Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain.
We have characterized a group of regulatory mutations that alter the activity of the outer dynein arms. Three mutations were obtained as suppressors 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 pfl6. Recombination analysis and complementation tests indicate that all five mutations are alleles at the SUP-PF-1/ODA4 locus and that each allele can restore motility to radial spoke and central pair defective strains. Restriction fragment length polymorphism analysis with a genomic probe for the beta-dynein heavy chain (DHC) gene confirms that this locus is tightly linked to the beta-DHC gene. Although all five mutant sup-pf-1 alleles alter the activity of the outer dynein arm as assayed by measurements of flagellar motility, only two alleles have a discernable polypeptide defect by SDS-PAGE. We have used photolytic and proteolytic cleavage procedures to localize the polypeptide defect to an approximately 100-kD domain downstream from the last putative nucleotide binding site. This region is encoded by approximately 5 kb of genomic DNA (Mitchell, D.R., and K. Brown. 1994. J. Cell Sci. 107:653-644). PCR amplification of wild-type and mutant DNA across this region identified one PCR product that was consistently smaller in the sup-pf-1 DNA. Direct DNA sequencing of the PCR products revealed that two of the sup-pf-1 mutations are distinct, in-frame deletions. These deletions occur within a region 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 interactions within the outer dynein arm. Since both the size and location of this domain have been conserved in all axonemal and cytoplasmic DHCs sequenced to date, it presumably performs a common function in all dynein isoforms.
Porter
M E
ME
Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis 55455.
Knott
J A
JA
Gardner
L C
LC
Mitchell
D R
DR
Dutcher
S K
SK
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002874:Chromosome Mapping; D004398:Dyneins / Q000235:genetics*; D005407:Flagella / Q000502:physiology; D017125:Genes, Protozoan; D008040:Genetic Linkage; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D013489:Suppression, Genetic
D004398:Dyneins