Chlamydomonas has two actin genes, one coding for a conventional actin and the other coding for a highly divergent actin. The divergent actin NAP (for "novel actin-like protein") is expressed only negligibly in wild-type cells but abundantly in a null mutant of conventional actin, the ida5 mutant. The presence of the dormant NAP gene suggests that NAP may also have its own function in wild-type cells under some conditions. However, no specific functions have been suggested. In this study, we examined the expression of actin and NAP in wild-type and ida5 cells under conditions where actin function has been shown to be important. We found that deflagellation induces the expression of NAP as well as that of actin in wild-type cells. The expressed NAP becomes localized to the regrown flagella, apparently without being associated with dynein. Mating of gametes also increased the expression of actin in wild-type cells and that of NAP in ida5 cells, resulting in accumulation of these proteins in flagella (in both wild-type and ida5 cells) and the fertilization tubule (only in wild-type cells). However, it did not induce significant NAP expression in wild-type cells. These and other observations suggest that the expression of actin and NAP mRNAs is controlled by two discrete mechanisms and that NAP plays a role in flagellar formation in wild-type cells.
Key words: dynein mutant/flagellar actin/actin-like protein/chimeric actin gene/fertilization tubule ABSTRACT. The ida5 mutant of Chlamydomonas, first isolated as a mutant lacking a subset of axonemal inner-arm dyneins, has recently been shown to lack conventional actin owing to a serious mutation in its gene. It lacks inner-arm dyneins probably because actin is an essential subunit for their assembly. In addition, 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 actin, 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-succinate lyase gene that suppresses the arg2 mutation yielded several transformants that displayed increased motility. All of them were found to have acquired the introduced actin gene in the genome and the product actin in the flagella, and regained the missing inner-arm dyneins and wild-type motility. In addition, most transformants also became able to grow the fertilization tubule whenmating reaction was induced. In addition to the wild-type actin gene, we also used a chimeric actin gene in which the N-terminal 12 amino-acid sequence of Chlamydomonasactin was replaced by that of the greatly divergent Tetrahymenaactin. Transformants with this gene also resulted in recovery of inner-arm dynein and 70-80% of the wild-type level of motility. These results established that the lack of inner-arm dynein and the fertilization tubule in ida5 are consequences of its loss of conventional actin. Furthermore, they demonstrate that Chlamydomonasoffers an excellent experimental system with which to study the structure-function relationship of actin by means of mutant analysis.
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