Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas.

Abstract

Abstract. Several flagellar dynein ATPase and radial spokehead genes have been isolated from a Chlamydomonas genomic expression library in Lgtll. The library was probed with polyclonal and monoclonal antibodies raised against purified flagellar polypeptides, and recombinant phage giving positive signals were cloned. In vitro translation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation of identity for three of the four clones. Evidence supporting the identification of the fourth, which encodes a dynein heavy chain, was provided by antibody selection; the fusion protein produced by this clone selected heavy chain-specific antibodies from a complex polyclonal antiserum recognizing many dynein determinants. One of the radial spoke sequences isolated here is of particular interest because it encodes the wild-type allele of a locus which was defined previously by temperature-sensitive paralyzed flagella mutation pf-26~ (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck, 1981, J. Cell Biol., 88:80-88). The cloned sequence was used to hybridselect mRNA from mutant pf-26~ cells, and when translated in vitro, the selected mRNA produced a mutant spokehead polypeptide with an altered electrophoretic mobility. This confirms that the pf-26~ mutation alters the primary structure of a radial spokehead polypeptide. To quantify spokehead and dynein mRNAs during flagellar regeneration, all of the cloned sequences were used as hybridization probes in RNA dot experiments. Levels increased rapidly and coordinately after deflagellation, peaked 3-10-fold above nondeflagellated controls, and then returned to control values within 2 h. This accumulation pattern was similar to that of flagellar ct-tubulin mRNA.

Mutations at twelve independent loci result in absence of outer dynein arms in Chylamydomonas reinhardtii.

“…For example, isolation of intragenic revertants from these strains may give rise to strains with variant outer dynein arms and thereby enable us to infer the function of various dynein subunits. Recently, gene clones for the a and 13 chains of outer arm dynein were obtained (Williams et al, 1986;Mitchell, D. R., and B. D. W'dliams, unpublished observations). These studies indicate that the structural and functional properties of dynein subunits can be studied with the power of genetic engineering in the near future.…”

The dynein genes of Paramecium tetraurelia: the structure and expression of the ciliary beta and cytoplasmic heavy chains.

“…The steady state concentration of axonemal dynein mRNA increases in response to deciliation/deflagellation where it has been examined, including in Chlamydomonas (Williams et al, 1986) and sea urchin embryos (Gibbons et al, 1992. In Paramecium, mRNA from DHC-6 but not DHC-8 was shown to be substantially increased during reciliation .…”

Polarity of flagellar assembly in Chlamydomonas.

“…Radial spokes are assembled onto the previously spokeless axonemes, restoring motility (Piperno et al, 1981). Using antibodies against radial spoke proteins (Williams et al, 1986), we have examined cells during the the process of dikaryon rescue to establish the temporal pattern of radial spoke protein appearance on the previously spokeless axon e m e s .…”

“…Chlamydomonas flagellar proteins, the cloning and sequencing of a number of genes encoding these proteins (Curry et al, 1992;Mitchell and Kang, 1991;Schloss et al, 1984;Silflow et al, 1985;Williams et al, 1986Williams et al, , 1989, and the ability to reintroduce these genes to form stable transformants (Kindle et al, 1989;Diener et al, 1990), it has become possible to study the polarity of assembly of specific flagellar structures. The general approach that we have taken has been to use cell fusion during mating to transfer a pool of traceable subunits from a donor cell into a recipient, and then cytologically, using specific antibodies, locate these subunits once they have been added into the structure of the recipient cell's flagella.…”

“…The epitope-tagged ot-tubulin was then localized using immunofluorescent and immunogold techniques using an epitope-specific mAb (Field et al, 1988). Similarly, the assembly of radial spokes onto spokeless, but full-length, axonemes during dikaryon rescue of a spokeless mutant was followed by immunofluorescent analysis using antibodies to specific radial spoke proteins (Williams et al, 1986).…”