Correspondence to: Mary E. Porter, Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Box 206, 420 Delaware St. SE, 4-102 Owre Hall, Minneapolis, MN 55455. Tel:(612) 626-1901 Fax:(612) 624-8118 E-mail:mary-p{at}biosci.cbs.umn.edu.
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Abstract |
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Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1 Dhc of the I1 inner dynein arm. The 1
Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH2- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1
Dhcs were recovered. The truncated 1
Dhc fragments lacked the dynein motor domain, but still assembled with the 1ß Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1
motor domain in the I1 structure and further revealed the role of the 1
Dhc in flagellar motility and phototactic behavior.
Key Words: motors, dynein, flagella, phototaxis, inner arm
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Introduction |
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THE movement of cilia and flagella is powered by axonemal dyneins, a family of mechanoenzymes that convert the energy derived from ATP binding and hydrolysis into the sliding of adjacent outer doublet microtubules (
The Chlamydomonas outer arm is one of the most well characterized dynein complexes; it is composed of three dynein heavy chains (Dhc)1 (, ß, and
), two intermediate chains (IC), and several light chains (LCs) (<20 kD), and repeats every 24 nm along the length of the axoneme (
The inner dynein arms share an overall structural similarity to the outer arms but are significantly more complex in both composition and function. Ion exchange chromatography and SDS-PAGE procedures have identified at least eight distinct inner arm Dhcs that are associated with specific ICs and LCs into seven different molecular complexes: one two-headed isoform (I1) and six single-headed isoforms (I2 and I3) (
Very little is also understood about the mechanism by which any Dhc is targeted to its specific location within the axoneme. In this report, we focus on the role of the 1 Dhc in the assembly and targeting of the inner arm isoform known as the I1 complex. The I1 dynein provides several advantages for the study of dynein targeting. First, it is a relatively simple complex composed of two Dhcs (1
and 1ß), three ICs of 140, 138, and 110 kD (
Dhc; mutations in this locus disrupt the assembly of the I1 complex and thereby alter flagellar motility (
To characterize the Dhc domains involved in the assembly and targeting of the I1 complex, we sequenced the complete Dhc1 transcription unit (>22 kb) and generated specific constructs of the Dhc1 gene. The constructs were used in cotransformation experiments to rescue the pf9 defects. These results represent the first full-length inner arm Dhc sequence to be described in any organism, and the first reported rescue of a Dhc mutation in Chlamydomonas. Our analysis of the Dhc1 transformants has also identified a subset of strains expressing truncated Dhc1 transcripts. The truncated transcripts encode NH2-terminal fragments of the 1 Dhc polypeptide that are capable of coassembly with other components of the I1 complex and rebinding to the proper axoneme location. These results indicate that domains within the NH2-terminal ~143 kD of the 1
Dhc are involved in the specific subunit interactions required for the assembly and targeting of the I1 complex. EM analysis of isolated axonemes has identified the position of the 1
Dhc motor domain within the structure of the I1 complex. The assembly of truncated 1
Dhcs in the flagella of the transformants also resulted in a new motility phenotype that has revealed the contribution of the 1
Dhc motor domain to flagellar motility and phototactic behavior. These findings have important implications for the regulatory mechanisms that control the activity of the I1 dynein motor.
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Materials and Methods |
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Origin of Genomic Clones and Sequence Analysis of the Dhc1 Gene
35 kb of genomic DNA in the region of the Dhc1 gene was recovered from a large insert, wild-type (21gr) Chlamydomonas library. The position of the Dhc1 transcription unit within this region was determined by probing Northern blots of wild-type RNA with selected subclones, and the Dhc1 transcription unit was thereby narrowed down to ~22 kb of genomic DNA (see Figure 1;
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Potential open reading frames were identified using the GCG program CodonPreference and a codon usage table compiled from the coding regions of 73 different Chlamydomonas nuclear sequences (
In five regions of the Dhc1 gene, the presence of multiple potential splice donor or acceptor sequences did not allow a confident prediction of the putative exons. In those cases, the splice junctions were determined directly by sequence analysis of reverse transcriptasePCR (RT-PCR) products generated from the Dhc1 transcript (see Figure 1). Total RNA was isolated from wild-type cells 45 min after deflagellation, and then 5 µg of total RNA was reverse transcribed using either a random primer or a sequence-specific reverse primer and the Superscript Preamplification System (GIBCO BRL) according to manufacturer's instructions. 5 µl of the resulting 25-µl cDNA product was used in a 100-µl PCR reaction with sequence specific primers. PCR reactions were performed using 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 2 mM deoxynucleotide triphosphates, 0.2 mM of each primer, and 2.5 U Taq polymerase (Life Technologies, Inc.). Some reactions also contained 3% DMSO. The PCR reactions were first denatured at 94°C for 3 min, followed by 30 cycles of 58°C for 1 min, 72°C for 3 min, and 94°C for 1 min, and then completed with a final cycle of 58°C for 1 min and 72°C for 5 min. The final reaction products were analyzed on agarose gels, and then purified using Wizard PCR preps (Promega Corp.) for direct sequencing with sequence-specific primers.
