| 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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| Protease Activity Assay in Fly Intestines
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Author:
Date:
2017-09-20
[Abstract] The intestine is a central organ required for the digestion of food, the absorption of nutrients and for fighting against aggressors ingested along with the food. Impairment of gut physiology following mucosal damages impacts its digestive capacities that consequently will affect growth, wellbeing or even survival of the individual. Hence, the assessment of intestinal functions encompasses, among others, the monitoring of its integrity, its cellular renewing, its immune defenses, the production of enteroendocrine hormones and its digestive capacities. Here, we describe in detail how to assess the activity of the proteases secreted in the intestinal lumen of adult Drosophila melanogaster flies. This method can also be used for larval intestines. The present protocol is adapted and ...
[摘要] 肠是消化食物所需的中枢器官，吸收营养物质，并与食物一起摄入的侵略者作斗争。 粘膜损伤后的肠道生理损伤影响其消化能力，从而影响个体的生长，健康甚至生存。 因此，肠功能的评估包括监测其完整性，细胞更新，免疫防御，肠内分泌激素的产生及其消化能力。 在这里，我们详细描述如何评估分泌在成年果蝇的肠腔中的蛋白酶的活性。 这种方法也可以用于幼虫肠。 本协议由“蛋白酶荧光检测试剂盒”（产品代码PF0100）中提出的Sigma-Aldrich公司的方案进行了改进和改进。
【背景】肠道受到诸如斋戒，禁食，化学物质，病原体，损伤等诸多压力的影响。肠道能够通过保持其体内平衡的生理平衡来克服这种压力。为了感知进入的压力并产生适应性的维持肠功能的答案，肠已经开发出强健和保守的机制，如局部先天免疫防御和组织再生（Royet和Charroux，2013; Bonfini等，2016）。然而，在某些情况下，肠道稳态的维持可能受到影响。例如，在老化期间，随着许多未成熟或分化不良的细胞的存在，组织稳态维持总体下降（Jasper，2015; ...
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| Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines
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Author:
Date:
2017-06-05
[Abstract] The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of ...
[摘要] 消除HIV-1患者的主要障碍是后整合延迟（Finzi等人，1999）。抗逆转录病毒治疗仅针对主动复制病毒，而具有低转录活性或无转录活性的潜伏感染仍未得到治疗（Sedaghat等人，2007）。为了消除病毒性水库，一项战略重点是通过“休克和杀死”来逆转HIV-1潜伏期（Deeks，2012）。该策略的基础是通过在抗逆转录病毒治疗下通过治疗性诱导病毒基因和蛋白质表达来克服HIV-1潜伏期的分子机制，并通过病毒的溶解性质或现在识别感染细胞的免疫系统引起选择性细胞死亡。最近，许多研究已经描述了药物抑制人类溴结构域蛋白质的溴结构域和末端（BET）家族的成员的治疗潜力（Filippakopoulos等人，2010; Dawson等人＆ / em>，2011; Delmore等人，2011），其包括BRD2，BRB3，BRD4和BRDT。小分子BET抑制剂，例如JQ1（Filippakopoulos et al。，2010; Delmore等人，2011），I-BET（Nicodeme等人＆lt; / ...
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