Flagellar mutants of Chlamydomonas : Studies of radial spoke-defective strains by dikaryon and revertant analysis

Abstract

The motility mutant of C. reinhardtii pf14 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a 2-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its 35S-labeled flagellar proteins. Six of these same proteins are missing in pf1, which lacks spokeheads. To determine whether any of the missing proteins represent the mutant gene product 2 experimental approaches were applied. The first makes use of the fact that gametes of either mutant strain when fused with a wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with 35S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The 2nd technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pf14 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pf1 only 2 radioactive proteins are restored; proteins 4, 6, 9 and 10 were not radioactive. Analysis of revertants of pf1 gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pf14, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.