Chlamydomonas reinhardtii flagellar motility mutant pf-14 fails to assemble radial spokes because of a deficiency for assembly-competent radial spoke protein 3 (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck. 1981. J. Cell Biol. 88:80-88). Here, we raise an antiserum to protein 3 and use it to isolate the corresponding structural gene from an expression library. Southern blot analysis indicates that the gene is single copy and has not undergone major rearrangement in mutant pf-14 cells. Northern blot analysis suggests that wild-type amounts of an apparently normal 2.3-kb transcript accumulate in mutant cells during flagellar regeneration. When this mutant RNA is hybrid selected and translated in vitro, however, it produces a slightly truncated polypeptide 3 with an altered charge. The mutant protein 3 fails to assemble into pf-14 flagella and is maintained within a cytoplasmic pool of unassembled radial spoke polypeptides, as indicated by immunoblot analysis of proteins from whole cells and isolated axonemes using antisera to several radial spoke polypeptides. Interestingly, amounts of the mutant protein are greatly diminished relative to other spoke components. Complete genomic and cDNA nucleotide sequences were determined, and the pf-14 mutation was identified. It is a C-to-T transition near the 5' end of the protein coding region, which changes codon 21 to the ochre termination signal UAA. The size and charge of the mutant protein, and its reduced levels in cells, suggest that it is produced by relatively inefficient translational initiation as codon 42. The unphosphorylated isoform of radial spoke protein 3 is identified, and the sequence similarities between intervening sequences of the radial spoke protein 3 gene and a conserved intervening sequence of the two Chlamydomonas beta-tubulin genes (Youngblom, J., J. A. Schloss, and C. D. Silflow. 1984. Mol. Cell. Biol. 4:2686-2696) are reported.

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FlagelHar radial spokes contribute to the regulation of dynein arm activity and thus the pattern of flagellar bending. We have sequenced the genes for radial spoke protein 4 (RSP4) and RSP6, two of the five proteins that make up the radial spoke head in Chlamydomonas reinhardtii. The two genes, which are tightly linked genetically (B. Huang, G. Piperno, Z. Ramanis, and D. J. L. Luck, J. Cell Biol. 88:80-88, 1981), are separated by only 435 bp. They encode proline-rich polypeptides of 49.8 kDa (RSP4) and 48.8 kDa (RSP6), which are 48%o identical to each other but do not resemble any previously sequenced proteins. The transcription start sites of these genes and an additional radial spoke protein gene, that for RSP3, were determined, and patterns of mRNA accumulation during flagellar regeneration were examined for the three radial spoke protein genes. These studies provide the molecular tools for a detailed analysis of radial spoke head function and assembly and for a determination of the mechanism by which the genes required to build a complex organelle are regulated.Genetic analysis of the unicellular biflagellate alga Chlamydomonas reinhardtii has revealed that mechanisms controlling dynein function reside in several flagellar structures, including the dynein arms themselves, the radial spokes, and structures projecting from the central pair of microtubules ( Fig. la; for reviews, see references 22, 32, and 39). The radial spoke consists of a thin stalk, which is attached to the A subfiber of the outer doublet microtubule, and a bulbous head, which is attached to the stalk and appears to interact with the projections from the central pair of microtubules. Cells lacking either functional radial spokes or central-pair complexes are paralyzed, but in certain genetic backgrounds, flagella can beat in the absence of these structures (5, 21) by using a symmetric stroke that resembles the movement of spermatozoon flagella, rather than the asymmetric, ciliary stroke that is used by wild-type C. reinhardtii for forward swimming.The polypeptide composition of radial spokes has been elucidated through two-dimensional (2D) gel analysis of flagellar proteins from wild-type and paralyzed flagella (pJ) mutants (40, 41). A comparison of the pf-14 mutant, which lacks radial spokes (55), with the wild type revealed that 17 polypeptides (designated radial spoke protein 1 [RSP1] through RSP17) are missing in the mutant (40, 41). A subset of five of these polypeptides (RSP1, RSP4, RSP6, RSP9, and RSP10) is missing in thepf-l andpf-17 mutants, which retain the spoke stalk but lack the spoke head (20, 40, 41; see also permits assembly but affects the function of RSP6 in a manner that depends on the conditions prevailing during assembly. Because of these intriguing assembly phenotypes, we decided to begin a sequence analysis of radial spoke head proteins with the genes for RSP6 and RSP4. A genomic clone for RSP6 had previously been isolated from an expression library (53), but no clones were identified for the RSP4 gene. However, gen...

The biflagellate alga Chlamydomonas has been used extensively in the genetic and biochemical analysis of flagellar assembly and motility. We have restored motility to a paralyzed-flagella mutant of Chlamydomonas by transforming with the corresponding wild-type gene. A nitrate reductasedeficient paralyzed-flagella strain, nitl-305 pf-14, carrying mutations in the genes for nitrate reductase and radial spoke protein 3, was transformed with wild-type copies of both genes. Two-thirds of the cells that survived nitrate selection also regained motility, indicating that they had been transformed with both the nitrate reductase and radial spoke protein 3 genes. Transformants typically contained multiple copies of both genes, genetically linked to each other, but not linked to the original mutant loci. Complementation of paralyzedflagella mutants by transformation is a powerful tool for investigating flagellar assembly and function.

Flagellar radial spokes contribute to the regulation of dynein arm activity and thus the pattern of flagellar bending. We have sequenced the genes for radial spoke protein 4 (RSP4) and RSP6, two of the five proteins that make up the radial spoke head in Chlamydomonas reinhardtii. The two genes, which are tightly linked genetically (B. Huang, G. Piperno, Z. Ramanis, and D.J.L. Luck, J. Cell Biol. 88:80-88, 1981), are separated by only 435 bp. They encode proline-rich polypeptides of 49.8 kDa (RSP4) and 48.8 kDa (RSP6), which are 48% identical to each other but do not resemble any previously sequenced proteins. The transcription start sites of these genes and an additional radial spoke protein gene, that for RSP3, were determined, and patterns of mRNA accumulation during flagellar regeneration were examined for the three radial spoke protein genes. These studies provide the molecular tools for a detailed analysis of radial spoke head function and assembly and for a determination of the mechanism by which the genes required to build a complex organelle are regulated.