Analyses of the Chlamydomonas Reinhardtii Genome: A Model, Unicellular System for Analyzing Gene Function and Regulation in Vascular Plants

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9975765GrossmanThe personnel involved in this project are Arthur Grossman (PI, Carnegie Institution of Washington), Paul Lefebvre and Carloyn Silflow (Co-Pls, University of Minnesota), John Davies (Co-PI, Iowa State University), Elizabeth Harris (Co-PI, Duke University), David Stem (Co-PI, Boyce Thompson Institute, Cornell University), and Ronald Davis (Co-PI, Stanford University).Chlamydomonas reinhardtii, a unicellular haploid green alga, has been and will continue to be a very important model system for elucidating basic biological processes in plants. Experimentation with Chlamydomonas is particularly important for the dissection of photosynthetic processes since this alga can be grown rapidly on an exogenous source of fixed carbon and is the only genetically tractable eukaryote for which mutations that affect all aspects of photosynthesis and carbon assimilation are conditional rather than lethal. The application of sophisticated genome-wide methodologies to studies of Chlamydomonas will augment its utility as a model for the analysis of photosynthetic function and regulation. Specifically, as a first goal, normalized Chlamydomonas CDNA libraries will be generated using RNA isolated from cells grown under a variety of environmental conditions. The 3' and 5' sequences of the cDNAs will help generate unique sets of ESTs that will be arrayed onto polylysine coated slides. These arrays will be used to examine global gene expression under conditions that markedly alter the activity and biosynthesis of the photosynthetic apparatus. The microarrays will also be used to analyze gene expression in those photosynthetic mutants that appear to be regulatory in nature (to identify potential gene targets for regulatory elements). Furthermore, full-length sequences will be generated for most of the cDNAs, which would represent almost the complete coding capacity of the Chlamydomonas nuclear genome. A second goal is to establish a partial physical map of the nuclear genome that is aligned with the genetic map with the aim of using the information to perform rapid and efficient map-based cloning of genes. The mapping studies will focus on specific regions of the nuclear genome that are centered around mutations that define genes having critical functions in photosynthesis. A third goal is to complete the sequence of the chloroplast genome, generate and analyze strains that are deleted for each of the chloroplast open reading frames, and use microarrays containing each of the chloroplast open reading frames to characterize global expression of chloroplast genes under different environmental conditions. The microarray technology will also be used to evaluate the abundance of all chloroplast mRNAs in nuclear mutants that exhibit aberrant transcriptional and post-transcriptional control of plastid gene expression. Much of the information and many of the tools that will be developed during the course of this work will allow studies of photosynthesis to attain a global dimension, which is critical for elucidating the dynamic nature of chloroplast function and the mechanisms involved in regulating chloroplast gene expression.