PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of mbcLink to Publisher's site
 
Mol Biol Cell. 1999 October; 10(10): 3507–3520.
PMCID: PMC25620

LC2, the Chlamydomonas Homologue of the t Complex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly

Tim Stearns, Monitoring Editor

Abstract

Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility.

INTRODUCTION

Dynein ATPases are microtubule-based molecular motors that provide force for a variety of important cellular processes (reviewed by Holzbaur and Vallee, 1994 blue right-pointing triangle; Witman et al., 1994 blue right-pointing triangle). Cytoplasmic dyneins are involved in vesicle transport, Golgi localization, nuclear migration, spindle formation and orientation, mitosis, and flagellar assembly. Inner arm and outer arm axonemal dyneins provide the force for flagellar and ciliary beating.

All characterized dyneins contain one or more heavy chains (HCs) that are associated with smaller polypeptides termed intermediate chains (ICs), light intermediate chains, and light chains (LCs). For example, Chlamydomonas outer arm dynein, which is the most well characterized axonemal dynein, contains three HCs, two ICs, and eight LCs (Table (Table1).1). Each HC has a globular head domain containing the site for ATP hydrolysis and a fibrous stem domain that extends to the base of the dynein, where the ICs and most of the LCs are located in a discrete complex. The heads of the HCs interact with the B-tubule of the opposing doublet microtubule to generate force, whereas the ICs are involved in anchoring the dynein to the A-tubule of the doublet microtubule (King et al., 1995 blue right-pointing triangle; Wilkerson et al., 1995 blue right-pointing triangle) and regulating dynein activity (Mitchell and Kang, 1993 blue right-pointing triangle). In contrast, little is known regarding the function(s) of the dynein LCs.

Table 1
Proteins associated with the outer dynein arm of Chlamydomonas

The need for more information on the dynein LCs has been underscored recently by 1) the discovery that LCs are associated with cytoplasmic dynein (King et al., 1996a blue right-pointing triangle), indicating that they are likely to have a universal role in dynein structure and/or function, and 2) the finding that two dynein LCs are encoded within a large region of mouse chromosome 17, which, in t mice, corresponds to the t haplotype (King et al., 1996b blue right-pointing triangle; Patel-King et al., 1997 blue right-pointing triangle). The t haplotype has four inversions relative to the wild-type chromosome; these inversions suppress recombination, so that mutations arising in the t haplotype are kept together (Silver, 1993 blue right-pointing triangle). This portion of chromosome 17 has been the subject of intense study because the t haplotype is inherited in a non-Mendelian manner. Heterozygous +/t males transmit the t-bearing chromosome to >95% of their offspring, a phenomenon known as transmission ratio distortion or meiotic drive. This is due to the concerted action of three or four t-encoded distorter genes acting on a t-encoded responder gene (Lyon, 1984 blue right-pointing triangle). In the presence of the t responder, the t distorters act in an additive manner to increase the percent of offspring that carry the t-bearing chromosome (for review, see Silver, 1985 blue right-pointing triangle, 1993 blue right-pointing triangle). Apparently the t-encoded versions of the distorter and responder genes contain mutations that give the t haplotype a selective advantage over the wild-type homologue. The identity and function of the responder gene product is unknown (Ewulonu et al., 1996 blue right-pointing triangle), but two of the putative distorter gene products (Tctex1 and Tctex2) are dynein LCs. Tctex1 (Lader et al., 1989 blue right-pointing triangle) is a homologue of a Chlamydomonas inner arm dynein LC (Harrison et al., 1998 blue right-pointing triangle) and also is a subunit of brain cytoplasmic dynein (King et al., 1996b blue right-pointing triangle). Tctex2 (Huw et al., 1995 blue right-pointing triangle) is a homologue of a Chlamydomonas outer arm dynein LC termed LC2 (Patel-King et al., 1997 blue right-pointing triangle). A third distorter gene product also may be a dynein subunit, because the Dnahc8 dynein HC (Vaughan et al., 1996 blue right-pointing triangle) maps at the site of the distorter gene Tcd2 (Harrison et al., 1998 blue right-pointing triangle). Consequently, it has been hypothesized that the non-Mendelian transmission of the t haplotype is due to its effect on sperm motility through dynein subunit interactions (Patel-King et al., 1997 blue right-pointing triangle; Harrison et al., 1998 blue right-pointing triangle). However, the mechanism by which this might work remains elusive.

Much information is now available on the structure of the Chlamydomonas outer dynein arm polypeptides, including the LCs. cDNAs encoding all 13 polypeptides in the outer arm have been isolated and sequenced; similarly, sequences have been obtained for the cDNAs that encode three subunits of the outer dynein arm docking complex (ODA-DC), a heterotrimeric structure closely associated with the outer arm and necessary for outer arm assembly (Takada and Kamiya, 1994 blue right-pointing triangle; Takada et al., 1996 blue right-pointing triangle; Koutoulis et al., 1997 blue right-pointing triangle; Casey et al., 1998 blue right-pointing triangle) (Table (Table1).1). In some cases, the sequences suggest possible functions for their respective gene products. However, more definitive information on the roles of the outer arm polypeptides has been obtained from analysis of mutants with defects in specific chains. Sixteen genes (ODA1–ODA14, PF13, and PF22) have been identified that affect outer arm assembly. Mutations in the ODA genes cause defects in the outer dynein arms and slow jerky swimming (Kamiya, 1988 blue right-pointing triangle), whereas defects in the PF genes cause outer arm defects and paralyzed flagella such that the cells are not motile (Huang et al., 1979 blue right-pointing triangle). Five of these genes encode the three dynein HCs and two ICs of the outer arm; three others encode subunits of the ODA-DC (Table (Table1).1). An additional gene, FLA14, encodes LC8, an Mr 8000 LC that is a subunit of outer arm dynein and also of cytoplasmic dynein, inner arm dynein I1, and myosin V (King and Patel-King, 1995b blue right-pointing triangle; King et al., 1996a blue right-pointing triangle; Espindola et al., 1996 blue right-pointing triangle; Harrison et al., 1998 blue right-pointing triangle; Pazour et al., 1998 blue right-pointing triangle). Mutations in most of the identified genes result in loss of the outer arm; deletion of FLA14 results in loss of intraflagellar transport (Rosenbaum et al., 1999 blue right-pointing triangle) and defects in the assembly of inner and outer arms and radial spokes. Unfortunately, no mutations have been identified in the genes that encode LCs specific for the outer arm dynein, so there is no genetic data on the roles of LC1–LC7 in outer arm assembly or function.

In an effort to learn more about the outer arm dynein LCs, we are taking a reverse genetics approach wherein we screen insertional mutants for defects in these chains. Insertional mutagenesis in Chlamydomonas is based on the fact that when Chlamydomonas is transformed, the exogenous DNA inserts at random into the nuclear genome and either disrupts a gene at the point of insertion or, more commonly, causes the deletion of a large block of DNA flanking the insertion site (Tam and Lefebvre, 1993 blue right-pointing triangle). In either case, the result is a restriction fragment length polymorphism (RFLP) that can be detected in Southern blots using a DNA probe for the affected gene. Inasmuch as cDNAs are available for all of the outer dynein arm LCs, it should be possible to use these cDNAs to identify mutants with defects in the LC genes. Indeed, we recently used this approach to identify the mutants in which LC8 was deleted (Pazour et al., 1998 blue right-pointing triangle).

