Detachment of the flagella of the bi-flagellate alga Chlamydomonas results in the regeneration of two 12Mu flagella in 90 min. Deflagellation induces a large increase in the synthesis and accumulation of flagellar protein mRNAs. These mRNAs rapidly fall back to pre-deflagellation levels once regeneration is complete, and this is due both to a decrease in synthesis and a shortening of mRNA half-life. Only detachment of the flagella is required for these events, as they will occur normally if flagellar regeneration is blocked (colchicine). The system is ideal for investigating the signals responsible for the coordinate control of gene transcription, translational controls, and problems of organelle protein assembly. Transcriptional control of the alpha-tubulin genes is being investigated by injecting constructs of the tubulin gene promotor DNA and chloramphenicol acetyl transferase gene (CAT) into the Xenopus oocyte nucleus and assaying for CAT. Proteins isolated from Chlamydomonas nuclei or cytoplasm are being coinjected with the constructs; they are identified as putative transcriptional control proteins by in vitro protein/DNA binding studies using DNA from the alpha-tubulin promotor region. In addition to the alpha and beta tubulin genes which have already been cloned and sequenced, we are isolating genes for radial link and dynein-ATPase peptides by use of polyclonal antibodies to these proteins and a lambda gt11 expression vector containing Chlamydomonas genomic DNA. The radial link genes and their 5' flanking regions will be sequenced and analyzed for commonalities. Radial link genes will also be used in studies on the transformation of paralyzed radial link mutants to the motile wild-type. Since tubulin mRNA half-life decreases upon completion of regeneration, the stability of radiolabeled in vitro-synthesized (SP6) tubulin mRNA will be examined in the reticulocyte lysate and by injection of the mRNA into Xenopus oocyte cytoplasm. Both flagellar alpha-tubulin mRNAs appear to be synthesized in response to deflagellation, but only one is used for flagellar protein synthesis. This apparent translational control will be analyzed using in vitro-synthesized tubulin mRNAs and the reticulocyte lysate; we will determine if the alpha tubulin mRNA which is not used for flagellar tubulin synthesis is used for the synthesis of mitotic tubulin. Other evidence suggests that the amount of tubulin dimers in the cell can control the amount of tubulin mRNA present. We will experimentally vary the amount of tubulin dimers in the pool and analyze the changes in tubulin mRNA synthesis and accumulation. The mechanism by which this control might be exerted will be investigated by RNA/protein cross linking studies. There is no feed back control between the amount of mRNA synthesized in responses to deflagellation and the amount already present. In studies on flagellar protein assembly, we will determine the role of the alpha-tubulin acetylation which occurs at the time of tubulin assembly at the flagellar tip. The possible role of acetylation in microtubule assembly/disassembly in vitro will be investigated as will be the calcium sensitivity of acetylated microtubules. The flagellar dynein-ATPase is prefabricated in the cytoplasm during flagellar regeneration and we will use dynein mutants to describe the steps in the assembly of the dynein polypeptides.
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