CAREER: Mechanisms that regulate activity of the plant DNA transposable element, mPing

这个项目试图发现调节转座元件运动的机制，也被称为“跳跃基因”。转座元件是在所有基因组中发现的丰富的DNA序列，它们能够将自己移动或复制到基因组中的新位置，从而导致新的遗传特征的变化。本研究的重点是水稻中的mPING元件，它属于一个转座元件家族，广泛分布于植物、动物、昆虫和真菌中。了解mPING的运动是如何控制的，为开发这些元件作为工具来识别与有益性状相关的基因提供了可能性，例如谷类作物的高产。该项目将通过提供遗传学和蛋白质化学实践经验的探究式生物化学实验室课程，吸引大量本科生参与，从而对劳动力发展产生广泛的教育影响。该项目还将让学生参与研究实验室，让他们直接参与发现过程的所有阶段，从实验到陈述和发表他们的发现。这项研究将使用遗传和生化分析来确定(1)mPING如何调节自己在基因组中的运动，(2)决定其插入位点偏好的因素，以及(3)mPING是如何复制的。这项研究建立在之前的三个观察结果的基础上，即高度活跃的mPING突变体的运动增加，对插入附近基因的偏好，以及随着时间的推移拷贝数增加的能力。通过确定调节活性转座复合体形成的因素，分析控制转座速率的机制。这包括体内结合分析(酵母单杂交和酵母双杂交)，以确定负责DNA-蛋白质复合体内相互作用的核苷酸和氨基酸残基。改变转座复合体形成的突变的效果将使用先前建立的酵母转座试验来测试。为了确定转座时间是否与细胞周期控制有关，将使用显微镜跟踪细胞分裂过程中转座酶蛋白定位的动态。MPING插入位点选择的机制将通过确定改变染色质结构如何改变插入模式来研究。同源重组修复在mPING复制中的作用也将通过测量对照和DNA修复突变体中的mPING复制来解决。总之，这些结果将定义控制这种活跃的转座元件运动的关键自我调节机制，从而提出可以做出哪些具体改变来增加这些元件作为基因组工程工具的效用。该奖项由生物科学局分子和细胞生物科学部遗传机制计划和刺激竞争研究实验计划(EPSCoR)共同资助。

This project seeks to discover mechanisms that regulate movement of transposable elements, also known as "jumping genes". Transposable elements are abundant DNA sequences found in all genomes, and their ability to move or copy themselves to new locations in the genome contributes to changes that can lead to new genetic traits. The focus of this research is the mPing element from rice, which belongs to a transposable element family that is broadly distributed in plants, animals, insects, and fungi. Understanding how movement of mPing is controlled offers the potential to exploit these elements as tools for identifying genes associated with beneficial traits, such as high grain yield in cereal crops. This project will have broad educational impacts relevant to workforce development by engaging a large number of undergraduate students through an inquiry-based biochemistry laboratory course that offers hands-on experience in genetics and protein chemistry. The project will also involve students in the research laboratory, providing them with direct participation in all phases of the discovery process, from experimentation to presentation and publication of their findings. This research will use both genetic and biochemical analyses to determine (1) how mPing regulates its own movement within the genome, (2) the factors that determine its insertion site preference, and (3) how mPing is replicated. This research builds on three previous observations, i.e., increased movement in hyperactive mPing mutants, preference for insertion into nearby genes, and the ability to increase in copy number over time. The mechanisms controlling transposition rate will be analyzed by determining the factors that regulate formation of the active transposition complex. This includes in vivo binding assays (yeast one-hybrid and yeast two-hybrid) to identify the nucleotides and amino acid residues responsible for interactions within the DNA-protein complex. The effects of mutations that modify transposition complex formation will be tested using a previously established yeast transposition assay. To determine if transposition timing is linked to cell cycle control, microscopy will be used to follow the dynamics of transposase protein localization during cell division. The mechanism of mPing insertion site selection will be investigated by determining how altering chromatin structure changes the insertion pattern. The role of homologous recombination repair in the replication of mPing will also be addressed by measuring mPing duplication in control and DNA repair mutants. Together, the results will define the critical self-regulatory mechanisms that control movement in this active transposable element and thereby will suggest what specific changes could be made to increase the utility of these elements as tools for genome engineering.This award was co-funded by the Genetic Mechanisms Program of the Division of Molecular and Cellular Biosciences in the Biological Sciences Directorate and by the Experimental Program to Stimulate Competitive Research (EPSCoR).