| Measurement of Lysosomal Size and Lysosomal Marker Intensities in Adult Caenorhabditis elegans
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Author:
Date:
2018-02-05
[Abstract] Assays have been developed to study trafficking in various tissues of Caenorhabditis elegans. Adult C. elegans intestinal cells are large and have extensive endocytic networks, thus making them a good system for deciphering the endocytic pathway using live imaging techniques. However, the presence of auto-fluorescent gut granules in adult intestine can interfere with the signals of endocytic compartment reporters, like GFP. Here we demonstrate a protocol adapted from the original method developed by the Grant laboratory to identify signals from reporters in adult intestinal ...
[摘要] 已经开发了用于研究线虫的各种组织中的贩运的测定法。 成人 C。 线虫肠细胞很大，具有广泛的内吞网络，因此使它们成为使用实时成像技术破译内吞途径的良好系统。 然而，成年肠道中存在自身荧光的肠道颗粒会干扰吞噬细胞记者的信号，如GFP。 在这里，我们展示了一个协议，从格兰特实验室开发的原始方法改编，以确定记者在成人肠细胞的信号。 该方案的目标是识别标记有荧光标记物的内吞室，而在秀丽隐杆线虫成虫肠细胞中没有背景自发荧光消化道颗粒的混杂效应。
【背景】秀丽隐杆线虫（Caenorhabditis elegans）是一种多细胞生物，已被用于研究内吞运输。最初开发了用于研究内吞的试验。线虫卵母细胞，胚胎和体细胞（清道夫细胞）。简而言之，卵母细胞和胚胎中的测定通过在逗号至1.5倍发育阶段测量肠隔室中含有蛋黄 - 蛋白 - 绿色荧光蛋白报道分子（VIT-2 :: GFP）的区室的强度和大小来进行Grant和Hirsh，1999; Schaheen等人，2006a）。在成年人中，在转基因成虫的体细胞中测量了含有GFP的隔室的强度和大小（从体壁肌细胞分泌进入囊泡并由体细胞胞吞）。表达肌肉myo-3 :: ...
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| 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 ...
[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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| DNA Fragmentation Analysis
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Author:
Date:
2014-08-05
[Abstract] DNA fragmentation with length corresponding to multiple integer of approximately 180 base pairs is a distinct feature of apoptosis in animals and programmed cell death in plants. This feature can simply be detected by DNA gel electrophoresis followed by ethidium bromide staining, although in some cases it is difficult to distinguish the DNA laddering. We herein describe a protocol to detect a programmed cell death-associated DNA laddering of plant tissues. After agarose-gel electrophoresis of genomic DNA, Southern hybridization using DIG-labeled genomic DNA probe is performed, that improves ...
[摘要] 具有对应于大约180个碱基对的多个整数的长度的DNA片段是动物中凋亡和植物中程序性细胞死亡的独特特征。 这个特征可以简单地通过DNA凝胶电泳随后溴化乙锭染色检测，尽管在一些情况下难以区分DNA梯状。 我们在本文中描述了用于检测植物组织的程序性细胞死亡相关DNA梯状的方案。 在基因组DNA的琼脂糖凝胶电泳后，进行使用DIG标记的基因组DNA探针的Southern杂交，其改善DNA梯状的检测。
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