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First published online 7 August 2007
doi: 10.1242/jcs.007369

Journal of Cell Science 120, 3075-3085 (2007)
Published by The Company of Biologists 2007
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Targeted gene disruption of dynein heavy chain 7 of Tetrahymena thermophila results in altered ciliary waveform and reduced swim speed

Christopher R. Wood1, Robert Hard2 and Todd M. Hennessey1,*

1 Department of Biological Sciences, State University of New York at Buffalo, Amherst, NY 14260, USA
2 Department of Anatomy, State University of New York at Buffalo, Amherst, NY 14260, USA


Figure 1
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  		Fig. 1. Disruption of the DYH7 gene. A 2.4 kb DNA fragment near the 5' end of the 14.5 kb DYH7 open reading frame (ORF) was cloned to pCR4-TOPO vector. The neo3 gene disruption cassette was inserted into ClaI and XmaI restriction sites that were introduced to this DYH7 fragment by site-directed mutagenesis. This disruption interrupts the predicted N-terminal tethering domain of DYH7 and is upstream of its P-loop domains. The resulting plasmid was linearized and used in biolistic transformation of vegetative CU427.4 Tetrahymena. The annealing sites for the DYH7 gene-specific primers (gsp) and neo3 cassette-specific primer (neo3BTU) are indicated.

 

Figure 2
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  		Fig. 2. Genotypic analysis of wild-type (CU427.4) and mutant (DYH7neo3) cell lines. (A) (Panel 1) Total genomic DNA was isolated and used as template for PCR with DYH7 gene-specific and neo3-specific primers. A 3.8 kb band and a 2.4 kb band were amplified from the mutant genomic DNA template using the gsp1 primer set (gsp1-5 and gsp1-3, see Fig. 1) specific for the 5' and 3' ends of the DYH7 fragment (lane 1). The 3.8 kb band, representing the presence of neo3 insertion, was absent when wild-type genomic DNA template was used (lane 3). A 2.8 kb band was amplified from the mutant genomic DNA template when a 5' gene-specific primer, gsp1-5, was paired with a neo3-specific primer, neo3BTU (lane 2). This band was absent when wild-type genomic DNA template was used (lane 4). (Panel 2) Total RNA was isolated and used as template for first-strand synthesis of cDNA with the gsp1-3 primer used above. The same DYH7 gene-specific primer set used in panel 1, gsp1-5/gsp1-3, amplified a 2.2 kb band from wild-type cDNA template (lane 6). This band was absent when mutant cDNA template was used (lane 5). (Panel 3) Primers specific for a DYH7 gene region upstream of the neo3 insertion site (gsp2-5 and gsp2-3) amplified a 300 bp band from the wild-type cDNA template (lane 9). This band was absent when mutant cDNA template was used (lane 7). As a positive control, primers specific for ribosomal protein gene RPL21 amplified a 370 bp band from both mutant and wild-type cDNA templates prepared using an oligo (dT) primer (lanes 8 and 10). (B) Real-time PCR analysis of mutant and wild-type cDNA. The same templates and primers from A (panel 3) were used in real-time PCR amplification with SYBR green-fluorescent probe. The fluorescent signal specific for expression of the endogenous DYH7 gene was observed with wild-type template (closed squares), but was absent with the mutant template (closed circles). The RPL21 control reactions, performed in separate tubes, produced a signal of similar threshold cycle with both mutant (open circles) and wild-type templates (open squares).

 

Figure 3
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  		Fig. 3. Swim paths of Tetrahymena. Examples of wild-type (CU427.4) and mutant (DYH7neo3) swim paths prepared by digital video microscopy of 2-second time intervals are shown. Mutants traverse a shorter path distance because they have a slower rate of forward propulsion.

