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Universal platform for quantitative analysis of DNA transposition

机译:用于DNA转座定量分析的通用平台

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Background Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition. Results Here we developed a universal in vivo platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable lacZ-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish Escherichia coli colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS903 transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne E. coli arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level. Conclusions The established universal papillation assay platform should be widely applicable to a variety of mobile elements. It can be used for mechanistic studies to dissect transposition and provides a means to screen or scrutinise transposase mutants and genes encoding host factors. In succession, improved versions of transposition systems should yield better tools for molecular biology and offer versatile genome modification vehicles for many types of studies, including gene therapy and stem cell research.
机译:背景技术已完成的基因组计划揭示了可转座遗传元件的惊人多样性,这暗示着尚待从多种生命形式中发现的新颖元件家族的存在。同时,已经将一些更好理解的转座子系统开发为分子生物学和基因组学应用中的有效工具。新的移动元件的表征和现有转座技术平台的改进保证了易于使用的测定法,用于DNA转座的定量分析。结果在这里,我们开发了一种通用的体内平台,用于分析II类移动元件(即DNA转座子)的转座频率。对于每个特定的转座子系统,转座子末端和同源转座酶基因的克隆,在三个连续的步骤中,生成了多功能质粒,该质粒驱动转座酶基因的诱导表达,并包含一个可移动的含lacZ的报道转座子。该测定法将转座事件评分为蓝色微菌落,乳头状,在指示板上原本为白色的大肠杆菌菌落中生长。我们开发了使用噬菌体Mu转座作为测试模型的检测方法,并使用各种MuA转座酶突变体验证了平台。为了进一步验证和说明通用性,我们将IS903转座系统组件引入了测定中。通过控制质粒携带的大肠杆菌阿拉伯糖启动子,可以将开发的测定法调节至所需的初始转座水平。实际上,通过改变生长培养基中阿拉伯糖或葡萄糖的浓度来调节转座频率。我们显示,可以分析易位水平的转座活性,从而可以对过度活跃或过度活跃的转座酶突变体进行简单的筛选,而不论原始的野生型活性水平如何。结论所建立的通用乳头法测定平台应广泛适用于各种移动元件。它可用于解剖转座的机理研究,并提供筛选或审查转座酶突变体和编码宿主因子基因的手段。相继,转座系统的改进版本将为分子生物学提供更好的工具,并为包括基因治疗和干细胞研究在内的许多类型的研究提供通用的基因组修饰载体。

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