The proposed translation start site was determined by the recovery of an RT-PCR product using a forward primer downstream of the TATA box sequence and a reverse primer in exon 3. The resulting RT-PCR product contained stop codons in all three frames immediately preceding the proposed start codon.
The predicted amino acid sequence encoded by the Dhc1 gene was analyzed using the GCG program Motifs. The programs Bestfit, Compare, and Pileup were used to compare the 1 Dhc sequence to Chlamydomonas outer arm Dhc sequences
, ß, and
(
-helical coiled coils were identified using the program COILS, version 2.2 (
Cosmid Library Screening and Construction of pD1SA
To identify clones that might contain a full-length Dhc1 gene, we screened two different Chlamydomonas cosmid libraries (
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A truncated version of the Dhc1 gene was constructed by fusing sequences from the 5' end to sequences from the 3' end. To recover the 5' end, a 19-kb SalI fragment was subcloned to form the plasmid pSM8 (see Figure 5 D). pSM8 contains ~1.7 kb of genomic DNA located 5' of the coding region, but ends in the middle of the Dhc1 transcription unit. pSM8 was digested with SalI and AscI to release the Dhc1 gene as an 11-kb fragment that is truncated before the region encoding the ATP hydrolytic site (P1). The 3' end of the Dhc1 gene was subcloned as a 4.3-kb SalI, EcoRI fragment to form the plasmid p14SE, which was digested with SalI and AscI to release the region 5' of the AscI site. The SalI-AscI fragment from pSM8 was ligated into the digested p14SE subclone to form the construct pD1SA (see Figure 5 D). pD1SA joins sequences from the 5' end of the Dhc1 gene to the 3' end at the AscI site. It is predicted to encode the first 1,956 amino acids of the 1 Dhc, and then terminate translation after adding nine novel amino acids (QCHGCGPGV) to the COOH terminus of the polypeptide.
Recovery of Bacterial Artificial Chromosome (BAC) Clones Containing the Dhc1 Gene
A modified pBELO BAC library containing Chlamydomonas genomic DNA was screened with selected subclones to identify large insert BAC clones containing the Dhc1 gene. This library was constructed by N. Haas and P. Lefebvre (University of Minnesota, St. Paul, MN) using genomic DNA from the cell-wall less strain cw92 and is currently available from Genome Systems, Inc. BAC DNA was isolated from positive clones using a modified version of the manufacturer's protocol available from C. Amundsen (University of Minnesota, St. Paul, MN) at the following URL: http://biosci.cbs.umn.edu/~amundsen/chlamy/methods/bac.html. The final pellet of BAC DNA was resuspended in 200 µl of TE and stored at -20°C. To identify clones containing full-length Dhc1 genes, 5 µl of BAC DNA was digested with the restriction enzyme SacI and analyzed on Southern blots using subclones from the 5' and 3' ends of the Dhc1 gene.
Nucleic Acid Analysis
Large-scale preparations of genomic DNA were isolated from wild-type and mutant transformant strains using CsCl gradients as described in
Cell Culture, Mutant Strains, and Cotransformation Experiments
The strains used in this study are listed in Table 1. All cells were maintained as vegetatively growing cultures at 21°C as previously described (
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The pf9-2 arg2 strain (
Analysis of Motility
Positive transformants were picked into 96-well plates and screened for motility on an inverted microscope (Olympus CK). Wells containing motile cells were streaked for single colonies and rescored on a phase-contrast microscope (Axioskop; Carl Zeiss, Inc.) using a 40x objective and a 10x eyepiece. The phenotypes of motile transformants were further analyzed by measuring forward swimming velocities and beat frequencies as previously described (
Transformants were tested for their ability to phototax using two different assays. In the first assay (
To verify that the rescued motility in the transformants was due to expression of the Dhc1 transgene and not a reversion event at the PF9 locus, the motile transformants were backcrossed to a pf28 allele (oda2), which lacks the outer arms but is wild-type at the PF9 locus (
Axoneme Isolation, Dynein Extracts, and Sucrose Gradients
Axonemes were prepared from large-scale (540 liters) liquid cultures of vegetative cells using procedures described by
SDS-PAGE and Immunoblot Analysis
Protein samples from whole axonemes and sucrose gradient fractions were separated on 5% polyacrylamide gels using the
Four different antibody preparations were used to probe the blots. The 1 Dhc antibody has been previously described in detail and is highly specific for the Dhc1 gene product (
Dhc polypeptide. The 1
Dhc antibody was affinity-purified on Western blots of dynein extracts, and then used at a 1:10 dilution. The IC140 antibody was provided by P. Yang and W. Sale (Emory University, Atlanta, GA). This antiserum was raised against a fusion protein containing a fragment of the 140-kD intermediate chain of the I1 complex (Yang and Sale, 1998), and it was typically used at a dilution of 1:3,000. The rabbit polyclonal antibody R5205, which was raised against a fusion protein of the human 14-kD dynein LC (
Electron Microscopy and Image Analysis
To view the I1 complex in strains with rescued motility, selected transformants were crossed to a pf9-3 strain to recover strains with rescued I1 complexes and the wild-type complement of outer dynein arms. Axonemes were prepared and processed for EM as previously described (
Recovery of Dhc1 Transgene from G3 After Transformation
To identify the 3' end of the Dhc1 transgene in the G3 transformant (which assembles the shortest 1 Dhc fragment), genomic DNA was isolated from wild-type and G3, digested with the restriction enzymes SacI and KpnI, and analyzed on Southern blots probed with Dhc1 subclones. A polymorphic 7.2-kb SacI-KpnI fragment was identified in G3 using subclone C. This polymorphic fragment was recovered from G3 genomic DNA by constructing a size-selected minilibrary, and then screening the library with subclone C. After single colony purification, the 3' end of the truncated Dhc1 transgene was sequenced with Dhc1 specific primers to determine the predicted amino acid sequence at the COOH terminus of the 1
Dhc fragment.