 

Figure 4
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  		Fig. 4. Swim speeds of intact wild-type (CU427.4) and mutant (DYH7neo3) cells. (A) Mutant cells were harvested for analysis from growth media containing paromomycin and cadmium (+ drug) or from growth media in which these selective agents were absent (– drug). Mutant cells consistently displayed swim speeds of almost half those of wild type. (B) Mutant and wild-type cell lines were cultured free of paromomycin and cadmium, and swim speeds were analyzed periodically over a span of 1 month. Mutant cells from standing tube (closed circles) and shaken flask (open circles) culturing methods are compared. Reversion to wild-type phenotype was not observed. The CU427.4 wild-type control cells (closed squares) were cultured in standing tubes. Each bar and data point represents the mean ± s.d. of three trials of 100-cell measurements.

 

Figure 5
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  		Fig. 5. Wild-type (CU427.4) and mutant (DYH7neo3) cells modulate their swim speed under the addition and removal of 8 mM KCl. (A) Mutant cells were harvested for swim speed analysis from growth media containing paromomycin and cadmium (+ drug) or from growth media in which these selective agents were absent (– drug). Despite their reduced basal swim speed relative to wild type, mutant cell lines are able to further decrease their swim speed under 8 mM KCl depolarizing stimulus in a way that mirrors that of wild type. (B) Swimming behavior was examined as cells were introduced to a range of KCl concentrations. Beyond 10 mM, cells began to display constant ciliary reversal behavior (CCR). Mutant cell lines cultured in the presence of selective agents (open circles) and the absence of selective agents (closed circles) retained their ability to display ciliary reversal with a concentration dependency similar to that of CU427.4 (closed squares). Each bar and data point represents the mean ± s.d. of three trials of 100-cell measurements.

 

Figure 6
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  		Fig. 6. Tetrahymena retain their body shape after detergent extraction of membrane systems. Differential-interference contrast microscopy of an intact wild-type cell (top) and a detergent-extracted cell model (bottom).

 

Figure 7
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  		Fig. 7. Swim speed of reactivated models. Mutant cells were harvested for analysis from growth media containing paromomycin and cadmium (+ drug) or from growth media in which these selective agents were absent (– drug). Detergent-extracted models of wild type (CU427.4) and mutant (DYH7neo3) were prepared. Forward swimming resumed when cell models of wild type and mutant were reactivated in the presence of 1 mM MgATP. Reactivated mutant models displayed a slower swim speed than that observed for wild-type models. Each bar represents the mean ± s.d. of three trials of 50-cell measurements.

 

Figure 8
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  		Fig. 8. Cilia on the aboral surface of wild-type and mutant cells observed using high-speed video microscopy techniques. Displayed left to right are six digitally traced frames extracted from video footage of one beat cycle of free-swimming wild type (CU427.4) and mutant (DYH7neo3). Each frame shows three adjacent cilia. Wild-type cilia displayed a consistent and highly organized metachronous waveform, whereas mutant cilia displayed aberrant waveforms and distorted metachrony.

 

Figure 9
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  		Fig. 9. Cell densities of wild-type (CU427.4) and mutant (DYH7neo3) cultures observed over time. (A) Mutant standing tube cultures (broken line) displayed a reduced growth rate when compared with wild-type CU427.4 standing tube cultures (solid line). (B) This growth-defect phenotype is rescued when the shaken flask culturing method is employed. Each bar and data point represents the mean ± s.d. of three trials of 100-cell measurements.

 

Figure 10
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  		Fig. 10. Phylogenetic analysis of Tetrahymena dynein genes. This phylogenetic tree was constructed from database amino acid sequences as described in the Materials and Methods. It represents an update of the original phylogenetic analysis of Tetrahymena dyneins by Asai and Wilkes (Asai and Wilkes, 2004Go), but it includes additional sequence information available from the current databases. The arrow points to DYH7 and shows that it is orthologous to the Chlamydomonas I1-beta. The inserted numbers represent the posterior probabilities and the scale bar represents 0.3 amino acid differences per residue.

 

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© The Company of Biologists Ltd 2007