The long term goal of the work described in this proposal is the elucidation of the mechanism by which the genes for the proteins comprising a single eukaryotic organelle--the flagellum--are regulated. Amputating the flagella of Chlamydomonas induces a massive change in gene expression, in which at least 30 genes for flagellar proteins are activated. Within 10 minutes, some signal travels from the cell surface to the nucleus and specifically stimulates transcription from the flagellar genes, and increased synthesis of the flagellar proteins. In the proposed research, mutants defective in the induction of flagellar gene expression after deflagellation will be isolated and characterized. An understanding of mechanisms which regulate gene expression in normal eukaryotic cells will be a prerequisite for understanding alterations in gene expression associated with diseases such as cancer. An additional health relatedness of the work comes from the extreme conservation of the structure and biochemistry of eukaryotic cilia and flagella. Insights gained in Chlamydomonas into the control of biosynthesis and assembly of ciliary proteins such as the dyneins should have relevance to human immotile cilia disorders, and sperm tail defects.
The specific aims of the work are as follows. First, a large number of mutants which cannot stimulate flagellar protein synthesis in Chlamydomonas will be isolated. In preliminary studies, such mutants have been identified among cells which cannot regenerate full length flagella after deflagellation. The synthesis defect is tested directly by in vivo labeling and SDS gel analysis of total cell protein.
The second aim i s to genetically characterize these mutants by dominance tests with wild type alleles, and complementation tests with other mutants. An example of the data sought would be an estimate of the number of mutable regulatory loci which can generate the observed phenotype. The number of complementation groups represented in a large sample of these mutants serves as an estimate of the complexity of the regulatory pathway. The third specific aim is to biochemically characterize mutants from each complementation group. For example, if a mutant is found which cannot stimulate flagellar protein synthesis, but after deflagellation the levels of mRNAs for flagellar proteins increase normally, a possible translational defect would be suggested for the mutant lesion.
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