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Results |
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Sequence Analysis of the Dhc1 Transcription Unit
In previous work, we identified a null mutation in the Dhc1 gene that resulted in the failure to assemble the I1 inner arm complex into the flagellar axoneme (
The predicted amino acid sequence of the encoded 1 Dhc contains 4,625 amino acid residues and corresponds to a polypeptide of 522,806 D (Figure 2). A search for potential nucleotide binding sites within the 1
Dhc sequence identified six consensus or near consensus phosphate-binding (P-loop) motifs with the sequence A/GXXXXGKT/S (
Dhc respectively; these appear to be unique to the 1
Dhc (Figure 2).
The predicted amino acid sequence of the 1 Dhc was compared with the three Dhc sequences (
, ß, and
) that form the outer dynein arm in Chlamydomonas (
Dhc also shares significant homology with the ß and
Dhcs of the outer arm (Figure 3, ~24% identity, ~56% similarity). Alignment of the Dhc sequences using the GCG program PILEUP confirmed that the presence of conserved domains within the NH2-terminal region, but also revealed several short stretches of unique peptide sequence in the 1
Dhc, including the region previously used to generate a monospecific 1
Dhc antibody (Figure 2;
The 1 Dhc sequence was also analyzed using programs that predict secondary structure to identify regions with the potential to form
-helical coiled-coil domains (
-helical coiled coils. The presence of limited coiled-coil domains separating the central portion of the Dhc from the NH2-terminal and COOH-terminal regions has been observed in other Dhc sequences (
Isolation of Dhc1 Transgenes
To better understand how the specific domains of the 1 Dhc polypeptide might be involved in the assembly and activity of the I1 inner arm dynein, we decided to analyze constructs of the Dhc1 gene in vivo in a pf9 mutant background. Because of the large size of the Dhc1 gene (~21 kb), two cosmid libraries and one BAC library were screened with probes representing the 5' and 3' ends of the Dhc1 gene to improve the chances of recovering clones that contain the full-length gene (Figure 5 A). The first cosmid library yielded a single clone, cA1, which was positive with both probes, but upon further analysis proved to be lacking a small portion at the 3' end of the gene (Figure 5 B). Screening the second cosmid library resulted in the recovery of a single clone, cW1, which contained the complete Dhc1 transcription unit as well as additional genomic sequences both 5' and 3' that might be required for proper expression in vivo (Figure 5 C). Four larger clones (100135 kb) containing the Dhc1 transcription unit were recovered from the BAC library; two of these clones were used in subsequent cotransformation experiments (Figure 5 E). We also constructed a truncated version of the Dhc1 transgene known as pD1SA by fusing an 11-kb region encoding the NH2-terminal 1,956 amino acids to a 1-kb region containing the 3' end of the gene (Figure 5 D). All of the Dhc1 transgenes were tested for their ability to rescue the pf9 mutant defects in vivo.
Rescue of pf9 Motility Defects by Transformation with Constructs of the Dhc1 Gene
Mutations at the PF9/IDA1 locus typically result in strains that have a slow, smooth swimming behavior ( Dhc gene that results in the failure to assemble the outer dynein arms (
The pf9-2 pf28 arg2 strain was first cotransformed with the selectable marker pARG7.8 and the cW1 cosmid containing the complete Dhc1 transcription unit. Positive transformants were selected by growth on solid medium lacking arginine, and then single colonies were picked into liquid media and screened for motility. Figure 5 C illustrates the combined results of several independent cotransformation experiments with the cW1 cosmid. Although arg+ transformants were recovered at expected frequencies (
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Given these preliminary findings, we decided to test the other Dhc1 transgenes for their ability to rescue the mutant phenotypes. The cosmid cA1 contains an incomplete copy of the Dhc1 transcription unit but also contains the ARG7 gene cloned within the bacterial vector sequences, thereby physically linking the selectable marker and the Dhc1 gene. Therefore, all arg+ transformants might be expected to contain the Dhc1 sequence integrated along with the ARG7 gene. However, the frequency of rescue with the cA1 cosmid (0.61%; Figure 5 B) was only slightly better than the cW1 cosmid. These observations indicated the Dhc1 transgenes were probably being fragmented during the transformation protocol, but the recovery of motile strains also suggested that a truncated version of the Dhc1 sequence was capable of restoring some function.