In this work, we report two insertional mutants with defects in the gene encoding LC2, an outer arm dynein LC that is the homologue of the mouse Tctex2 protein. This gene previously was named ODA12 (Koutoulis et al., 1997 blue right-pointing triangle), but its product was not identified. Complete deletion of the LC2 gene results in complete loss of the outer dynein arm and impaired motility; transformation of the deletion mutant with the cloned LC2 gene restores the outer arm and rescues the motility phenotype. Therefore, LC2 has an essential role in outer arm assembly. This is the first mutation to be identified in an outer arm-specific LC, and the first evidence that loss of a single dynein LC can have a deleterious effect on flagellar function. The results suggest a specific model in which mutant (t haplotype-encoded) and wild-type dyneins differentially bind to axonemes of t-bearing or wild-type sperm, with resulting differences in their motility that ultimately lead to non-Mendelian transmission of the t locus.

MATERIALS AND METHODS

Strains

Chlamydomonas reinhardtii strains used in this work include g1 (nit1, agg1, mt+) (Pazour et al., 1995 blue right-pointing triangle), 1330.1 (ac14, nit1, NIT2, mt−) (Pazour, unpublished data), 137c (nit1, nit2, mt+), H8− (arg7, mt−), H11+ (arg7, mt+) (Tam and Lefebvre, 1993 blue right-pointing triangle), CC2290 (mt−) (Gross et al., 1988 blue right-pointing triangle), CC2236 (oda5, mt+) (Kamiya, 1988 blue right-pointing triangle), CC2240 (oda7, mt+) (Kamiya, 1988 blue right-pointing triangle), CC2242 (oda8, mt+) (Kamiya, 1988 blue right-pointing triangle), CC2492 (pf13a, mt+) (Huang et al., 1979 blue right-pointing triangle), and CC1382 (pf22, mt+) (Huang et al., 1979 blue right-pointing triangle). Strains produced in the course of this study include F56 (oda12-1::NIT1, ac14, nit1, mt−) and V3 (oda12-2::NIT1, nit1, mt+), generated by insertional mutagenesis of 1330.1 and g1, respectively, 2081.2 (oda12-1::NIT1, mt−; offspring of F56 × 137c cross), 2567.1 (oda12-1::NIT1, arg7; offspring of 2081.2 × H11+ cross), and transformants S1, S2, S3, S4, S5, and S20 obtained by transforming 2567.1 with ODA12 genomic clones and pARG7.8 (Debuchy et al., 1989 blue right-pointing triangle).

Growth Medium

Chlamydomonas was grown in the following media: M (Sager and Granick [1953] medium I altered to have 0.0022 M KH2PO4 and 0.00171 M K2HPO4), M − N (M medium without nitrogen), R (M medium plus 0.0075 M sodium acetate), R + Arg (R medium plus 50 μg/ml arginine), SGII/NO3 (Sager and Granick [1953] medium II modified to have 0.003 M KNO3 as the nitrogen source), and M − N + KNO3 (M − N medium plus 0.003 M KNO3).

Transformation

Transformation was performed using the glass bead method of Kindle (1990) blue right-pointing triangle as described by Pazour et al. (1995) blue right-pointing triangle. The original insertional mutant library was made by transforming strains g1 and 1330.1 with the linearized plasmid pGP505 containing the NIT1 gene (Fernandez et al., 1989 blue right-pointing triangle) as described previously (Pazour et al., 1995 blue right-pointing triangle; Koutoulis et al., 1997 blue right-pointing triangle). Strain 2567.1, an arg7 derivative of F56, was cotransformed with the pARG7.8 plasmid containing the ARG7 gene (Debuchy et al., 1989 blue right-pointing triangle) and phage or plasmid clones containing the LC2 gene. NIT1 transformants were selected on SGII/NO3 medium; ARG7 transformants were selected on R medium.

Analysis of Swimming Speeds

Swimming speed was calculated using an ExpertVision Motion Analysis (Santa Rosa, CA) system. Cells were observed with dim red illumination, and their positions were recorded every 67 ms by the MotionAnalysis system (Moss et al., 1995 blue right-pointing triangle). Subsequently, paths were determined, and the speed of individual cells was calculated using the speed operator. The final result is the average of >100 cells.

Genetic Analysis

Mating and tetrad analyses were performed as described by Levine and Ebersold (1960) blue right-pointing triangle and Harris (1989) blue right-pointing triangle. Cells of each mating type were grown on solid medium (R or R + Arg) and resuspended in M − N liquid medium. After pellicles became apparent in 1 or 2 d, the mixture was plated on solid M medium; the plates were allowed to dry and placed in the dark for 6–10 d. Zygotes were hatched on solid R or R + Arg medium and dissected using a glass needle. The meiotic progeny were allowed to grow for 3–5 d and then transferred to 5 ml of liquid R or R + Arg medium. Cells were allowed to grow for 2–5 d and then scored for motility by microscopic observation of cells illuminated with dim red light. The arg7 phenotype was scored by comparing cell growth on R versus R + Arg medium.

Electron Microscopy

Cells were fixed in glutaraldehyde (Hoops and Witman, 1983 blue right-pointing triangle) and processed as described by Wilkerson et al. (1995) blue right-pointing triangle.

Axoneme Isolation, Electrophoresis, and Immunoblotting

Wild-type and oda12 strains were deflagellated with dibucaine, and the resulting flagella were isolated by standard procedures (Witman, 1986 blue right-pointing triangle). After demembranation with Nonidet P-40, axonemes were placed in SDS sample buffer and heated at >95°C for several minutes. All samples were electrophoresed in 5–15% acrylamide gradient gels (King et al., 1986 blue right-pointing triangle). Gels were blotted to polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Waters, Milford, MA) by the two-step procedure of Otter et al. (1987) blue right-pointing triangle. To detect dynein polypeptides, blots were probed with monoclonal antibody 1878A (specific for IC1; King et al., 1985 blue right-pointing triangle), rabbit polyclonal EU51 (specific for IC140; Yang and Sale, 1998 blue right-pointing triangle), or affinity-purified rabbit polyclonal antibody R5391 (specific for LC2; Patel-King et al., 1997 blue right-pointing triangle) as described by Pazour et al. (1998) blue right-pointing triangle.

Cloning the ODA12 Locus

To obtain genomic clones of the ODA12 locus, Chlamydomonas genomic DNA was partially digested with Sau3AI and size fractionated by NaCl gradient centrifugation. The fraction containing DNA fragments in the 10- to 20-kbp range was ligated into the BamHI site of Lambda DashII (Stratagene, San Diego, CA) and packaged in vitro using Gigapack II extracts (Stratagene). The library was screened by hybridization with the LC2 cDNA, and three phage clones (λODA12#8, λODA12#17, and λODA12#20) were obtained. The EcoRI insert of λODA12#17 was subcloned into the EcoRI site of pKS+ (Stratagene) and named pHS44. pBD14 was constructed by cloning the 3.1-kbp HindIII fragment of pHS44 into the HindIII site of pKS+. pBD17 was constructed by cloning the 5.7-kbp KpnI–SalI fragment of pHS44 into pKS+ that had been cut with SalI and KpnI.

Other Procedures

DNA was isolated by digesting ~0.3 ml of packed cells with 0.5 ml of proteinase K (1 mg/ml) in 5% sodium lauryl sulfate, 20 mM EDTA, and 20 mM Tris, pH 7.5, at 50°C for 12-16 h. Ammonium acetate was added to 1.5 M, the mixture was extracted once with 50% phenol and 50% chloroform and once with chloroform, and then the DNA was precipitated with isopropyl alcohol. DNA was resuspended in 10 mM Tris and 1.0 mM EDTA, pH 8.0, and digested with PstI or PvuII. Gel electrophoresis and Southern blotting were performed according to standard procedures (Sambrook et al., 1987 blue right-pointing triangle).

