Detachment or shortening of the flagella of the bi-flagellate alga Chlamydomonas induces the cell to turn on flagellar protein synthesis. The synthesis of these proteins in vivo can be followed by SDS gel electrophoretic analysis of the cytoplasm of regenerating cells. It was shown that flagellar regeneration is not required for the turning on of flagellar protein synthesis; rather, the absence of the flagellum is all that is necessary. Indeed, flagellar protein synthesis is turned on and off at the proper time even when flagellar assembly is blocked by colchicine. Flagellar shortening can be induced by lowering the calcium and raising the sodium or potassium content of the medium; only a small amount of flagellar shortening is required for full turn-on of flagellar protein synthesis. It is shown by 2-D gel analysis that the alpha tubulin subunit of flagellar microtubules is post translationally modified; current studies are designed to show the build-up of this precursor in the cell prior to modification and insertion into flagellar microtubules, and to determine at what stage in the procss - immediately after translation or at the time of assembly - the procssing of the alpha tubulin subunit occurs. The alpha tubulin precursor is slightly more basic than the mature alpha tubulin which appears in the flagellum. Studies are designed to show the nature of the modification - eg. phosphorylation, acetylation. Messenger RNAs for both alpha and beta tubulins have been isolated and shown to be active in cell-free synthesis of the tubulins. The synthesis is quantitated by use of specific antibodies made to Chlamydomonas flagellar alpha and beta tubulins. The antibodies do not cross interact with each other or with brain tubulins. The mRNAs, which are 80% pure, are being used to make cDNA for insertion into plasmids, which will then be selected for carrying alpha and beta tubulin genes. The cDNA isolated from these plasmids will then be used to quantitate mRNA formation during flagellar elongation and shortening, and for analysis of the tubulin genes.
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