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

University Of South Carolina At Columbia, Columbia SC

Investigators

Abstract

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).