RESULTS

Identification of Mutants with Defects in the LC2 Gene

We previously described the isolation of a large number of Chlamydomonas insertional mutants with defects in phototaxis, cell motility, and flagellar assembly (Pazour et al., 1995 blue right-pointing triangle, 1998 blue right-pointing triangle; Koutoulis et al., 1997 blue right-pointing triangle). Because the mutations in these cell lines usually result in RFLPs that can be detected in Southern blots probed with cDNAs encoding parts of the affected genes, it has opened the door to a reverse genetics approach wherein it is possible to identify mutants with specific defects in cloned genes (Wilkerson et al., 1995 blue right-pointing triangle; Pazour et al., 1998 blue right-pointing triangle, 1999 blue right-pointing triangle).

There are eight outer dynein arm LCs. cDNA clones encoding each of these have been isolated via protein sequence, but to date only one LC gene, FLA14, encoding LC8 (Pazour et al., 1998 blue right-pointing triangle), has been disrupted. We were particularly interested in finding a mutation that affected LC2, the homologue of the mouse Tctex2 protein. In an attempt to identify such a mutant, we screened our entire collection of Chlamydomonas insertional mutants by Southern blotting using the LC2 cDNA as a probe. Chlamydomonas contains a single copy of the LC2 gene, which is cut once by PvuII (Patel-king et al., 1997 blue right-pointing triangle). Thus, the LC2 cDNA detects two bands (0.9 and 3.5 kbp) on Southern blots of wild-type genomic DNA cut with this enzyme (Figure (Figure1A).1A).

Figure 1
Southern blot showing RFLPs in F56 and V3. (A) Genomic DNA was isolated from slow-swimming insertional mutants, cut with PvuII, and analyzed by Southern blotting using the LC2 cDNA as a probe. This probe detects 0.9- and 3.5-kbp bands in most cell lines, ...

One strain, F56, was missing both hybridizing bands (Figure (Figure1A),1A), indicating that the gene encoding LC2 is completely deleted in this strain. Previously, strain F56 had been briefly reported to contain a mutation that caused complete loss of the outer dynein arm and a slow-swimming phenotype (Koutoulis et al., 1997 blue right-pointing triangle). This mutant complemented oda1–oda11, pf13, and pf22 in stable diploids, indicating that it was defective in a new ODA gene, which was termed ODA12 (Koutoulis et al., 1997 blue right-pointing triangle; see below). The product of this gene was not identified, because no RFLPs were observed in Southern blots of F56 DNA probed with cDNAs encoding outer arm dynein HCs or ICs (Koutoulis et al., 1997 blue right-pointing triangle). We similarly did not detect RFLPs in Southern blots of F56 DNA probed with cDNAs encoding ODA-DC polypeptides or LCs other than LC2 (Pazour and Witman, unpublished data). Therefore, F56 does not appear to have a defect in any outer dynein arm-associated protein other than LC2.

Another cell line, V3, was missing the smaller of the two hybridizing bands (Figure (Figure1A).1A). V3 also had a slow-swimming phenotype (see below). To determine which part of the gene is deleted in V3, probes specific to each end of the LC2 cDNA were made by PCR amplification of the LC2 cDNA with T3 and T7 primers, digesting the product with PvuII, and gel-purifying the resulting 0.6- and 0.4-kbp bands. PvuII cuts the LC2 cDNA at only one site, between G614 and C615 in the ~550-bp 3′ untranslated region (UTR) (see Patel-King et al., 1997 blue right-pointing triangle, their Figure 1). Therefore, the 0.6-kbp probe corresponds to the 5′ UTR, the entire coding sequence, and a small amount (~60 bp) of the 3′ UTR; the 0.4-kbp probe corresponds to the remainder of the 3′ UTR. The 5′ probe detected the larger band in DNA from V3 cells and cells that were wild-type for LC2 (Figure (Figure1B).1B). The 3′ probe detected the smaller band in DNA from cells that were wild-type for LC2 but did not hybridize with any band in DNA from the V3 cells (Figure (Figure1B).1B). This indicates that the 3′ end of the gene is deleted in V3. Because the promoter and amino-terminal coding region are retained, these cells have the potential to produce at least a portion of LC2.

The LC2 Locus Is Tightly Linked to ODA12

To determine whether the slow-swimming phenotype was linked to the disruption of LC2, the F56 and V3 lines were back-crossed to wild-type cells. The swimming defect appears to be caused by a single nuclear mutation as the Oda phenotype segregated 2:2 in 25 tetrads obtained from a back-cross of F56. DNA was isolated from 15 offspring of the F56 cross and 19 offspring of the V3 cross and examined by Southern blotting using the LC2 cDNA as a probe. Figure Figure22 shows that whenever the LC2 gene was disrupted, the cells had an Oda-swimming phenotype. This strongly suggests that the ODA12 gene encodes LC2 of the outer dynein arm. The total deletion allele has been designated oda12-1; the partial deletion allele has been designated oda12-2.

Figure 2
The motility defect in the F56 and V3 strains segregates with the defect in the LC2 gene. (A) The initial F56 isolate was crossed to 137c, and a slow-swimming offspring was isolated and crossed again to 137c. Twenty-five tetrads were dissected from this ...

The discovery that the LC2 locus is tightly linked and probably identical to ODA12 provided an opportunity to obtain independent proof that ODA12 is a novel gene. CC2290 is a wild-type C. reinhardtii isolate that is highly interfertile with 137c-derived lab strains but divergent enough that it is easy to identify RFLPs between the two strains (Gross et al., 1988 blue right-pointing triangle). We identified an RFLP detected by the LC2 cDNA probe in DNA of CC2290 versus the 137c-derived strains (Figure (Figure3).3). We then compared the segregation of this RFLP with the segregation of uncloned genes that affect the outer arm in crosses between CC2290 and cell lines (derived from 137c) carrying mutations in the latter genes. Uncloned genes affecting the outer arm include ODA5, ODA7, ODA8, PF13, and PF22 (ODA1 [Takada et al., 1996 blue right-pointing triangle] and ODA10 [Pazour, Koutoulis, and Witman, unpublished data] have been cloned but not yet published). Tetrads from the crosses were dissected, the progeny were scored for motility, and DNA was isolated from one product of 10 different tetrads. The segregation of the LC2 PvuII RFLP was scored by Southern blotting and compared with the segregation of the motility defect. If the oda12 mutation is an allele of (or is tightly linked to) one of these genes, all offspring that have motility defects will have the 137c version of the LC2 PvuII RFLP. However, if oda12 is not an allele of one of these genes, the motility defect will segregate independently from the LC2 PvuII RFLP. As can be seen in Table Table2,2, the LC2 PvuII RFLP segregated independently of oda5, oda7, oda8, pf13, and pf22, confirming that ODA12 is a novel gene.

Figure 3
LC2 cDNA detects an RFLP between 137c and CC2290 strains. DNA from the lab strain 137c and the wild isolate CC2290 was cut with PvuII and analyzed by Southern blotting using the LC2 cDNA as a probe. This probe detects two bands of ~3.5 and ~0.9 ...
Table 2
Segregation of the LC2 RFLP

Phenotype of oda12 Cells

Most of the known defects in ODA1–ODA10 cause loss of the outer dynein arms. As a result, the cells swim in a jerky manner at ~30% of the normal speed. These mutant cells also have an altered photoshock response. During photoshock, which is induced by a flash of bright light, wild-type cells stop swimming, switch to a flagellar waveform, and swim backward for a few milliseconds (Schmidt and Eckert, 1976 blue right-pointing triangle). In contrast, oda mutants stop in response to the flash but do not swim backward (Mitchell and Rosenbaum, 1985 blue right-pointing triangle).

oda12-1 cells swim in a typical oda-like manner. They are slower than normal and have a jerky appearance as they swim forward. The swimming pattern of oda12-2 cells is not as slow or jerky as that of oda12-1 or other oda mutant cells. Swimming speeds were quantitatively measured with an ExpertVision Motion Analysis system (Figure (Figure4).4). The swimming speeds of wild-type (g1) cells are broadly distributed with a mean of 114 μm/s. The mean swimming speed of oda12-1 cells is reduced to 39 μm/s, whereas the speed of oda12-2 cells is slightly faster at 49 μm/s. Both oda12-1 and oda12-2 cells show an altered photoshock response; when flashed with light, they stop briefly and then resume forward swimming without a period of backward swimming.

