Work from our laboratory has shown that the tubulin gene family in the unicellular eukaryote Chlamydomonas reinhardtii consists of 2 beta tubulin genes coding for identical protein products, and 2 alpha tubulin genes coding for slightly different gene products. Why this haploid organism maintains 23 genes for each of the tubulin types, and what roles are played by each of the genes during the life cycle, are questions we can now address. Three approaches using molecular biology and genetics will be used to understand the functions of the tubulin genes. 1) To determine the degree of coordination in the expression of the genes, specific DNA probes will be used to quantitate the level of transcripts from each of the genes under a variety of conditions in which tubulin protein assembly and tubulin gene expression is regulated. These conditions include passage through the cell cycle, which triggers tubulin gene expression at a specific stage; amputation of the flagella, which triggers a massive increase in the expression of the tubulin (and other flagellar genes); and induced depolymerization of flagellar or cytoplasmic microtubules, which decreases expression of the tubulin genes. 2) To locate regions of the tubulin genes important for regulation of expression, transformation of modified tubulin-lacZ fusion genes into, Chlamydomonas will be used to examine their expression under the conditions described above. These transformation experiments will make it possible to examine possible control regions of the genes both 3' and 5' to the coding sequence. Of particular interest as a possible control region will be the third intervening sequence of the beta tubulin genes, which is 89% conserved between the 2 genes. 3) Resistance to anti-microtubule drugs will be used to select mutants in the tubulin genes. Examination of mutants will allow us to determine the function of each of the tubulin genes by analyzing the cell process disrupted in individual mutants. Comparison of altered and wild type genes should help identify regions of the tubulins required for drug binding. A number of tubulin-binding drugs, such as colcemid and vinblastine, have found widespread use as chemotherapeutic agents. The proposed research should provide significant insight into the action of anti-tubulin drugs, as well as the more generally important question of the regulation of gene expression in a eukaryotic system.
