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. 1997 Jun 2;137(5):1069-80.
doi: 10.1083/jcb.137.5.1069.

The Chlamydomonas Reinhardtii ODA3 Gene Encodes a Protein of the Outer Dynein Arm Docking Complex

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Free PMC article

The Chlamydomonas Reinhardtii ODA3 Gene Encodes a Protein of the Outer Dynein Arm Docking Complex

A Koutoulis et al. J Cell Biol. .
Free PMC article

Erratum in

  • J Cell Biol 1997 Aug 11;138(3):729

Abstract

We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the uter ynein rm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737- 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83. 4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme.

Figures

Figure 1
Figure 1
Structure of outer dynein arm. (A) Diagrammatic representation of C. reinhardtii outer dynein arm, which consists of three DHCs (α, β, and γ), two ICs (69 and 78), and at least 10 LCs (8, 11, 14, 16, 18, 19, 20, and 22) (modified from King et al., 1995). (B–E) Electron micrographs of cross-sections of C. reinhardtii flagella. (B) Wild-type (g1). Note outer dynein arms (arrows) and inner dynein arms (arrowheads). The arms occupy precise positions on the A-tubule of the doublet microtubule. (C) oda9 insertional mutant lacking outer dynein arms (Wilkerson et al., 1995). Tiny projections at sites normally occupied by outer dynein arms are putative ODA-DCs (arrow). (D) oda3-4 insertional mutant (V40) lacking outer dynein arms. The profiles of the doublet microtubules at the sites normally occupied by the outer dynein arms are rounder than in oda9, indicating that this mutant lacks the ODA-DC (Takada and Kamiya, 1994). (E) oda3-4 insertional mutant rescued by transformation with the cosmid pK001. The outer dynein arms are completely restored.
Figure 1
Figure 1
Structure of outer dynein arm. (A) Diagrammatic representation of C. reinhardtii outer dynein arm, which consists of three DHCs (α, β, and γ), two ICs (69 and 78), and at least 10 LCs (8, 11, 14, 16, 18, 19, 20, and 22) (modified from King et al., 1995). (B–E) Electron micrographs of cross-sections of C. reinhardtii flagella. (B) Wild-type (g1). Note outer dynein arms (arrows) and inner dynein arms (arrowheads). The arms occupy precise positions on the A-tubule of the doublet microtubule. (C) oda9 insertional mutant lacking outer dynein arms (Wilkerson et al., 1995). Tiny projections at sites normally occupied by outer dynein arms are putative ODA-DCs (arrow). (D) oda3-4 insertional mutant (V40) lacking outer dynein arms. The profiles of the doublet microtubules at the sites normally occupied by the outer dynein arms are rounder than in oda9, indicating that this mutant lacks the ODA-DC (Takada and Kamiya, 1994). (E) oda3-4 insertional mutant rescued by transformation with the cosmid pK001. The outer dynein arms are completely restored.
Figure 3
Figure 3
Restriction maps of cosmid pK001 insert and plasmid inserts, and ability of the inserts to rescue motility in oda3. The number of Oda+ transformants out of the total number of Nit+ transformants examined is given in parentheses. The pK144 insert was the smallest piece of genomic C. reinhardtii DNA to rescue oda3. A 1-kb HindIII fragment was used to isolate cDNA clones. (Asterisk) A plasmid (pK143) was constructed that contained an insert with an inverted 6-kb XhoI fragment (arrow pointing right). In cotransformation experiments using pK143, one cell line was generated in which the rate of swimming was restored to near wild-type levels. EM indicated that outer dynein arms also were restored. However, in crosses to g1, mixed motility phenotypes were recovered, and the results could not be interpreted in terms of Mendelian segregation of a single gene (data not shown). Further study will be necessary to understand the basis for apparent rescue in this transformant.
Figure 2
Figure 2