Figure 4
Swimming speed of wild-type and oda12 mutant cells. The Speed Operator of the ExpertVision Motion Analysis system was used to calculate the swimming speed of individual cells. The histograms show the distribution of speeds within each sample. These are ...

LC2 Is Required for Assembly of the Outer Dynein Arm

Previously, we briefly reported that the oda12-1 mutant was lacking the outer dynein arms (Koutoulis et al., 1997 blue right-pointing triangle), a defect consistent with the swimming phenotype described above. Electron microscopic analysis indicates that whereas oda12-1 cells lack all of the outer dynein arms, oda12-2 cells are much more variable (Figure (Figure5A).5A). Some flagellar cross-sections have no outer dynein arms, whereas others have significant numbers. This indicates that the partial deletion does not completely abolish the assembly of outer arms.

Figure 5Figure 5
LC2 is required for assembly of outer dynein arms. (A) Electron micrographs of cross-sections of flagella of wild-type g1 cells show prominent outer dynein arms (arrows) on all of the doublet microtubules except for doublet one (arrowhead), which does ...

Western blot analysis was used to more quantitatively assess the loss of outer dynein arms in oda12-1 and oda12-2 cells. Axonemes were isolated from wild-type cells and from each of the mutants. Equal amounts of protein (standardized to tubulin) were separated by SDS-PAGE and transferred to a PVDF membrane. The blot was first probed with an antibody to an inner dynein arm subunit (IC140) that should not be affected by the oda12 mutations. All three lanes had similar amounts of this antigen, confirming that equal numbers of axonemes had been loaded in each lane on the gel (Figure (Figure5B).5B). The blot was then probed with antibodies to outer dynein arm subunits IC1 and LC2 (Figure (Figure5B).5B). Both proteins were readily detected in axonemes of wild-type cells. In contrast, neither protein was detected in axonemes of oda12-1 cells, confirming that LC2 and the outer dynein arm is completely missing in this mutant. However, small amounts of IC1 and LC2 were detected in oda12-2 cells; the oda12-2 LC2 appeared to migrate with the same relative mobility as the wild-type LC2. Therefore, oda12-2 cells produce small amounts of apparently full-length LC2. This is sufficient to permit assembly of some outer arms, although the loss of outer arms appears to be more severe than indicated by electron microscopy.

The oda12 Phenotype Can Be Rescued by Transformation with DNA Encoding LC2

To determine whether the oda12 phenotype is due specifically to loss of LC2, we assayed the ability of the LC2 gene to restore the wild-type phenotype to oda12 mutant cells. Three genomic clones encoding LC2 were isolated from a λ phage library using the LC2 cDNA clone as a probe. Two of these, λODA12#17 and λODA12#20, had very similar restriction patterns. The third, λODA12#8, was shifted along the chromosome slightly from the first two (Figure (Figure6A).6A). The LC2 coding region (as determined by hybridization) is contained completely within a 3.1-kbp HindIII fragment common to all three clones (Figure (Figure6A,6A, dark bar). DNA from each of these three λ phage was transformed into the cell line 2567.1 (which carries oda12-1 and arg7 mutations), along with DNA containing the ARG7 gene. Individual ARG7 transformants were screened by light microscopy to determine whether the oda12 motility phenotype had been rescued as a result of cotransformation with the phage DNA. All three phage clones were able to rescue the defect (Figure (Figure6A),6A), indicating that each carried the complete ODA12 gene. The insert from λODA12#17 was subcloned into a plasmid vector and named pHS44; as expected, this also was able to rescue the motility defect. The locus was further refined by subcloning the 5.7-kbp KpnI-SalI and the 3.1-kbp HindIII fragments, which were termed pBD17 and pBD14, respectively. Both of these fragments complemented the oda12 defect (Figure (Figure6A),6A), indicating that ODA12 is located within the 3.1-kbp fragment that also contained the LC2 coding region as determined by hybridization. This data provide very strong evidence that the outer dynein arm defect of oda12 is the result of the LC2 deletion.

Figure 6
Rescue of oda12 by transformation. (A) Map of the ODA12 locus. Three genomic λ phage clones (λODA12#8, λODA12#17, and λODA12#20) were isolated and mapped. The EcoRI sites at the ends of the clones ...

The swimming speed of five independent transformants was measured. All swam faster than the oda12-1 mutant line and had recovered the ability to swim backward when photoshocked. Transformants S3 (from λODA12#20), S4 (from λODA12#20), and S5 (from λODA12#8) were similar to wild-type in both mean swimming speed and the distribution of swimming speeds (cf. Figures Figures44 and and6B).6B). Transformant S2 (from λODA12#17) was similar to wild-type except for a greater number of very slow cells that reduced its mean speed to 82 μm/s (Figure (Figure6B).6B). The swimming speeds of transformant S1 (from λODA12#17) were very broadly distributed around a mean of 81 μm/s (Figure (Figure6B).6B). The mixed distribution of swimming speeds seen in the S1 and S2 populations was not the result of an impure culture, because the cells were cloned from a single cell before analysis. More likely, it was the result of gene silencing, whereby newly integrated genes in some Chlamydomonas cells become transcriptionally inactivated (Cerutti et al. 1997 blue right-pointing triangle).

To confirm that the rescue of the Oda− phenotype was due to integration of the cloned DNA and not suppression of the mutant phenotype, transformant S20 (obtained by transformation of 2507.1 with pHS44), which has wild-type motility, was crossed to an oda12-1 line and the resultant tetrads dissected. Motility of the offspring was scored, and DNA was isolated and analyzed by Southern blotting using the LC2 cDNA as a probe (Figure (Figure6C).6C). This probe revealed that the two PvuII restriction fragments observed in wild-type cells, but missing in the oda12-1 line, were restored in the rescued cell lines. Analysis of one full tetrad and single products of 12 additional tetrads showed that whenever the two PvuII bands were present, the cells swam with wild-type speeds. This indicates that restoration of the phenotype is due to the cloned DNA.

Transformants S1 and S3 were examined by electron microscopy to determine whether the outer dynein arms had been restored (Figure (Figure6D).6D). Transformant S3, which had a distribution of swimming speeds similar to that of wild type, had a full complement of outer dynein arms. In contrast, cells of the transformant S1, which had a broader distribution of swimming speed and a lower mean swimming speed, showed a corresponding variability in the restoration of arms. Most of the flagellar cross-sections showed intermediate numbers of arms, but in a few, no arms were present, suggesting that the intermediate swimming speed was due to restoration of only some of the outer dynein arms.

