Recovery of Flagellar Inner-arm Dynein and the Fertilization Tubule in Chlamydomonas ida5 Mutant by Transformation with Actin Genes.

Abstract

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.

Impaired Flagellar Regeneration Due to Uncoordinated Expression of Two Divergent Actin Genes in Chlamydomonas

“…2). As shown previously (Ohara et al ., 1998), dynein recovery is due to the expression of CrA from the chimeric genes. However, only a small amount of CrA mRNA was transcribed in the TF5 cells, and, unlike in wild-type cells, NAP mRNA also was expressed (Fig.…”

“…The flagellar deficiency seems to be confined to the flagellation process, as flagellated cells recovered dynein composition and motility to the same degree as transformants with wildtype CrA (Fig. 2 and Table 1; Ohara et al, 1998). In contrast, TF10, in which the transforming construct comprises the CrA 5'-UTR region plus the NAP coding region, has normal flagellation activity (Fig.…”

“…The Chlamydomonas reinhardtii actin-less mutant, ida5 (Kato et al ., 1993) and a double mutant, ida5/arg2 (Ohara et al ., 1998), were used. The arginine-requiring mutant arg2 was obtained from Dr. E. Harris of the Chlamydomonas Genetic Center (Department of Botany, Duke University, Durham, NC).…”

“…In ida5 cells transformed with the genomic CrA gene, the level of NAP message is inversely related to the level of expressed actin protein (Ohara et al ., 1998). These observations led to the hypothesis that the expression of NAP is negatively regulated by actin.…”

CMF70 is a subunit of the dynein regulatory complex

“…The C. reinhardtii CMF70 ortholog is located on genome scaffold 44, which is encompassed by linkage group XIV (Merchant et al, 2007). The only mapped mutation overlapping with scaffold 44 is the ida5 locus (Merchant et al, 2007), which encodes actin (Ohara et al, 1998).…”