The unicellular green alga Chlamydomonas can swim with two very different flagellar waveforms. Forward swimming is accomplished using an asymmetric beat, resembling the beat of cilia. Reverse swimming is accomplished by a symmetrical waveform, similar to classic flagellar beating. The transition between the two forms is controlled by the contraction of Ca++ around the axoneme. Two different classes of mutants in Chlamydomonas have been isolated which have lost the ability to generate the asymmetric waveform: central pair apparatus defective mutants and move backward only mutants. The recent development of insertional mutagenesis in this organism has made it possible to rapidly generate mutants in many genes regulating flagellar function and to clone the affected genes. This technique will be used in this project to begin a comprehensive molecular, biochemical and ultrastructural analysis of the regulation of flagellar waveforms.
The specific aims are: 1) to use insertional mutagenesis to identify and clone genes whose products are required for generation of an asymmetric waveform in Chlamydomonas flagella; 2) to identify the gene products of each cad and mbo gene by sequencing genomic and cDNA clones; 3) to localize each gene product within the flagellum; 4) to address the function of each gene product by detailed characterization of mutant phenotypes. Chlamydomonas is the primary genetic system available for the study of flagellar structure and function. Dozens of mutants affecting flagellar function have been well- characterized, and many of the genes coding for components of the microtubules, radial spoke assembly and dynein arms have been isolated. The central apparatus is perhaps the least understood region of the axoneme. This product will identify and characterize many of the proteins involved in the generation of the asymmetric flagellar waveform. This research will advance our understanding of human biology in several important ways, given the important role of cilia in respiratory function and the role of flagella in male fertility.