DISCUSSION

In this report we describe, for the first time, mutations in the C. reinhardtii gene encoding LC2, an LC that is specific for the outer dynein arm. Deletion of this gene leads to loss of the outer dynein arm and impaired flagellar motility. The LC2 gene is the Chlamydomonas homologue of mouse Tctex2 (Patel-King et al., 1997 blue right-pointing triangle), which has been identified as one of the distorter genes of the t haplotype (Huw et al., 1995 blue right-pointing triangle). The finding that LC2 is essential for outer arm assembly supports the hypothesis that some of the phenotypes expressed by t haplotypes are due to interactions between defective dynein polypeptides and suggests a specific model by which this might occur.

Function of LC2

Although mutations are known for each of the HCs and ICs of the Chlamydomonas outer dynein arm, previously a mutation has been identified in only one gene (FLA14) that encodes an outer arm LC, LC8 (Pazour et al., 1998 blue right-pointing triangle). However, LC8 also is a component of inner arm dynein, cytoplasmic dynein, and myosin (Espindola et al., 1996 blue right-pointing triangle; King et al., 1996a blue right-pointing triangle; Harrison et al., 1998 blue right-pointing triangle), and knockout of the LC8 gene has pleiotropic effects that are difficult to interpret in terms of the specific role of LC8 in the outer dynein arm. In contrast, LC2 appears to be specific for the outer arm. Complete deletion of the LC2 gene causes a complete loss of the outer dynein arm, with no other structural changes in the flagellar axoneme. Loss of the outer arms in this mutant causes a decrease in swimming speed to ~34% of that of wild-type cells, a reduction similar to what has been observed for other mutations that cause complete loss of the outer dynein arm (Mitchell and Rosenbaum, 1985 blue right-pointing triangle; Kamiya, 1988 blue right-pointing triangle). Loss of the outer arm also causes loss of the ability to swim backward during the photoshock response, as reported previously for a mutant with a defect in the outer arm (Mitchell and Rosenbaum, 1985 blue right-pointing triangle).

It is most remarkable that LC2, which has a mass of only 15.8 kDa (King and Patel-King, 1995a blue right-pointing triangle), is absolutely essential for assembly of the outer dynein arm, which contains at least 13 different polypeptides and has a total mass of ~2 MDa (Table (Table1;1; Witman et al., 1983 blue right-pointing triangle). Two independent lines of evidence indicate that LC2 is a component of the IC–LC complex (Mitchell and Rosenbaum, 1986 blue right-pointing triangle; King and Witman, 1990 blue right-pointing triangle; Witman et al., 1992 blue right-pointing triangle), which is located at the base of the dynein (King and Witman, 1990 blue right-pointing triangle). First, LC2 is associated with the β subunit of the outer dynein arm (Pfister and Witman, 1984 blue right-pointing triangle) and is retained in the Chlamydomonas mutant oda4-s7, which expresses only the NH2-terminal one-third of the β HC (Sakakibara et al., 1993 blue right-pointing triangle). This portion of the β HC forms the stem and base of the outer arm dynein (Witman et al., 1994 blue right-pointing triangle) and binds the IC–LC complex (King and Witman, 1989 blue right-pointing triangle). Second, after dissociation of the outer arm dynein with nonionic detergent, LC2 was immunoprecipitated as part of an aggregate with IC1, IC2, and another LC (Mitchell and Rosenbaum, 1986 blue right-pointing triangle). Therefore, by virtue of its location in the arm, LC2 is in a position to interact directly with tubulin or the ODA-DC and might be required for binding a preassembled outer dynein arm to the doublet microtubule. Alternatively, LC2 might be required for preassembly of the outer dynein arm in the cytoplasm (Fowkes and Mitchell, 1998 blue right-pointing triangle), for stability of the preassembled complex, or even for transport of the preassembled complex into the flagellum. Whatever the precise reason, the current findings demonstrate that LC2 has an important role in dynein assembly. It is likely that the LCs of other dyneins have equally important roles in the complexes of which they are a part.

LC2 is Encoded by ODA12

We previously identified a novel Chlamydomonas gene, ODA12, that was necessary for outer arm assembly (Koutoulis et al., 1997 blue right-pointing triangle); however, the product of this gene was not determined. The following evidence now indicates that ODA12 encodes LC2: 1) Southern hybridization showed that the gene encoding LC2 is completely deleted in the original oda12 mutant strain; 2) the defining phenotype of oda12 cells segregated with the LC2 deletion in crosses between oda12 and wild-type cells; and 3) the oda12 phenotype was rescued by transformation of oda12 cells with a small (3.1-kbp) genomic DNA clone containing the LC2 gene. Thus, the phenotype observed for oda12 cells is due to the deletion of the LC2 gene.

The current findings also confirm that ODA12 is a novel gene, distinct from any of the other ODA or similar genes that have been reported. Previously, we reported that cDNA clones encoding ODA1, ODA2, ODA3, ODA4, ODA6, ODA9, and ODA11 detected no RFLPs in oda12 versus wild-type strains, and that oda12 complemented oda1, oda3, oda5, oda7, oda8, oda10, pf13, and pf22 in stable diploids (Koutoulis et al., 1997 blue right-pointing triangle). Similarly, we determined that cDNA clones encoding the unpublished genes ODA13 and ODA14 (Table (Table1)1) did not detect RFLPs in oda12 versus wild-type strains (Pazour and Witman, unpublished results). We now have used the LC2 cDNA as a physical marker for the ODA12 gene and find that an RFLP, detected by the cDNA in CC2290 versus 137c-derived strains, segregates independently of oda5, oda7, oda8, pf13, and pf22. These results provide additional evidence that ODA12 cannot be the same as any of those genes.

With the discovery that the ODA12 gene product is an outer dynein arm LC, only four known ODA genes (ODA5, ODA7, ODA8, and ODA10) and two known PF genes (PF13 and PF22) that affect outer arm assembly have yet to have their products identified. It will be of great interest to determine the products of these genes and their roles in outer arm assembly. It may not be coincidental that the products of six ODA or ODA-like genes are still unknown, whereas mutations have not yet been reported for an identical number of outer dynein arm LCs (Table (Table1).1). The current findings underscore the possibility that some of these other LCs also could be critical for dynein assembly. The ease with which a reverse genetics approach may be applied to Chlamydomonas should greatly facilitate the identification of mutants with defects in these polypeptides.

oda12-2 Is a Hypomorphic Allele

The results presented here show that the oda12 mutation originally reported by Koutoulis et al. (1997) blue right-pointing triangle is a complete deletion of the LC2 gene; this allele has been designated oda12-1. Here we also describe a second allele (oda12-2) in which only the 3′ end of the LC2 gene is deleted. Ultrastructural analysis revealed that the oda12-2 mutant assembles only a few outer arms on its doublet microtubules (Figure (Figure5A).5A). Presumably because some outer arms are present, oda12-2 cells swim slightly faster than oda12-1 cells (Figure (Figure4A).4A). Western blot analysis confirmed that the outer dynein arm polypeptide IC1 is greatly diminished but not entirely missing in axonemes of oda12-2 (Figure (Figure5B).5B). Interestingly, a small amount of approximately full-length LC2 also is present in the oda12-2 axoneme. That an apparently full-length product is synthesized suggests that the oda12-2 deletion removes most or all of the 3′ UTR but little if any of the coding sequence. The 3′ UTR of eukaryotic mRNAs has several important functions, including control of mRNA stability (Beelman and Parker, 1995 blue right-pointing triangle; Decker and Parker, 1995 blue right-pointing triangle; Wickens et al., 1997 blue right-pointing triangle) and mRNA localization (Wilhelm and Vale, 1993 blue right-pointing triangle; Decker and Parker, 1995 blue right-pointing triangle). A defect in either of these functions likely would result in the production of reduced amounts of LC2, which in turn would permit assembly of reduced numbers of outer arms. The incorporation of small amounts of LC2 along with equally small amounts of IC1 into the oda12-2 axonemes is consistent with LC2 being absolutely essential for outer dynein arm assembly.

