SGER - Global Analysis of Functional Units of Plant Chromosomes: DNA Replication, Domain Structure, and Transcription

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Scientific objectives and approachesPlants have many advantages for studying eukaryotic DNA replication origins and their relationship to structural and transcriptional domains. Rice and Arabidopsis have small genomes that have been or will soon be fully sequenced. Plant tissues at different stages of development and rapidly proliferating, synchronizable cultured cells are readily accessible in quantities amenable for biochemical analysis. The long term goal of this research is to exploit these properties to construct and overlay chromosomal maps of replication origins, matrix attachment regions, modified histones, DNA methylation levels and transcription. As a step toward this long term goal, this SGER award will allow evaluation of two approaches to mapping replication origins, both of which recently have been used successfully in yeast. One approach uses in vivo labeling of newly replicated DNA in a synchronized cell population. The feasibility of synchronizing cells and obtaining the levels of BrdU incorportation required for this procedure will be tested. The other approach uses chromatin immunoprecipitation (ChIP) to identify sequences associated with proteins of the ORC and MCM origin-binding complexes. It does not depend on synchronous cell division, but it does require specific antibodies and sensitive immunoprecipitation procedures. This funding will be used to develop and test additional antibodies and the necessary ChIP procedures for origin mapping by this approach.