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...