Development of Molecular Biology Approaches to Arabidopsis Thaliana

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It is proposed to clone, sequence and map transcriptionally inducible genes from Arabidopsis thaliana. These genes will be the nitrate reductase gene inducible by nitrate; chitinase inducible by ethylene and pathogens, and gibberellic acid inducible genes. The cis acting sites will be mapped by deletion analysis, linker scanning and small fragment insertion into a test promoter. In order to facilitate gene isolation and mapping a complete physical map will be constructed for Arabidopsis. The Arabidopsis chromosomes will be physically separated and isolated using an apparatus applying a contour-clamed homogeneous electric field with a transverse alternating angle of 120o. The NotI fragments from each chromosome will then be separated and correlated with the RFLP genetic map by hybridization with each RFLP probe. Each fragment will also be cloned in a newly developed yeast vector. We will focus attention on the cloning of an Arabidopsis centromere. These will be used in the construction of an artificial chromosome shuttle vector between yeast and Arabidopsis. Arabidopsis thaliana has great advantages over other plants for molecular studies. The results of this work will greatly benefit other investigators using this plant, particularly the integration of the RFLP map with NOTI fragments and the development of an artificial chromosome shuttle vector between yeast and Arabidopsis.