Previous study of an outer arm mutation, oda4-s7, had indicated that the NH2-terminal third of the ß Dhc polypeptide is sufficient for assembly of a dynein complex ( Dhc, we cotransformed the pf9 pf28 mutant with the smaller pD1SA construct, which encodes ~40% of the 1
Dhc sequence. These experiments yielded seven motile strains (Figure 5 D), which represented only a modest (0.87%) increase in the frequency of rescue, but these rescues confirmed that truncated Dhc1 transgenes could restore partial motility. To see if it was possible to completely rescue the motility defects, we also transformed the pf9 pf28 mutant with two BAC clones that contained the full-length Dhc1 gene located in the middle of ~100135-kb genomic inserts (Figure 5 E). The frequency of rescue (~0.1%) was still quite low, but the motility phenotypes of the rescued strains were very similar to pf28 (see below).
The recovery of motile isolates after cotransformation could also be due to an intragenic reversion event at the PF9 locus during the course of transformation and/or selection. To confirm that the motility of the transformants was due to the successful expression of the Dhc1 transgene and not a reversion event, two of the cW1 transformants (E2 and G4) were crossed with oda2, another mutant allele at the PF28/ODA2 locus, and the progeny from 11 complete tetrads were analyzed for each cross. If the rescued motility was due to reversion of the pf9-2 mutation, all the resulting tetrad progeny would be motile and swim with a pf28/oda2like motility phenotype. However, if the rescued motility was due to the presence of the Dhc1 transgene, then the restored motility phenotype would be expected to segregate independently of the pf9-2 mutation, and a class of immotile progeny with the original pf9-2 pf28 genotype should be recovered. Surprisingly, we observed three different motility phenotypes in the tetrad progeny. The first class swam with a motility phenotype that was indistinguishable from either pf28 or oda2. The second class swam with a jerky motion like the pf28/oda2 strains but appeared slower. The third class was immotile with short, stumpy flagella. The recovery of aflagellate strains demonstrated that the original pf9-2 mutation was still present in the genetic background of the two transformants and that the rescued motility was due to the presence of the Dhc1 transgene.
Motility Phenotypes of the Dhc1 Transformants
Although the frequency of rescue was low, it was clear that the rescued motility was due to the presence of the different Dhc1 transgenes, and so the motility phenotypes of the Dhc1 transformants were analyzed in greater detail. More specifically, we measured the flagellar beat frequency, the forward swimming velocity, and the ability to phototax (Table 2). Transformants with complete rescue of the pf9 mutation would be expected to have a swimming phenotype nearly identical to that of pf28. The flagellar beat frequencies of the Dhc1 transformants were almost identical to the beat frequency of pf28, but measurements of forward swimming velocities clearly indicated that most of the transformants swam more slowly than pf28 (Table 2). In particular, the swimming velocities of the rescued strains obtained by transformation with the cosmid clones and pD1SA were slower than those obtained by transformation with the BAC clones. These results suggested that there were still some inner arm defects in most of the Dhc1 transformants.
We next tested if the Dhc1 transformants had recovered the ability to phototax.
To examine the motility of the transformants in the presence of outer arms, two strains, G4 and E2, were crossed to pf9-3 and tetrad products containing the Dhc1 transgene in a wild-type outer arm background (G4+OA and E2+OA) were recovered. The two strains have beat frequencies almost identical to wild type, but their swimming velocities are intermediate in speed between pf9 and wild type (Table 2). Moreover, in the presence of the outer arms, the two strains could photoaccumulate as effectively as wild type. These results suggest that the outer arms can compensate in some way for the phototaxis defects in the Dhc1 transformants.
Truncated 1 Dhcs in the Dhc1 Transformants
The swimming behavior of the Dhc1 transformants obtained with the cosmid clones demonstrated that the introduction of these Dhc1 clones resulted in only a partial rescue of the pf9 motility defects. Given the large size of the Dhc1 transcription unit (>22 kb), we were initially concerned that these transgenes might not be expressing wild-type levels of the Dhc1 gene product. To address this question, we isolated axonemes from the Dhc1 transformants and analyzed the components of the I1 complex. Previous work has shown that the I1 complex is composed of eight polypeptides, two Dhcs (1 and 1ß), three ICs (IC140, IC138, and IC110) (
Dhc, which can be identified on Western blots using antibody directed against a peptide epitope in the NH2-terminal region (
Dhc antibody. The 1
Dhc antibody identified the ~520 kD 1
Dhc in the pf28 control sample, but polypeptides significantly smaller than the 1
Dhc were identified in all of the motile strains obtained by transformation with the Dhc1 cosmids. In contrast, all of the axoneme samples prepared from rescued strains obtained by transformation with the Dhc1 BAC clones contained full-length 1
Dhc polypeptides. These results indicated that the partial rescue phenotype seen with the Dhc1 cosmid clones was not due to low levels of expression of a full-length 1
Dhc, but instead due to the expression of truncated 1
Dhcs, ranging in size from ~165 to ~300 kD.
