Reversion analysis of dynein intermediate chain function.

The ODA6 locus of Chlamydomonas reinhardtii encodes a 70 kDa intermediate chain protein of the flagellar outer row dynein ATPase, and mutations at this locus prevent assembly of the entire outer row dynein arm complex. To initiate a structure-function analysis of the 70 kDa protein, we used transformation with chimeric mutant/wild-type genes to localize the defect in one assembly mutation, oda6-95. Sequence analysis revealed a frame-shift mutation in codon 53, which is followed by a stop codon after 13 amino acids in the new reading frame. By selecting intragenic pseudorevertants of this mutation we obtained 11 new oda6 alleles. Many of these pseudorevertants encode intermediate chain proteins that permit assembly of outer row arms but do not restore full wild-type motility. Revertant strains fall into two phenotypic classes, one with average beat frequencies of 54 Hz (similar to wild type) and one with average frequencies of 27 Hz (compared with 24 Hz for oda6-95) during normal forward swimming. Low beat frequency strains also display abnormalities during photophobic reversal (symmetric waveform). Amplification and sequence analysis of revertant alleles indicated that each reversion caused a second frame-shift, within a 115 nt interval, which restored the original reading frame, and that phenotypic severity was related to both direction (5' or 3') and distance between the original mutation and the reversion event. On the basis of immunoblot analysis of outer arm proteins, we conclude that revertant motility defects do not correlate with deficits in assembly of a specific dynein heavy chain or intermediate chain polypeptide, and electron microscopy confirms that revertants have normal outer arm structures. These results suggest that the 70 kDa intermediate chain plays a direct role in outer arm function distinct from its role in the assembly process.