title: Recovery of flagellar inner-arm dynein and the fertilization tubule in Chlamydomonas ida5 mutant by transformation with actin genes.
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 when mating 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 Chlamydomonas actin was replaced by that of the greatly divergent Tetrahymena actin. 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 Chlamydomonas offers an excellent experimental system with which to study the structure-function relationship of actin by means of mutant analysis.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Ohara
authors.forename: T
authors.initials: T
authors.lastname: Kato-Minoura
authors.forename: R
authors.initials: R
authors.lastname: Kamiya
authors.forename: M
authors.initials: M
authors.lastname: Hirono
mesh_terms: D000199:Actins / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002465:Cell Movement; D002696:Chlamydomonas / Q000235:genetics / Q000502:physiology*; D004252:DNA Mutational Analysis; D004398:Dyneins / Q000235:genetics / Q000378:metabolism*; D005306:Fertilization; D005407:Flagella / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic
chemical_list: D000199:Actins; D011993:Recombinant Fusion Proteins; C117538:actin-like protein, NAP; D004398:Dyneins