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To determine if other I1 subunits were associated with the truncated 1 Dhcs, Western blots of isolated axonemes were probed with an antiserum raised against the 140-kD intermediate chain (Yang and Sale, 1998). This antibody detects the IC140 in wild-type axonemes, but not in I1 mutant axonemes. As shown in Figure 6 C, the IC140 antibody recognized a single polypeptide of ~140 kD in pf28 and each rescued transformant, but did not detect the IC140 in any of the pf9 mutant strains. Similar results were seen using the antibody directed against the 14-kD Tctex1 light chain (data not shown).
Assembly of I1 Complexes in Dhc1 Transformants
To confirm that the other polypeptide subunits were assembled into an I1 complex, we isolated whole axonemes from large-scale cultures of two Dhc1 transformants, E2 and G4, as well as from control pf28 cells. Partially purified I1 complexes were obtained by high salt extraction of the isolated axonemes followed by sucrose density gradient centrifugation. The resulting fractions were analyzed by both SDS-PAGE and Western blotting. Figure 7 A shows the 19S region of a sucrose gradient that was loaded with the pf28 dynein extract. The two Dhcs and three ICs of the I1 isoform cosediment as a complex that peaks in fraction number 4. Duplicate samples tested on Western blots probed with the 1 Dhc antibody confirmed the presence of the 1
Dhc in the 19S region (right). Figure 7 B shows four fractions from the sucrose gradient that was loaded with the G4 dynein extract. The gel on the left reveals that the 1ß Dhc and the three intermediate chains of the I1 complex have shifted and now cosediment at ~16S, peaking in fraction 6. A novel polypeptide of ~183 kD cosediments in the same region (see asterisks). Western blot analysis with the 1
Dhc antibody identified this novel band as the truncated 1
Dhc (Figure 7 B, blot). Identical results were observed with dynein extracts isolated from the E2 strain (data not shown). The truncated 1
Dhcs in the Dhc1 transformants therefore form stable complexes with the other polypeptides of the I1 complex, but the resulting mutant complexes sediment more slowly than wild-type complexes.
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Structural Analysis of Axonemes from Dhc1 Transformants Reveals Defects in the I1 Complex
To analyze the structure of the I1 complex in the Dhc1 transformants, we prepared purified axonemes from wild-type and mutant strains for thin section EM. To facilitate the analysis of the images, the transformants G4 and E2 were crossed to a pf9-3 strain to recover the Dhc1 transgene in a wild-type outer arm background (G4+OA and E2+OA, see Materials and Methods). Figure 8 a shows the grand average of the 96-nm repeat from wild-type axonemes, and Figure 8 b indicates the corresponding densities. Previous work has shown that the inner dynein arms repeat as a complex group of structures every 96 nm in register with the radial spokes ( Dhcs. Lobes 1 and 3 of the I1 complex are present in the axonemes from these samples, but lobe 2 is still missing. Difference plots between these images and wild type confirms that the loss of lobe 2 is the only significant defect in the two Dhc1 transformants (Figure 8f and Figure h). These images demonstrate that the I1 complex is assembled and targeted to the appropriate axoneme location. In addition, these images suggest that the region of the 1
Dhc that is missing in the Dhc1 transformants corresponds to lobe 2 of the I1 structure.
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The Dhc1 Transcripts in the Cosmid Transformants Lack the 3' End of the Gene
Although the initial cotransformation experiments involved the use of a full-length or near full-length Dhc1 cosmid clones, all of the motile transformants recovered with these clones assemble partially functional I1 complexes with truncated 1 Dhcs (Figure 6 and Table 2). To understand how the 1
Dhc fragments were related to the Dhc1 sequence, we analyzed the Dhc1 transcripts from several of the rescued transformants on Northern blots. Total RNA was first isolated from the G4+OA and E2+OA strains, which contain the Dhc1 transgene in the pf9-3 null mutant background. This background facilitated our analysis because the pf9-3 mutation is a large deletion (~13 kb) in the Dhc1 gene and does not generate an endogenous Dhc1 transcript (
Figure 9 A shows a partial restriction enzyme map of the Dhc1 gene and the subclones that were used as probes to analyze the Dhc1 transcripts. As shown in Figure 9 B, probe A3', which spans the Dhc1 transcription start site, identified a single, large (>13 kb) transcript in wild-type RNA. However, in G4+OA and E2+OA, the transcripts recognized by the A3' probe were significantly smaller than the wild-type Dhc1 transcript, but these smaller transcripts were still upregulated in response to deflagellation (compare lanes 0 and 45). Identical results were observed with the next two subclones, probes B and C. Probe D, which includes the conserved region encoding the primary ATP hydrolytic site, hybridized to the truncated transcripts in E2+OA, but it did not recognize the truncated transcripts in G4+OA. Probes EG did not hybridize with any transcripts in the transformants (Figure 9 B, right panel, and data not shown). The Dhc1 transcripts in G4 and E2 are therefore truncated from the 3' end of the Dhc1 gene, and G4 is truncated before E2.
