Identification of all dynein heavy chain gene products in Chlamydomonas
Toshiki Yagi1, Masahide Kikkawa1, and Ritsu Kamiya2
1) Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, 113-0033, Tokyo, Japan
2) Department of Biological Science, Graduate School of Science, University of Tokyo, 113-0033, Tokyo, Japan
Chlamydomonas genome has 16 DHC genes, 15 of which have been correlated with particular DHC proteins: one cytoplasmic DHC that functions in retrograde intra-flagellar transport (IFT); three DHCs of outer-arm dynein; two DHCs of two-headed inner-arm dynein (dynein f); six DHCs of major single-headed inner-arm dynein (dynein a, b, c, d, e, and g); and three DHCs of recently identified minor single-headed inner-arm dynein (Yagi et al., 2009). Thus, one DHC gene product remains to be identified. This remaining DHC gene codes for a DHC with a significantly diverged amino-acid sequence and was previously thought to be a cytoplasmic type (Porter et al., 1999), but a recent phylogenetic analysis assigned it to an axonemal type (Wikstead and Gull, 2007). Here, we determined the cDNA sequence upstream of the AAA1 domain of this DHC, and raised a polyclonal antibody againist the corresponding polypeptide. Immunoblot analysis showed that this DHC is present in flagella. It was present in the mutant axoneme missing outer-arm dynein, but not in the axoneme of ida4, a mutant that has a mutation in the gene of an inner-arm light chain, p28, and lacks four inner-arm DHCs (DHC2, 6, 9, and 11). This finding suggests that the unassigned DHC is an inner-arm dynein. Our study has thus completed the assignment of all DHC genes in Chlamydomonas. This is the first time that the products of all DHC genes have been identified in any organism with cilia/flagella.
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