The tubulin gene family in the flagellated unicellular eukaryote, Chlamydomonas reinhardtii, consists of two beta tubulin genes coding for identical proteins, and two alpha tubulin genes coding for slightly different products. Why this haploid organism maintains duplicate genes for the tubulins, and what rules are played by the genes in the life cycle, are questions to be addressed. The isolation of mutants resistant to anti-microtubule herbicides has identified both tubulin and non- tubulin genes that may be important for microtubule organization and function. Techniques of molecular biology and genetics will be used to understand the functions of these genes. Mutant lines resistant to anti- microtubule drugs will be characterized. Comparison of the distribution, stability, and assembly properties of microtubules in mutant and wild- type cells, in the presence and absence of the anti-microtubule drugs, will be obtained by using immunofluorescence techniques and by examining flagellar assembly and disassembly. Conditional-lethal mutants will be examined to determine the cell cycle stage at which they are blocked. The role of the genes in chromosome transmission will be examined. Genetic techniques will be used to identify other genes whose products interact with alpha tubulin in the cell. Isolation of genes identified by mutational analysis will be accomplished using transformation to complement Chlamydomonas mutations with cloned Chlamydomonas genes. Transposon mutagenesis will also be developed as a tool for gene isolation. Transformation will be used to identify cis-acting sequence elements important for transcriptional chemotherapeutic agents. The proposed research will provide insight into the action of anti-tubulin drugs, as well as the more generally important question of identifying genes involved in microtubule function in eukaryotic systems.