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To characterize the Dhc1 transcripts present in other transformants, RNA was isolated from three additional strains: G3, G9, and A2. G3 and G9 are the cW1 transformants that assemble the smallest and largest 1 Dhc fragments respectively, whereas A2 is a cA1 transformant that assembles the largest (>300 kD) 1
Dhc fragment obtained thus far (Figure 6). As shown in Figure 9 C, in each strain, probe C hybridized to a truncated transcript that is significantly smaller than the endogenous Dhc1 transcript derived from the pf9-2 mutant background. However, probe D, which corresponds to the region encoding the ATP hydrolytic site, failed to hybridize with the truncated transcripts present in the G3, A2, and G9 samples. Similar results were seen with probes E and F (data not shown). Therefore, all three strains encode 1
Dhc fragments that are truncated before the proposed motor domain.
Recovery of a Modified Transgene from a Dhc1 Transformant
To identify the sites where the Dhc1 cosmid clones were being modified during transformation, we isolated genomic DNA from the transformants and analyzed the structure of the integrated Dhc1 transgenes on Southern blots. Figure 10 A shows a blot of SacI digested genomic DNA that was hybridized with a probe for subclone C. As expected, this probe hybridized to the endogenous Dhc1 gene present in the pf9-2 mutant background of the transformants. However, for each transformant, a second polymorphic band could be detected using either probe C (Figure 10 A) or probe D (data not shown). From these and other blots, we concluded that the Dhc1 cosmids were being rearranged during integration into genomic DNA. In addition, the region of the Dhc1 gene encoding the conserved motor domain appeared to be the most common target for disruption during these integration events.
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Because the G3 transformant assembles the smallest 1 Dhc fragment identified thus far (Figure 6), we recovered the modified Dhc1 transgene using probe C to screen a mini-library made from genomic DNA of the G3 transformant (see Materials and Methods). The Dhc1 transgene was then sequenced with Dhc1 specific primers to identify the junction between the Dhc1 sequence and the site of integration in G3 genomic DNA. The Dhc1 sequence in G3 is fused to an unidentified DNA sequence, and the resulting hybrid gene is predicted to encode up to amino acid residue 1,249 of the 1
Dhc, followed by the addition of 17 novel amino acids before encountering a stop codon (Figure 10 B). Sequence analysis of an RT-PCR product derived from G3 RNA has confirmed the presence of this hybrid transcript. The polypeptide encoded by the modified transgene would, therefore, correspond to a 1
Dhc fragment of ~143 kD that is truncated just COOH-terminal to the epitope recognized by the 1
Dhc antibody (Figure 10 C). The recovery of this fragment in G3 axonemes (Figure 6) reveals that the NH2-terminal coiled-coil domains of the 1
Dhc are not required for I1 complex assembly.
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Discussion |
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16 different Dhc genes have been identified in Chlamydomonas: 2 cytoplasmic Dhc sequences ( Dhc in detail.
Sequence Analysis of Dhc1 Transcription Unit
As described in Figure 1, we have determined the nucleotide sequence for the complete Dhc1 transcription unit and used this information to obtain the predicted amino acid sequence of the 1 Dhc (Figure 2). To our knowledge, this is the first full-length, inner arm Dhc sequence to be reported in any organism. Comparisons to other full-length Dhc sequences indicate that the 1
Dhc is most similar to the ß and
Dhcs of the outer arm, and that these sequence similarities extend into the NH2-terminal region (Figure 3). As the NH2-terminal region of the Dhc is thought to be involved in the association with isoform specific IC and LC subunits, these observations suggest that the 1
Dhc and the ß and
Dhc contain conserved sites for the binding of accessory subunits. Indeed, recent studies have revealed that the I1 complex and the outer arm do contain similar IC and LC components, such as the 8-kD LC, the Tctex1 and Tctex2 LCs, and a family of WD-repeat containing ICs (
Dhc sequence is the presence of a P-loop motif (Pn) at amino acid residues 960967 (Figure 2). A weakly conserved P-loop motif has also been identified within the first 200 residues of the ß Dhc (
Dhc is a bona fide nucleotide binding site is unknown, but the future sequence analyses of 1
Dhc homologues in other organisms should indicate whether the Pn motif is a conserved feature of this class of Dhc.