Dynein Defects and t Haplotype Phenotypes

The mouse t haplotype is an ~30-Mb region of chromosome 17 containing numerous genes and defined by the presence of four inversions relative to the wild-type homologue, so that recombination is suppressed and the entire region usually segregates as a single unit (Olds-Clarke and Johnson, 1993 blue right-pointing triangle; Silver, 1993 blue right-pointing triangle). One of the most intriguing phenotypes of the t haplotype is that heterozygous +/t males transmit the t haplotype-bearing version of chromosome 17 to >95% of their progeny. Analysis of partial t haplotypes resulting from rare recombination events within the t complex indicate that this transmission ratio distortion, or meiotic drive, is the result of two or three distorter genes, located in different parts of the t haplotype, acting on a single responder gene, termed Tcr (t complex responder) (Lyon, 1984 blue right-pointing triangle). The distorter genes can act cis or trans on the responder gene, and their effects are additive. The result of these interactions is to increase the probability of transmission of the chromosome carrying the t haplotype responder gene.

The discovery that some of the candidate distorter genes encode dynein LCs has prompted a model that explains the t haplotype phenotype on the basis of interactions between dyneins and other axonemal components (Patel-King et al., 1997 blue right-pointing triangle; Harrison et al., 1998 blue right-pointing triangle). The current finding that LC2 is important for dynein assembly permits the model to be described in more specific terms (Figure (Figure7).7). In this model, the responder gene encodes a protein (Tcr) that is expressed in the haploid nucleus and remains associated with that nucleus throughout the remainder of spermiogenesis. Because soluble components can diffuse through the intercellular bridges that connect developing spermatids, this protein must become rapidly associated with some structural component of the spermatid, such as the basal body or the elongating axoneme, to prevent its diffusion to neighboring spermatids. Ultimately, Tcr must interact with the dynein arms. For the sake of simplicity, we will assume that Tcr is part of an axonemal component such as the ODA-DC (Takada and Kamiya, 1994 blue right-pointing triangle; Koutoulis et al., 1997 blue right-pointing triangle) or the dynein regulatory complex (Gardner et al., 1994 blue right-pointing triangle; Piperno et al., 1994 blue right-pointing triangle) that interacts directly with the dynein arms, but it could interact with the arms indirectly, as through a basal body-associated templating activity that determines the location of the outer dynein arm binding sites on the doublet microtubule. In contrast, the distorter proteins, which include subunits of the inner and outer dynein arms, diffuse through the intercellular bridges, so that both wild-type and mutant (t haplotype-encoded) dyneins are preassembled in the cytoplasm of both spermatids bearing the wild-type responder (Tcr+) and spermatids bearing the t haplotype-encoded responder (Tcrt).

Figure 7
Model for mouse t phenotypes based on dynein–axoneme interactions. In all cases, the distorter gene (d) products, which are dynein subunits, are free to diffuse (arrows) through the intercellular bridges that connect developing spermatids. However, ...

The model predicts that Tcrt binds wild-type dynein strongly but mutant dynein less efficiently. In +/t mice (Figure (Figure7A),7A), both types of dyneins will be present in the cytoplasm of the developing spermatid. The wild-type dyneins will out-compete the mutant dyneins for binding sites on the Tcrt-containing axoneme, with the result that these axonemes will assemble a nearly normal complement of arms and have motility that is only slightly impaired because of the presence of Tcrt (see below). In contrast, the model predicts that Tcr+ binds both wild-type and mutant dyneins equally, with the result that both types of dyneins will be assembled into the Tcr+-containing axonemes. Because of the presence of many mutant dyneins in these latter axonemes, the resulting sperm will be much less efficient than those containing Tcrt in some critical aspect of motility, resulting in transmission ratio distortion.

A second phenotype of the t haplotype that is readily accounted for by this model is that t/t males are sterile (Figure (Figure7B).7B). t/t males will produce only mutant dynein, which has a low affinity for Tcrt. As a result, few arms will be assembled into the axoneme, and if they are assembled they will be defective. The resulting sperm will have aberrant or no motility and be unable to fertilize an egg.

In an analysis of partial t haplotypes, it was observed that if a t-encoded responder was present on one chromosome, with no t-encoded distorters acting on it cis or trans, then that chromosome was transmitted at a low ratio (11–17%) (Lyon, 1984 blue right-pointing triangle). This suggests that the mutant responder itself impairs the motility of Tcrt-bearing sperm. This also is compatible with our model (Figure (Figure7C).7C). In spermatids of such mice, only wild-type dynein is present, and it binds to both Tcrt- and Tcr+-containing axonemes. The resulting Tcrt-containing sperm will have slightly impaired motility as a result of having Tcrt in their axonemes. This motility will not be so compromised that the sperm are unable to fertilize an egg and not as defective as that of Tcr+-containing spermatids with a mixture of mutant and wild-type dyneins (see Figure Figure7A).7A). Nevertheless, they will have a competitive disadvantage, compared with Tcr+-containing sperm with only wild-type dyneins, in reaching and fertilizing the egg.

The sperm motility phenotypes predicted by this model are in good agreement with observations of sperm from +/t and t/t mice (summarized by Harrison et al., 1998 blue right-pointing triangle). For example, sperm from t/t mice rarely exhibit a regular beat pattern and have little progressive motility, whereas the mean swimming speed of sperm from +/t mice is intermediate between that of t/t and +/+ mice (Olds-Clarke and Johnson, 1993 blue right-pointing triangle). Moreover, sperm from +/t mice separate into two subpopulations, one having nearly normal motility and the other having abnormal motility (Olds-Clarke and Johnson, 1993 blue right-pointing triangle); the former may correspond to Tcrt-containing sperm, and the latter may correspond to Tcr+-containing sperm. Certainly these motility defects could be accounted for by defects in dynein arm assembly and/or function. As indicated by our analysis of the oda12-2 mutant compared with oda12-1 and wild-type cells, intermediate levels of outer dynein arms can result in intermediate swimming speeds. Furthermore, Chlamydomonas cells with defects in both the inner and outer arms have a more severe phenotype than those with a defect in either arm alone (Kamiya et al., 1989 blue right-pointing triangle), which is consistent with the additive effects of the distorter genes in mice (Lyon, 1984 blue right-pointing triangle). Finally, at least some of the motility defects observed in sperm of +/t mice are dependent on Ca2+ (Olds-Clarke and Johnson, 1993 blue right-pointing triangle), and both inner and outer dynein arms are involved in Ca2+-mediated changes in flagellar waveform. For example, in Chlamydomonas, the inner arms are involved in phototactic steering (King and Dutcher, 1997 blue right-pointing triangle), and mutants with defects in the outer arm are defective in backward swimming during photoshock (Mitchell and Rosenbaum, 1985 blue right-pointing triangle; this report); both of these responses are mediated by Ca2+.

Conclusion

The above model should be readily testable by further molecular genetic and biochemical studies of t haplotype-bearing mice. Of particular importance will be identification of the products of the Tcr gene and of the remaining distorter genes. In any case, the findings presented here demonstrate for the first time that a defect in a dynein LC can have a profound effect on outer arm assembly. It is likely that the LCs have equally important roles in other dyneins. Because oda12-1 is a null mutant, it should now be possible to investigate LC function by in vitro mutagenesis of the ODA12 gene, followed by transformation of the modified gene into the oda12-1 mutant, where the modified gene product should be assembled into an outer dynein arm. The effects of the LC modification on flagellar movement and dynein activity in vitro could then be determined.

