THE 78,000 M(R) INTERMEDIATE CHAIN OF CHLAMYDOMONAS OUTER ARM DYNEIN IS A WD-REPEAT PROTEIN REQUIRED FOR ARM ASSEMBLY

We have isolated and sequenced a full-length cDNA clone encoding the 7 8,000 M(r) intermediate chain (IC78) of the Chlamydomonas outer arm dy nein. This protein previously was shown to be located at the base of t he solubilized dynein particle and to interact with alpha tubulin in s itu, suggesting that it may be involved in binding the outer arm to th e doublet microtubule. The sequence predicts a polypeptide of 683 amin o acids having a mass of 76.5 kD. Sequence comparison indicates that I C78 is homologous to the 69,000 M(r) intermediate chain (IC69) of Chla mydomonas outer arm dynein and to the 74,000 M(r) intermediate chain ( IC74) of cytoplasmic dynein. The similarity between the chains is grea test in their COOH-terminal halves; the NH2-terminal halves are highly divergent. The COOH-terminal half of IC78 contains six short imperfec t repeats, termed WD repeats, that are thought to be involved in prote in-protein interactions. Although not previously reported, these repea ted elements also are present in IC69 and IC74. Using the IC78 cDNA as a probe, we screened a group of slow-swimming insertional mutants and identified one which has a large insertion in the IC78 gene and seven in which the IC78 gene is completely deleted. Electron microscopy of three of these IC78 mutants revealed that each is missing the outer ar m, indicating that IC78 is essential for arm assembly or attachment to the outer doublet. Restriction fragment length polymorphism mapping p laces the IC78 gene on the left arm of chromosome XII/XIII, at or near the mutation oda9 which also causes loss of the outer arm. Mutants wi th defects in the IC78 gene do not complement the oda9 mutation in sta ble diploids, strongly suggesting that ODA9 is the structural gene for IC78.