The central and COOH-terminal thirds are the most highly conserved regions of the 1 Dhc, and our transformation experiments are consistent with previous proposals that this region corresponds to the dynein motor domain (
Dhc sequence, but the same amino acid substitution was found in the
Dhc sequence (
The COOH-terminal third of the 1 Dhc also contains a small region ~340 amino acids downstream from P4 that is predicted to form a limited coiled-coil domain (Figure 4). A similar region in cytoplasmic Dhc sequences has been identified recently as the stalk structure that extends from the globular head domain and forms the microtubule binding site (
Dhc sequence, ~280 amino acids downstream from the proposed microtubule binding site. The function of this sixth P-loop in the 1
Dhc is unknown, but its position downstream from the microtubule binding site is intriguing. Recent sequence analysis has suggested that all dyneins may contain six ATPase-like repeat regions: the four central P-loops previously identified and two additional, less well conserved repeats after the COOH-terminal coiled-coil domains (
Rescue of the pf9 Mutant Phenotype by Transformation with Dhc1 Constructs
Using the Dhc1 sequence information, we recovered two BAC clones and two cosmid clones containing full-length or near full-length Dhc1 genes, and then used these constructs to rescue the pf9 motility defects (Figure 5). 20 independent transformants with rescued motility were recovered. Backcrossing the transformants confirmed that the rescued motility was due to the presence of the Dhc1 transgene and not to a reversion event at the PF9 locus. However, analysis of the Dhc1 transformants produced two unexpected results. First, the frequency of rescue (<1%) was much lower than previously observed with other flagellar genes (510%) ( Dhcs were truncated in most of the motile transformants recovered thus far.
One reason that the cotransformation frequencies were so low might be due to the large size of the Dhc1 transcription unit, which could make the Dhc1 transgenes more susceptible to damage during the transformation protocol. Exogenous DNA sequences often undergo deletions as they integrate into the Chlamydomonas genome ( Dhc fragments (Figure 6) indicated that the Dhc1 cosmids were being disrupted during the transformation protocol, and both Southern and Northern blot analyses of the rescued transformants (Figure 9 and Figure 10) have confirmed that deletions from the 3' end of the Dhc1 cosmids did occur. The presence of additional genomic DNA flanking the Dhc1 transcription unit in the BAC clones may have served to protect the Dhc1 transgenes and thereby permitted the full-length rescues observed with these clones (Figure 5 and Figure 6).
Another reason for the low frequency of rescue might be the relatively small amount of genomic DNA present on the 5' end of the Dhc1 gene in certain constructs (Figure 5). If this region was randomly deleted, the resulting cotransformants would not retain the sequences necessary for expression of the Dhc1 transcript and rescue of the mutant phenotype. Recent experiments with constructs encoding the IC140 subunit have indicated that sufficient DNA upstream from the 5' end of the IC140 gene is essential for efficient rescue of the ida7 mutation (
The NH2-terminal 143-kD of the 1 Dhc Is Sufficient for Complex Assembly
The second unexpected result was the frequency with which we recovered motile transformants expressing only NH2-terminal fragments of the 1 Dhc (Figure 6 B). Northern and Southern blot analyses have shown that the rescued strains retained the 5' sequence elements required for regulated expression of the Dhc1 transcripts, but several of these strains lacked the 3' end of the transgene (Figure 9 and Figure 10). Therefore, the truncated 1
Dhcs represent those NH2-terminal fragments that were competent to assemble with other subunits into the I1 complex (Figure 6B and Figure C, and Figure 7).
The observation that none of the 1 Dhc fragments is smaller than ~143 kD may indicate that this is the shortest NH2-terminal fragment capable of complex assembly. Studies of outer arm mutants have identified a novel ß Dhc mutation with similar properties. The oda4-s7 mutant expresses a 160-kD fragment of the ß Dhc that is capable of coassembly with other outer arm subunits at the correct axoneme location (
Although the NH2-terminal third of the 1 Dhc is the most variable region, secondary structure programs have identified a region just before P1 that is predicted to form a limited coiled-coil domain (Figure 4). This domain, which has been identified in nearly all Dhcs sequenced to date (
Dhc fragment (Figure 6 B). Sequence analysis of the truncated Dhc1 transgene demonstrated that the 1
Dhc sequence terminates before the region predicted to form the NH2-terminal coiled-coil domain (Figure 10). Given that this 1
Dhc fragment still assembles with other I1 components into the flagellar axoneme (Figure 6), other sites within the NH2-terminal region must be required for complex formation. We plan to analyze additional Dhc1 constructs to further delineate the domains required for specific subunit interactions and complex assembly.