Genetic analysis of ODA3. (A) Complementation in stable diploids demonstrates that V40 is an oda3 allele (referred to as oda3-4). Cell line 27D, a plus mating type (mt+) derivative of the insertional mutant V40, was mated to oda1 (mt−) and oda3-1 (mt−) mutants to generate stable diploids D93 and D98, respectively. Complementation (Oda+ phenotype) was observed in D93 but not in D98, indicating that the ODA1 gene is functional in V40, while the ODA3 gene is defective. To confirm that cell lines D93 and D98 were true stable diploids, genomic DNA from the indicated cell lines was digested with BamHI and probed in Southern blots with a 1.6-kb XbaI fragment of λ phage QK7 that is tightly linked to the mt locus. mt+ and mt− correlate with hybridization to ∼9- and ∼5-kb fragments, respectively, when using this probe. Hybridization to both ∼9- and ∼5-kb fragments in D93 and D98 indicates that these cell lines contain both mating types and thus are stable diploids. (B) Linkage between inserted DNA and Oda− phenotype in oda3-4. The insertional mutant oda3-4 (V40) (Oda− and Nit+ phenotypes) was crossed to B214 (Oda+ and Nit− phenotypes). Progeny were scored for motility (Oda+/−) and ability to grow on nitrate as sole nitrogen source (Nit+/−). Genomic DNA was isolated from the progeny, from oda3-4 (V40), and from the untransformed parental cell line (g1). The DNA was digested with PstI and probed in Southern blots with pUC119, which contains only the plasmid sequence used for transformation. The pUC119 sequence (3.5-kb fragment) is present in oda3-4 but not g1; in the progeny, the plasmid sequence segregates with the Oda− and Nit+ phenotypes, indicating that the Oda− phenotype is due to the inserted DNA. Data are shown for four products (27A–27D, bracketed) from one tetrad and individual products (28.1–33.1) from five other tetrads. (C) Linkage between rescuing DNA (pK001) and restored Oda+ phenotype. Cell line 98A (a nit2 derivative of oda3-4) was cotransformed with pMN68 (containing NIT2) and the cosmid pK001. One of the rescued transformants, T278, was crossed to oda3-4, and progeny were scored for motility (Oda+/−). Genomic DNA from 98A, T278, and the progeny was digested with PvuII and probed in Southern blots with the ODA3 probe, which flanked the original insertion in oda3-4 and is contained in pK001. The probe hybridizes to an ∼3.2-kb endogenous fragment, as well as to new fragments of ∼3.2 and ∼1.8 kb in T278. The new bands cosegregate with the Oda+ phenotype in the progeny from the cross of T278 and oda3-4, indicating that the Oda+ phenotype is linked to stably inserted pK001 DNA. Data are shown for 14 meiotic products (178A–183C) from six different zygotes.
Figure 4
Figure 4
Expression of ODA3. (A) The ODA3 gene is induced after deflagellation. Northern blot of C. reinhardtii wild-type (g1) mRNA isolated from nondeflagellated cells (Control) and from cells 30 min after deflagellation (Deflag.). The blot was probed on two different occasions and the resultant autoradiographs were superimposed. The 3.1-kb band represents mRNA hybridizing to the 1-kb HindIII fragment of pK001 (Fig. 3), which contains part of the ODA3 sequence; this mRNA is strongly induced in the deflagellated cells. The 1.5-kb band represents mRNA hybridizing to a PTX2 cDNA clone (pG557 insert, provided by G.J. Pazour); PTX2 is not induced by deflagellation (Pazour, G.J., and G.B. Witman, manuscript in preparation) and is used here to demonstrate that similar amounts of mRNA were loaded into each lane. (B) The ODA3 gene occurs once in the C. reinhardtii genome. Southern blot of C. reinhardtii wild-type (g1) genomic DNA digested with five different restriction enzymes and probed with a full-length ODA3 cDNA clone (pK101 insert). A single band was detected in each lane. (C) ODA3 produced by translation in vitro migrates as an ∼105,000-M r protein. Synthetic mRNA was prepared from ODA3 cDNA (pK101) and translated in a rabbit reticulocyte lysate system. An autoradiograph of an SDSpolyacrylamide gel of the product is shown. M r standards were β-galactosidase (116), phosphatase b (97), and BSA (66).
Figure 5
Figure 5
Nucleic acid and deduced amino acid sequence of ODA3. The numbers on the lefthand and righthand sides refer to nucleotides and amino acid residues, respectively. In-frame stop codons before and after the open reading frame are marked with asterisks. Underlined amino acids correspond to sequence obtained directly from tryptic fragments of the ∼105-kD polypeptide of the ODA-DC. Dotted underlines mark the imperfect 11– amino acid tandem repeat. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF001309.
Figure 6
Figure 6
Graphical representation of the probability of coiledcoil regions in the ODA3 gene product as determined by the NEWCOILS program (Matrix = MTDIX; with a 2.5-fold weighting of positions a and d).

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