2016-08-20
[Abstract] VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhou et al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to verify that any changes are mediated by VPS34, particularly as alternate pathways of PI(3)P production are possible such as via class II PI3Ks (Devereaux et al., 2013). Assaying VPS34 activity ...
[摘要] VPS34是哺乳动物细胞中唯一的III类磷脂酰肌醇-3-激酶（PI3K），并且产生绝大多数细胞磷脂酰肌醇-3-磷酸[PI（3）P]。 PI（3）P是一个关键的信号脂质，在诸如内吞作用和自噬的过程中起许多膜运输的作用。 VPS34是关键的细胞调节剂，功能丧失可具有灾难性作用并且是胚胎致死的（Zhou等人，2011）。 细胞PI（3）P的水平可以通过荧光染色技术确定，并且可以用于监测对VPS34活性的影响，然而重要的是验证任何变化由VPS34介导，特别是作为PI（3）的替代途径， P生产是可能的，例如通过II类PI3K（Devereaux等人，2013）。 直接在体外测定VPS34活性可以是描绘特定刺激的作用的关键阶段。
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| Analysis of Telomeric G-overhangs by in-Gel Hybridization
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Author:
Date:
2016-04-05
[Abstract] Telomeric DNA in majority of eukaryotes consists of an array of TG-rich tandem repeats. The TG-rich DNA strand is oriented with its 3’ end towards chromosome termini and is usually longer than its complementary CA-rich strand, thus forming 3’ single stranded overhang (G-overhang). G-overhangs arise from incomplete replication of chromosome termini by the lagging strand mechanism and post-replicative nucleolytic processing. The G-overhang is important for telomere protection as it serves as a binding platform for specific proteins and is required for t-loop formation. Hence, structure of telomeric G-overhang is an important indicator of telomere maintenance and functionality. Here we describe a method for analysis of G-overhangs in a model plant Arabidopsis thaliana by in-gel ...
[摘要] 在大多数真核生物中的端粒DNA由富含TG的串联重复阵列组成。富含TG的DNA链以其3'末端朝向染色体末端定向，并且通常比其互补的富含CA的链更长，从而形成3'单链突出端（G突出端）。 G突出由染色体末端的不完全复制通过滞后链机制和复制后核酸水解加工产生。 G突出端对于端粒保护是重要的，因为其用作特异性蛋白质的结合平台并且是t环形成所需的。因此，端粒G突出端的结构是端粒维持和功能的重要指标。在这里我们描述了通过凝胶内杂交技术在模拟植物拟南芥中分析G突出端的方法。该方法允许单链端粒DNA的量的相对定量。短端粒探针放射性标记并在非变性条件下与DNA杂交以特异性检测ssDNA。可以使用在相同凝胶中的变性条件测量总端粒DNA，并且该程序通常在非变性凝胶内杂交之后。 ssDNA的末端性质通过核酸外切酶处理来验证。这种技术最初是在酵母中开发的，现在被用作从人类到植物的多种生物体中的G突出端分析的主要工具。
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