MUTATIONS IN THE SUP-PF-1 LOCUS OF CHLAMYDOMONAS-REINHARDTII IDENTIFYA REGULATORY DOMAIN IN THE BETA-DYNEIN HEAVY-CHAIN

We have characterized a group of regulatory mutations that alter the a ctivity of the outer dynein arms. Three mutations were obtained as sup pressors of the paralyzed central pair mutant pf6 (Luck, D. J. L., and G. Piperno. 1989. Cell Movement. pp. 49-60), whereas two others were obtained as suppressors of the central pair mutant pf16. Recombination analysis and complementation tests indicate that all five mutations a re alleles at the SUP-PF-1/ODA4 locus and that each allele can restore motility to radial spoke and central pair defective strains. Restrict ion fragment length polymorphism analysis with a genomic probe for the beta-dynein heavy chain (DHC) gene confirms that this locus is tightl y linked to the beta-DHC gene. Although all five mutant sup-pf-1 allel es alter the activity of the outer dynein arm as assayed by measuremen ts of flagellar motility, only two alleles have a discernable polypept ide defect by SDS-PAGE. We have used photolytic and proteolytic cleava ge procedures to localize the polypeptide defect to an similar to 100- kD domain downstream from the last putative nucleotide binding site. T his region is encoded by similar to 5 kb of genomic DNA (Mitchell, D. R., and K. Brown. 1994. J. Cell Sci. 107:653-644). PCR amplification o f wild-type and mutant DNA across this region identified one PCR produ ct that was consistently smaller in the sup-pf-1 DNA. Direct DNA seque ncing of the PCR products revealed that two of the sup-pf-1 mutations are distinct, in-frame deletions. These deletions occur within a regio n that is predicted to encode a small alpha-helical coiled-coil domain of the beta-DHC. This domain may play a role in protein-protein inter actions within the outer dynein arm. Since both the size and location of this domain have been conserved in all axonemal and cytoplasmic DHC s sequenced to date, it presumably performs a common function in all d ynein isoforms.