Recently it has become possible to transform a paralyzed radial spoke mutant of the bi-flagellate alga Chlamydomonas with the wild-type flagellar radial spoke gene, thereby restoring flagellar motility. This transformation system will permit the study of many aspects of flagellar protein synthesis, assembly and function. Before carrying out these studies, however, it will be necessary thoroughly to describe the transformation of Chlamydomonas with flagellar genes. These initial studies will be done using the gene for radial spoke-3 (RSP-3) and the paralyzed flagella mutant, pf-14 in which this gene is defective, causing the lack of radial spokes. Using both genetic and molecular procedures it will be determined how many copies of the gene are present, whether the genes are integrated into the chromosomes and, if so, where the integration occurs. An effort will be made to optimize the targeting of the transforming gene to its homologue in the cell (homologous recombination). This will then make it possible to obtain mutants in radial spoke proteins by gene disruption. Finally, because the efficiency of transformation of Chlamydomonas is relatively high, additional flagellar genes will be cloned by complementation. Radial spokes, composed of 17 individual proteins are structural components of flagella, the organelles by which the unicellular green alga, Chlamydomonas, swims. Each of these proteins is essential for normal flagellar beating. Certain mutations affecting these proteins result in flagella that lack radial spokes and are immotile. Newly developed techniques for genetically transforming Chlamydomonas now make it possible to study in detail the roles of the individual proteins in flagellar motility. The proposed research represents the first step toward such studies. Flagellar beating is one of the mechanisms by which cells move. Understanding the structure, function, and interactions of flagellar proteins is fundamental to our understanding of this form of cellular motility.
