Recovery of flagellar inner-arm dynein and the fertilization tubule in Chlamydomonas ida5 mutant by transformation with actin genes

The ida5 mutant of Chlamydomonas, first isolated as a mutant lacking a subs et of axonemal inner-arm dyneins, has recently been shown to lack conventio nal actin owing to a serious mutation in its gene. It lacks inner-arm dynei ns probably because actin is an essential subunit for their assembly. In ad dition, male gametes of ida5 are unable to produce the fertilization tubule , a structure that contains a core of actin filament bundles. To establish that those observed deficiencies are solely attributable to the loss of act in, and to provide a basis for future studies on the actin function in this organism, we examined in this study whether transformation of this mutant with cloned actin genes can rescue the mutant phenotypes. Cotransformation of the double mutant ida5arg2 with the wild-type actin gene and arginino-su ccinate lyase gene that suppresses the arg2 mutation yielded several transf ormants that displayed increased motility. All of them were found to have a cquired the introduced actin gene In the genome and the product actin in th e flagella, and regained the missing inner-arm dyneins and wild-type motili ty. In addition, most transformants also became able to grow the fertilizat ion tubule when mating reaction was induced. In addition to the wild-type a ctin gene, we also used a chimeric actin gene in which the N-terminal Y2 am ino-acid sequence of Chlamydomonas actin was replaced by that of the greatl y divergent Tetrahymena actin. Transformants with this gene also resulted i n recovery of inner-arm dynein and 70-80% of the wild-type level of motilit y. These results established that the lack of inner-arm dynein and the fert ilization tubule in ida5 are consequences of its loss of conventional actin . Furthermore, they demonstrate that Chlamydomonas offers an excellent expe rimental system with which to study the structure-function relationship of actin by means of mutant analysis.