ACKNOWLEDGMENTS

We thank Dr. J. Aghajanian (Worcester Foundation for Biomedical Research, Shrewsbury, MA) for the electron microscopy presented in this study and Drs. P. Yang and W. Sale (Emory University, Atlanta, GA) for the gift of the anti-IC140 antibody. These studies were supported by grants from the National Institutes of Health (GM30626 to G.B.W. and GM51293 to S.M.K.) and the Campbell and Hall Charity Fund (to G.B.W.).

Abbreviations used:

HC
heavy chain
IC
intermediate chain
LC
light chain
ODA-DC
outer dynein arm docking complex
PVDF
polyvinylidene difluoride
RFLP
restriction fragment length polymorphism
UTR
untranslated region

REFERENCES

  • Beelman CA, Parker R. Degradation of mRNA in eukaryotes. Cell. 1995;81:179–183. [PubMed]
  • Benashski SE, Patel-King RS, King SM. Light chain 1 from the Chlamydomonas outer dynein arm is a leucine-rich repeat protein associated with the motor domain of the gamma heavy chain. Biochemistry. 1999;38:7253–7264. [PubMed]
  • Bowman AB, Patel-King RS, Benashski SE, McCaffery M, Goldstein LSB, King SM. Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility and mitosis. J Cell Biol. 1999;146:165–180. [PMC free article] [PubMed]
  • Casey DM, Pazour GJ, Wilkerson CG, Inaba K, Koutoulis A, Takada S, Kamiya R, Witman GB. Identification and insertional mutagenesis of a new Chlamydomonas reinhardtii gene, oda 14, that encodes the 25-kd subunit of the outer dynein arm docking complex. Mol Biol Cell. 1998;9:155a.
  • Cerutti H, Johnson AM, Gillham NW, Boynton JE. A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression. Genetics. 1997;145:97–110. [PubMed]
  • Debuchy R, Purton S, Rochaix J-D. The arginosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J. 1989;8:2803–2809. [PubMed]
  • Decker CJ, Parker R. Diversity of cytoplasmic functions for the 3′ untranslated region of eukaryotic transcripts. Curr Opin Cell Biol. 1995;7:386–392. [PubMed]
  • Espindola FS, Cheney RE, King SM, Suter DM, Mooseker MS. Myosin-V and dynein share a similar light chain. Mol Biol Cell. 1996;7:372a.
  • Ewulonu UK, Schimenti K, Kuemerle B, Magnuson T, Schimenti J. Targetted mutagenesis of a candidate t complex responder gene in Mouse t haplotypes does not eliminate transmission ratio distortion. Genetics. 1996;144:785–792. [PubMed]
  • Fernandez E, Schnell R, Ranum LPW, Hussey SC, Silflow CD, Lefebvre PA. Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA. 1989;86:6449–6453. [PubMed]
  • Fowkes ME, Mitchell DR. The role of preassembled cytoplasmic complexes in assembly of flagellar dynein subunits. Mol Biol Cell. 1998;9:2337–2347. [PMC free article] [PubMed]
  • Gardner LC, O’Toole E, Perrone CA, Giddings T, Porter ME. Components of a “dynein regulatory complex” are located at the junction between the radial spokes and the dynein arms in Chlamydomonas flagella. J Cell Biol. 1994;127:1311–1325. [PMC free article] [PubMed]
  • Gross CH, Ranum LPW, Lefebvre PA. Extensive restriction fragment length polymorphisms in a new isolate of Chlamydomonas reinhardtii. Curr Genet. 1988;13:503–508. [PubMed]
  • Harris EH. The Chlamydomonas Sourcebook. San Diego: Academic Press; 1989.
  • Harrison A, Olds-Clarke P, King SM. Identification of the t complex-encoded cytoplasmic dynein light chain Tctex1 in inner arm I1 supports the involvement of flagellar dynein in meiotic drive. J Cell Biol. 1998;140:1137–1147. [PMC free article] [PubMed]
  • Holzbaur ELF, Vallee RB. DYNEINS: molecular structure and cellular function. Annu Rev Cell Biol. 1994;10:339–372. [PubMed]
  • Hoops HJ, Witman GB. Outer doublet heterogeneity reveals structural polarity related to beat direction in Chlamydomonas flagella. J Cell Biol. 1983;97:902–908. [PMC free article] [PubMed]
  • Huang B, Piperno G, Luck DJL. Paralyzed flagella mutants of Chlamydomonas reinhardtii defective for axonemal doublet microtubule arms. J Biol Chem. 1979;254:3091–3099. [PubMed]
  • Huw L-Y, Goldsborough AS, Willison K, Artzt K. Tctex2: a sperm tail surface protein mapping to the. t-complex. Dev Biol. 1995;170:183–194. [PubMed]
  • Kamiya R. Mutations at twelve independent loci result in absence of outer dynein arms in Chlamydomonas reinhardtii. J Cell Biol. 1988;107:2253–2258. [PMC free article] [PubMed]
  • Kamiya R, Kurimoto E, Sakakibara H, Okagaki T. A genetic approach to the function of inner and outer arm dynein. In: Warner FD, Satir P, Gibbons LR, editors. Cell Movement, vol. I: The Dynein ATPases. New York: Alan R. Liss; 1989. pp. 209–218.
  • Kindle KL. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA. 1990;87:1228–1232. [PubMed]
  • King SJ, Dutcher SK. Phosphoregulation of an inner dynein arm complex in Chlamydomonas reinhardtii is altered in phototactic mutant strains. J Cell Biol. 1997;136:177–191. [PMC free article] [PubMed]
  • King SM, Barbarese E, Dillman JF, III, Patel-King RS, Carson JH, Pfister KK. Brain cytoplasmic and flagellar outer arm dyneins share a highly conserved Mr 8,000 light chain. J Biol Chem. 1996a;271:19358–19366. [PubMed]
  • King SM, Dillman JF, III, Benashski SE, Lye RJ, Patel-King RS, Pfister KK. The mouse t-complex-encoded protein Tctex-1 is a light chain of brain cytoplasmic dynein. J Biol Chem. 1996b;271:32281–32287. [PubMed]
  • King SM, Otter T, Witman GB. Characterization of monoclonal antibodies against Chlamydomonas flagellar dyneins by high-resolution protein blotting. Proc Natl Acad Sci USA. 1985;82:4717–4721. [PubMed]
  • King SM, Otter T, Witman GB. Purification and characterization of Chlamydomonas flagellar dyneins. Methods Enzymol. 1986;134:291–306. [PubMed]
  • King SM, Patel-King RS. Identification of a Ca2+-binding light chain within Chlamydomonas outer arm dynein. J Cell Sci. 1995a;108:3757–3764. [PubMed]
  • King SM, Patel-King RS. The Mr = 8,000 and 11,000 outer arm dynein light chains from Chlamydomonas flagella have cytoplasmic homologues. J Biol Chem. 1995b;270:11445–11452. [PubMed]
  • King SM, Patel-King RS, Wilkerson CG, Witman GB. The 78,000-Mr intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein. J Cell Biol. 1995;131:399–409. [PMC free article] [PubMed]
  • King SM, Witman GB. Molecular structure of Chlamydomonas outer arm dynein. In: Warner FD, Satir P, Gibbons LR, editors. Cell Movement, vol. I: The Dynein ATPases. New York: Alan R. Liss; 1989. pp. 61–75.
  • King SM, Witman GB. Localization of an intermediate chain of outer arm dynein by immunoelectron microscopy. J Biol Chem. 1990;265:19807–19811. [PubMed]
