Centrioles are complex microtubule structures. They are present at the mitotic spindle poles of many eukaryotic cells. In interphase cells they define the centrosphere; an organizing center for microtubules. In cells bearing cilia or flagella they form the proximal region of the basal body, a nucleating structure for microtubules of the flagellar axonemes. It is proposed to study the function and replication of centrioles/basal bodies in the model system Chlamydomonas reinhardtii. In this organism a number of mutants affecting basal body function and assembly are clustered on an unusual linkage group that has Mendelian segregation patterns but gives a circular linkage map. The physical counterpart of this linkage group is a 6-9 megabase linear DNA that has been localized to basal bodies. In its mitotic and meiotic cycle Chlamydomonas manifests the classical motifs to centriolar morphogenesis: replicative formation, de novo origin and interconversion to basal bodies. The proposed studies focus on the basal body associated DNA. It is proposed to study further the structure of this DNA to understand the origin of the circular genetic map and to determine its localization in the cell at times in the meiotic cycle when centriole/basal bodies are absent. A major effort will be made to clone and characterize unique sequence segments of this DNA corresponding to a limited region of the genetic map. The proposed studies are strongly supported by features of the model system. Lineages derived from interspecific crosses with C. smithii make restriction fragment length polymorphism linkage mapping efficient. The basal body associated DNA can be isolated as a chromosome by pulsed field gel electrophoresis thus providing an enriched source of DNA fragments. The linkage group segment to be analyzed has already been indexed by ten phenotypic markers and six molecular markers. Methods have been devised to synchronize events in the meiotic and mitotic life cycles for isolation of RNAs. The proposed transcription analysis, testing of homology to DNAs from other cells, and DNA sequence analysis are expected to reveal the function of this DNA and indicate whether it is a feature of other centriole/basal body containing cells.