Assembly of the Dynein Motor Domain
If the Dhc1 transgenes were deleted randomly from the 3' end, we would expect to recover a broad distribution of Dhc fragments ranging in size from the minimum required to assemble the I1 complex to nearly full-length. Therefore, why are almost all of the 1 Dhc fragments smaller than ~217 kD (Figure 6 B)? One possibility may be that larger 1
Dhc fragments are unstable and prevent assembly of the I1 complex into the axoneme. Studies in Dictyostelium have shown that constructs of cytoplasmic Dhc lacking significant portions of the COOH terminus are expressed poorly as compared with other constructs that contain the entire motor domain (
Dhc fragments with partial motor domains may inhibit flagellar motility. If so, we would not recover such transformants in our screen, which was based on the rescue of a motility defect. Indeed, Northern blot analysis of the two transformants (A2 and G9) that assemble the largest 1
Dhc fragments demonstrates that the sequences encoding the dynein motor domain are not present in the associated Dhc1 transcripts (Figure 9). The NH2-terminal regions of these larger 1
Dhc fragments must, therefore, be fused to other protein sequences. Interestingly, the absence of motile transformants with partial motor domains is consistent with previous reports that nucleotide binding by the cytoplasmic Dhc is inhibited by deletion of the COOH terminus, leading to the formation of rigor complexes (
Implications for the Structure of the I1 Complex
Structural studies of the isolated I1 complex by negative staining or rotary shadowing have shown that it is a two-headed isoform ( Dhc (Figure 7), we can now identify the position of the 1
motor domain within the I1 structure. EM analysis of axonemes isolated from the E2 and G4 transformants has revealed that lobe 2 of the I1 structure corresponds to the missing 1
motor domain (Figure 8). Lobe 2 is close to the first radial spoke, in a position that may permit direct signaling between the radial spoke and the 1
Dhc motor domain. We predict that the remaining two lobes of the I1 structure represent the positions of the 1ß Dhc motor domain and stem region containing the I1 ICs and LCs respectively. We are currently transforming other I1 mutants with the genes for other I1 subunits to identify the polypeptide components that are located within these structural domains (
The Role of the 1 Dhc Motor Domain in Motility and Phototaxis
The recovery of transformants missing only the 1 Dhc motor domain but containing the other components of the I1 complex has allowed us to analyze the specific role of the 1
Dhc motor domain in flagellar motility. I1 mutants lacking both the 1
and 1ß Dhcs have a slow, smooth swimming phenotype with an altered flagellar waveform (
Dhcs swim faster than I1 mutants but slower than control strains containing both Dhcs (Table 2). The 1
Dhc motor domain, therefore, contributes directly to force production during motility.
The I1 complex is also an essential component of the phototaxis response in Chlamydomonas. Strains that have defects in outer dynein arms, the dynein regulatory complex, or other inner arm isoforms can phototax, but mutants lacking the I1 complex cannot (
To assess the specific role of the 1 Dhc motor domain in phototaxis, we compared the swimming behavior of the Dhc1 transformants to that of control cells in response to a directional light source. pf28 cells were clearly phototactic, but the Dhc1 transformants with truncated 1
Dhcs remained uniformly dispersed during the time course of our assays. These observations indicate that the motor activity of the 1
Dhc contributes to phototaxis, at least in the absence of the outer arms. However, if the outer arms were present, the Dhc1 transformant strains could undergo phototaxis, whereas I1 mutant strains could not (Table 2). This difference in behavior in the presence or absence of outer arms suggests that there are cooperative interactions between the I1 complex and the outer arms during the phototaxis response.
Previous studies have demonstrated that differences in the activity of the cis and trans flagellum are the basis of the phototaxis response ( Dhc (
Dhc motor domain.
These observations raise several interesting questions about the mechanism by which changes in the phosphorylation state of IC138 might contribute to phototaxis. For example, where is IC138 located in the axoneme relative to the motor domains of the two I1 Dhcs and the three outer arm Dhcs? Is it in lobe 3 of the I1 structure, which is also in close proximity to at least one outer arm per axoneme repeat? Does IC138 interact directly with either the 1 or 1ß Dhc? We are planning to address these questions by analyzing subunit interactions within the I1 complex. Other important questions concern the identity and location of the axonemal kinases and phosphatases that modulate the phosphorylation state of IC138. Work from other laboratories has indicated that many of these regulatory components are tightly bound to the flagellar axoneme (
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We wish to thank other members of the Porter laboratory for their advice and support during the course of this project. We are also grateful to the members of the laboratories of C. Silflow, P. Lefebvre, and R. Linck (University of Minnesota) for their helpful suggestions at our weekly group meetings. Parts of this work were completed by S.H. Myster in partial fulfillment of the requirements for a Ph.D. degree at the University of Minnesota.
This work was supported by a grant from the National Institutes of General Medical Sciences (GM 55667) to M.E. Porter and S.H. Myster was supported in part by a research training grant from the National Science Foundation for Interdisciplinary Studies on the Cytoskeleton (DIR91134444). E. O'Toole was supported by a National Institutes of Health Biotechnology Resource (RR00592) grant to J.R. McIntosh.
Submitted: May 27, 1999; Revised: July 19, 1999; Accepted: July 20, 1999.
used in this report: BAC, bacterial artificial chromosome; Dhc, dynein heavy chain; IC, intermediate chain; LC, light chain; OA, outer arm; P-loop, phosphate binding motif; P1-P4, Pn, Pc, P-loops 1-4, NH2- and COOH-terminal P-loop; RT-PCR, reverse transcriptase PCR
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