  • Koutoulis A, Pazour GJ, Wilkerson CG, Inaba K, Sheng H, Takada S, Witman GB. The Chlamydomonas reinhardtii ODA3 gene encodes a protein of the outer dynein arm docking complex. J Cell Biol. 1997;137:1069–1080. [PMC free article] [PubMed]
  • Lader E, Ha H-S, O’Neill M, Artzt K, Bennett D. tctex-1: a candidate gene family for a mouse t complex sterility locus. Cell. 1989;58:969–979. [PubMed]
  • Levine RP, Ebersold WT. The genetics and cytology of Chlamydomonas. Annu Rev Microbiol. 1960;14:197–216. [PubMed]
  • Lyon M. Transmission ratio distortion in mouse t-haplotypes is due to multiple distorter genes acting on a responder locus. Cell. 1984;37:621–628. [PubMed]
  • Mitchell DR, Brown KS. Sequence analysis of the Chlamydomonas alpha and beta dynein heavy chain genes. J Cell Sci. 1994;107:635–644. [PubMed]
  • Mitchell DR, Brown KS. Sequence analysis of the Chlamydomonas reinhardtii flagellar α dynein gene. Cell Motil Cytoskeleton. 1997;37:120–126. [PubMed]
  • Mitchell DR, Kang Y. Reversion analysis of dynein intermediate chain function. J Cell Sci. 1993;105:1069–1078. [PubMed]
  • Mitchell DR, Rosenbaum JL. A motile Chlamydomonas flagellar mutant that lacks outer dynein arms. J Cell Biol. 1985;100:1228–1234. [PMC free article] [PubMed]
  • Mitchell DR, Rosenbaum JL. Protein-protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms. Cell Motil Cytoskeleton. 1986;6:510–520. [PubMed]
  • Moss AG, Pazour GJ, Witman GB. Assay of Chlamydomonas phototaxis. Methods Cell Biol. 1995;47:281–287. [PubMed]
  • Olds-Clarke P, Johnson LR. t haplotypes in the mouse compromise sperm flagellar function. Dev Biol. 1993;155:14–25. [PubMed]
  • Otter T, King SM, Witman GB. A two-step procedure for efficient electrotransfer of both high-molecular (>400,000) and low-molecular-weight (<20,000) proteins. Anal Biochem. 1987;162:370–377. [PubMed]
  • Patel-King RS, Benashski SE, Harrison A, King SM. Two functional thioredoxins containing redox-sensitive vicinal dithiols from the Chlamydomonas outer dynein arm. J Biol Chem. 1996;271:6283–6291. [PubMed]
  • Patel-King RS, Benashski SE, Harrison A, King SM. A Chlamydomonas homologue of the putative murine t-complex distorter Tctex-2 is an outer arm dynein light chain. J Cell Biol. 1997;137:1081–1090. [PMC free article] [PubMed]
  • Pazour GJ, Dickert BL, Witman GB. The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly. J Cell Biol. 1999;144:473–481. [PMC free article] [PubMed]
  • Pazour GJ, Sineshchekov OA, Witman GB. Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii. J Cell Biol. 1995;131:427–440. [PMC free article] [PubMed]
  • Pazour GJ, Wilkerson CG, Witman GB. A dynein light chain is essential for the retrograde particle movement of intraflagellar transport (IFT) J Cell Biol. 1998;141:979–992. [PMC free article] [PubMed]
  • Pfister KK, Witman GB. Subfractionation of Chlamydomonas 18 S dynein into two unique subunits containing ATPase activity. J Biol Chem. 1984;259:12072–12080. [PubMed]
  • Piperno G, Mead K, LeDizet M, Moscatelli A. Mutations in the “dynein regulatory complex” alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes. J Cell Biol. 1994;125:1109–1117. [PMC free article] [PubMed]
  • Rosenbaum JL, Cole DG, Diener DR. Intraflagellar transport: the eyes have it. J Cell Biol. 1999;144:385–388. [PMC free article] [PubMed]
  • Sager R, Granick S. Nutritional studies with Chlamydomonas reinhardi. Ann NY Acad Sci. 1953;56:831–838. [PubMed]
  • Sakakibara H, Mitchell DR, Kamiya R. A Chlamydomonas outer arm dynein mutant missing the α heavy chain. J Cell Biol. 1991;113:615–622. [PMC free article] [PubMed]
  • Sakakibara H, Takada S, King SM, Witman GB, Kamiya R. A Chlamydomonas outer arm dynein mutant with a truncated β heavy chain. J Cell Biol. 1993;122:653–661. [PMC free article] [PubMed]
  • Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1987.
  • Schmidt JA, Eckert R. Calcium couples flagellar reversal to photostimulation in Chlamydomonas reinhardtii. Nature. 1976;262:713–715. [PubMed]
  • Silver LM. Mouse t haplotypes. Annu Rev Genet. 1985;19:179–208. [PubMed]
  • Silver LM. The peculiar journey of a selfish chromosome: mouse t haplotypes and meiotic drive. Trends Genet. 1993;9:250–254. [PubMed]
  • Takada S, Kamiya R. Functional reconstitution of Chlamydomonas outer dynein arms from α-β and γ subunits: requirement of a third factor. J Cell Biol. 1994;126:737–745. [PMC free article] [PubMed]
  • Takada S, Wilkerson CG, Kamiya R, Witman GB. The ODA1 gene encodes a 62-kDa component of a complex necessary for outer arm dynein docking on the doublet microtubules of Chlamydomonas flagella. Mol Biol Cell. 1996;7:568a.
  • Tam L-W, Lefebvre PA. Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis. Genetics. 1993;135:375–384. [PubMed]
  • Vaughan KT, et al. Multiple mouse chromosomal loci for dynein-based motility. Genomics. 1996;36:29–38. [PubMed]
  • Wickens M, Anderson P, Jackson R J. Life and death in the cytoplasm: messages from the 3′ end. Curr Opin Genet Dev. 1997;7:220–232. [PubMed]
  • Wilhelm JE, Vale RD. RNA on the move: the mRNA localization pathway. J Cell Biol. 1993;123:269–274. [PMC free article] [PubMed]
  • Wilkerson CG, King SM, Koutoulis A, Pazour GJ, Witman GB. The 78,000 Mr intermediate chain of Chlamydomonas outer arm dynein is a WD-repeat protein required for arm assembly. J Cell Biol. 1995;129:169–178. [PMC free article] [PubMed]
  • Wilkerson CG, King SM, Witman GB. Molecular analysis of the γ heavy chain of Chlamydomonas flagellar outer-arm dynein. J Cell Sci. 1994;107:497–506. [PubMed]
  • Witman GB. Isolation of Chlamydomonas flagella and flagellar axonemes. Methods Enzymol. 1986;134:280–290. [PubMed]
  • Witman GB, Johnson KA, Pfister KK, Wall JS. Fine structure and molecular weight of the outer arm dyneins of. Chlamydomonas. J Submicrosc Cytol. 1983;15:193–197.
  • Witman GB, King SM, Moss AG, Wilkerson C. The intermediate chain/light chain complex: an important structural entity of outer arm dynein. In: Baccetti B, editor. Comparative Spermatology 20 Years After. New York: Raven Press; 1992. pp. 439–443.
  • Witman GB, Wilkerson CG, King SM. The biochemistry, genetics, and molecular biology of flagellar dynein. In: Hyams JS, Lloyd CW, editors. Microtubules. New York: Wiley-Liss; 1994. pp. 229–249.
  • Yang P, Sale WS. The Mr 140,000 intermediate chain of Chlamydomonas flagellar inner arm dynein is a WD-repeat protein implicated in dynein arm anchoring. Mol Biol Cell. 1998;9:3335–3349. [PMC free article] [PubMed]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology