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1 Graduate School of Natural Science and Technology;
2 Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Multiple forms of Tn subunits have been identified in fast skeletal (Sheng et al. 1992), slow skeletal (Grand & Wilkinson 1977) and cardiac muscles (Grand & Wilkinson 1976). Specific isoforms probably play important roles in determining the distinctive functional properties of the various muscle types. TnI isoforms are expressed in two patterns: a tissue-specific and a developmentally regulated pattern. An approach to understanding the role of TnI in the regulation of contraction is to study functional differences between various TnI isoforms. The most important molecular genetic mechanisms for generating protein isoform variants are alternative splicing from a single gene or transcription from different genes in a multigene family (Baldwin et al. 1985; Kobayashi et al. 1989; Barbas et al. 1993). TnI isoforms in Drosophila are produced from a single gene (Barbas et al. 1993) but those in vertebrates are produced from a multigene family (Hastings 1997). Interestingly, the two TnI isoforms of the protochordate ascidian, Ciona intestinalis, are encoded by a single gene (MacLean et al. 1997). It is likely that the alternative splicing mechanism is the ancestral type in the TnI genes of invertebrates, and that during vertebrate evolution, this mechanism has been abandoned in favor of transcriptional regulatory mechanisms (Hastings 1997). However, contrary to the alternative splicing mechanism of TnI genes in other invertebrates, there are four TnI genes encoding four different isoforms in C. elegans (WormBase).
After a new gene appears by gene duplication, an additional functional variant of the product can be produced by mutation and selective pressure (Gilbert 1978). Gene duplication is generally considered the primary engine of isoform diversity in the evolution of multigene families (MacLean et al. 1997). Differences in exon numbers, splicing sites and expression profiles are helpful in solving the evolutionary history of closely related genes (Baldwin et al. 1985). In globular proteins, it is known that modular structural units are often separated by different exons (Go 1983). Examination of the splicing pattern often reveals the relationships derived by the gene duplication process.
It is also of interest to know how molecular interactions within the Tn complex affect animal behavior. A genetic approach to the study of Tn functions is a powerful means by which to understand muscle function and animal behavior. Mutations in the Drosophila TnI and TnT genes produce muscle hypercontraction and have flying, walking, crawling and jumping defects (Fyrberg et al. 1990; Nongthomba et al. 2003, 2004).
The nematode C. elegans has two primary muscle types: body wall muscle for locomotion and pharyngeal muscle for feeding (Waterston 1988; Moerman & Fire 1997). These correspond to skeletal and cardiac muscles in vertebrates, respectively. In the worm pharyngeal muscle, development starts at the comma stage and body wall muscle elongation follows depending on progression after the twofold stage. Deficiencies of body wall TnC or Tm function in C. elegans causes the Pat (paralyzed arrest at embryonic twofold stage) phenotype (Williams & Waterston 1994) and in TnT, it causes Mup (muscle position abnormal) phenotype (Myers et al. 1996). The mup-2/tnt-1 TnT gene is abundantly expressed in body wall muscle but at least four TnT genes are expressed in the worm (WormBase). Two different genes: pat-10/tnc-1 and tnc-2 encode the two isoforms of TNC that are expressed in body wall and pharyngeal muscles, respectively (Terami et al. 1999; our unpublished observation).
Nothing is known of the functional differences between the TnI isoforms in C. elegans or their evolutionary relationships. Investigation of these isoforms will provide important insights into the evolutionary divergence of these proteins but also contribute to solving problems regarding mechanisms of muscle filament assembly and functional interactions within the Tn complex. What was the evolutionary history of the original TnI gene that produced four genes? What is the evidence that this has led to functional diversity as detected by different patterns of gene expression and changes in in vivo and biochemical function? We can study the CeTnI genes as a model to investigate these questions in detail for a single protein family in a genetically amenable organism.
In this study, we cloned cDNA and genomic fragments of the tni-2/unc-27 gene and completed the genome structures of three additional genes, tni-1, tni-3 and tni-4, based on the C. elegans genome sequence database, WormBase. Tissue-specific expression patterns of these genes were studied by injecting promoter::lacZ/gfp/rfp gene fusion constructs. We found that three of four CeTNI isoforms are expressed in the body wall muscle tissues at different levels and that tni-4 is expressed specifically in the pharynx. We have used RNAi to study the functions of the four TnI isoforms during muscle development. Finally, we performed in vitro protein overlay assays between four CeTNI and two CeTNC isoforms using anti-CeTNI and anti-CeTNC antibodies. Functional differences and evolutionary relationships among the four TnI isoforms in the worm are discussed. This is the first stage of characterizing all the isoforms of a troponin subunit in one animal.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Three positive clones were isolated by immunoscreening of a 
ZAPII cDNA library using antibody against Ascaris TnI (Nakae & Obinata 1993). One clone contained a 0.8 kb fragment, which showed a high homology to the tni-2 gene and had the same sequence as the cDNA clones, cm15e4 and cm20c1 (Waterston et al. 1992) corresponding to the ZK721.2 ORF at the position of the unc-27 gene. The other two clones were the partial transcripts missing the 5'end of the same gene. These results indicate that cDNA clones of the gene, tni-2/unc-27 are abundant in the library. The unc-27 (e155) mutation results in a stop at codon Gln10 (CAG
TAG; QStop, arrowhead in Fig. 2). Together with the tni-2 gene, the 5' ends of another three genes were defined by RT-PCR experiments (see Experimental procedures). The complete maps and genome organization of the four TnI genes are shown in Fig. 1. All four TnI gene transcripts are trans-spliced by the SL1 spliced leader (Fig. 1B). The N-terminal amino acid sequences were deduced from the sequences obtained following RT-PCR (Fig. 2).
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The deduced amino acid sequences of the four TNI isoforms of C. elegans were aligned with those of Drosophila (Barbas et al. 1993), crayfish (Kobayashi et al. 1989), rabbit cardiac (Grand & Wilkinson 1976), slow (Grand & Wilkinson 1977) and fast muscles (Sheng et al. 1992). N-terminal extensions are common in the TnIs of invertebrates, those from the heart of Ciona and the cardiac TnI isoforms of vertebrates (Figs 2 and 3A). This region may have functional homology and indicate an evolutionary relationship of these muscles (Grand & Wilkinson 1976). Comparison of the TnC-binding site sequence of the TnIs showed more than 60% homology between TNI-1, -2, -3 and -4 isoforms of the C. elegans, Drosophila and crayfish but only about 20% homology with body wall and heart muscle isoforms of Ciona, cardiac, and the slow and fast muscles of rabbits (Figs 2 and 3A). Detailed inspection showed that the actin/TnC-binding sites CeTNI-1 and CeTNI-2 were identical and shared more than 82% homology with C. elegans, Drosophila and crayfish, but only around 50% homology to the isoforms of the body wall and heart muscles of Ciona and the cardiac, slow and fast muscles of rabbits (Fig. 3A). C-terminal extensions consisting of repeated glutamate residues are present in CeTNI-1, CeTNI-2 and CeTNI-3, but absent in CeTNI-4 (Fig. 3A). The presence of the C-terminal extension in the three isoforms was of particular interest because of their expression in body wall muscles. A phylogenetic tree was constructed from the alignment of TNIs from C. elegans, Drosophila, crayfish, Ciona body wall and heart, rabbit cardiac, slow and fast muscles (Fig. 3B). Gene structures, functional differences and evolutionary relationships of these TnIs are discussed below.
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The expression patterns of tni-1/-2/-3/-4::lacZ, tni-2::gfp and tni-1/-3::rfp constructs were observed (Fig. 4) and the results are summarized in Fig. 4A and Table 1. Expression of tni-1/-2/-3::lacZ began at the comma stage (Fig. 4B, panel e, f, g) and continued from the L1 to adult stages in body wall muscles (Fig. 4B, panel a, b, c). The tni-1::lacZ animals showed only weak expression in the body wall muscles but the tni-2::lacZ construct gave strong expression in all the body wall muscles (Fig. 4B, panel a, b). The detailed localization of the expression of both genes was confirmed by co-expression experiments using tni-1::rfp and tni-2::gfp (Fig. 4C, see later). The tni-3::lacZ construct was expressed in all the body wall muscles from the L1 to adult stages (Fig. 4B, panel c). In the adult stage, tni-3::lacZ expression in body wall muscles became weaker with time although strong expression persisted in the head and vulval muscles.
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tni-4::lacZ was expressed only in the pharynx from the twofold to adult stages (Fig. 4B, panel d, h). We searched the B and C subelements in the upstream region of tni-4 gene and tni-4::lacZ construct that are responsible for the pharyngeal expression of the myosin heavy chain gene, myo-2 (Okkema & Fire 1994). Among the 31 nucleotide sequences of B subelement, the CEH-22 binding site (AAGTG) was conserved in tni-4. A second unknown binding site (AAAATG) in the B subelement was also found in the upstream region of the tni-4 genes of C. elegans and the related nematode C. briggsae (Fig. 5B). Nucleotide alterations in the AAAATG region (Bmut4) reduce transcriptional activity of pharyngeal expression of myo-2 (Okkema & Fire 1994). CEH-22, together with a second unknown factor could control the pharyngeal expression of tni-4 in C. elegans. PHA-4 and PEB-1 are two binding proteins of the C subelement (Kalb et al. 2002). The 5' upstream regions of the tni-4 genes of both C. elegans and C. briggsae have a partial homology to the PHA-4 binding site of myo-2 (Fig. 5C) suggesting that PHA-4 could also regulate tni-4 pharyngeal expression.
TNI localizations were also detected by indirect immunofluorescence staining of wild-type animals using anti-CeTNI-2 and anti-CeTNI-4 antibodies (Fig. 4D). The anti-CeTNI-2 antibody strongly stained the body wall muscle (Fig. 4D panel a), and faintly the pharyngeal muscle (Fig. 4D, panel b, resulting from the cross reactivity of anti-CeTNI-2 antibody, shown in Fig. 7A, left panel), vulval muscles (Fig. 4D, panel c) and anal muscles (Fig. 4D, panel d). At high magnification the anti-CeTNI-2 antibody stained the thin filaments in the I-band regions that also contain actin (Fig. 4D, panel e). The anti-CeTNI-4 antibody stained only the pharynx muscles (Fig. 4D, panel f, g). These results confirm the expression patterns determined using the reporter gene constructs.
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Single or multiple losses of TnI function were assessed by RNAi experiments. Because muscles are required for most activities of C. elegans, observations were made of body morphology, locomotion, egg laying and viability of eggs. Seventy percent of tni-1 (RNAi) (n = 28) animals at L1 stage could not elongate their bodies and were unable to move, especially their posterior parts. Animal motility is more clearly detectable in liquid medium at the adult stage. Wild-type (n = 20) and tni-1 (RNAi) animals showed 120 (± 4.5 s.d.) and 32.4 (± 3.6 s.d.) waves/min, respectively. tni-2 (RNAi) (n = 25) animals showed only 1.5 (± 0.5 s.d.) waves/min and 80% of these animals were without sinusoidal trails on NGM (Nematode Growth Medium) plate (Fig. 6B). The null unc-27 (e155) (n = 20) animals showed 24.8 (± 2.7 s.d.) waves/min, and tni-2 (RNAi) towards unc-27 (e155) (n = 20) animals showed 9.4 (± 2.8 s.d.) waves/min. tni-2 (RNAi) animals exhibited a more severe uncoordinated motility (Fig. 6B) than that of the unc-27 (e155) animals. The TNI-band intensity of unc-27 (e155) animals was detected as 10% of the wild-type in Western analysis (not shown). This result is consistent with the deformed muscle morphology of the unc-27 (e155) mutant animals (Burkeen et al. 2004). These authors also reported that the null allele unc-27 (e155) does not produce the most severe defects compared to the weak alleles. This means that mis-sense mutations (especially those classifiable as dominant negative mutants) or RNAi treatments (perhaps resulting from incomplete isoform specificity) can have more serious effects on filament assembly and muscle formation than null mutations. We cannot rule out the possibility that a severe functional defect of tni-2 (RNAi) animals on motility may arise from some interference of other isoforms as a result of nonspecific binding by related RNAi species.
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More than 90% of animals injected with the tni-1/-2/-3 (RNAi) mixture (n = 27) exhibited from the L1 stage abnormal body morphology, prominent swelling near the vulva and intestines with similar phenotypes to those of tni-3 (RNAi) animals observed at the L4 stage (Fig. 6G). tni-1/-2/-3 (RNAi) animals with many vacuoles near the vulva, intestine and anus, and were severely constipated, moved uncoordinatedly and exhibited the Egl phenotype at the adult stage (not shown) similar to tni-3 (RNAi) animals. The cuticles of young adults with the Egl phenotype had burst and were actually punctured. Compared to tni-2 (RNAi) and tni-3 (RNAi) animals, the head region of tni-1/-2/-3 (RNAi) animals had abnormal muscle cells with deformed pharynxes (Fig. 6I). Phenotypic effects in these RNAi animals appeared mainly restricted to muscles expressing these genes (Fig. 4B,C).
tni-4 (RNAi)-treated (n = 20) animals produced 70% dead embryos before the comma stage (Fig. 6K–M). This is in contrast to wild-type control worms injected only with buffer (n = 21) which showed 3% dead embryos. Embryonic cells totally degraded between 400 and 500 min and by approximately early 400 min, embryos had shown developmental arrest at gastrulation, the 28-cell stage (Fig. 6K). Initially, arrested embryos exhibited a severe shrinkage as a result of loss of water (Fig. 6L) but by 500 min further degradation decreased cell size with more cell loss (Fig. 6M). Fifteen percent of surviving tni-4 (RNAi) animals showed no pharynx pumping but grew normally. Generally, at about 350 min, cell proliferation largely ceases and the completely formed internal cylinder of the pharynx starts to pump at about 760 min. tni-4 (RNAi)-treated embryos arrested development before the pharynx pumping was initiated. Despite the presence of transcription factors such as pha-4 (Kalb et al. 2002), peb-1 (Kalb et al. 2002), ceh-22 (Okkema & Fire 1994) and other muscle genes known to be expressed in the pharynx, the embryonic arrest indicates that completion of normal pharynx formation requires tni-4 function. This is consistent with previous RNAi experiments with two pharynx Tm isoforms, CeTMIII and CeTMIV, which showed that developmental effects appeared at a much earlier stage than body wall muscle development (Anyanful et al. 2001).
Using the RNAi feeding (Kamath et al. 2003; Simmer et al. 2004) and soaking methods (Maeda et al. 2001), tni-1 (RNAi) animals showed dumpy, Egl and Unc phenotypes. tni-2 and tni-3 (RNAi) animals appeared wild type and tni-4 (RNAi) animals grew slowly. Our RNAi study (by microinjection) gave much more severe phenotypes and was consistent with the tissue expression patterns (Table 1, Figs 4 and 6), suggesting that this method more accurately reflects the results of other approaches to determine gene function.
Interaction: In vitro molecular interaction between four CeTNI and two CeTNC isoforms
Anti-CeTNI-2 antibody cross-reacted with four CeTNIs but did not cross-react with the CeTNCs (Fig. 7A, left panel, Fig. 7B). The smaller band of CeTNI-2 may have come from limited proteolysis of CeTNI-2 in bacteria or from a second initiation product of the construct. Anti-CeTNI-4 antibody specifically reacted only with CeTNI-4 (Fig. 7A, right panel, Fig. 7B). CeTNI-2 and CeTNI-4 were overlaid on to two CeTNCs and were detected using anti-CeTNI-2 and anti-CeTNI-4 antibodies, respectively. The overlay indicated that CeTNI-2 bound to both CeTNC-1 and CeTNC-2 (Fig. 7C, left panel), but that CeTNI-4 specifically bound to CeTNC-2 (Fig. 7C, right panel).
After determining the specificity of anti-CeTNC-1 and anti-CeTNC-2 antibodies by Western analysis (Fig. 7D), we performed overlay assays of each CeTNC on the four CeTNIs. CeTNC-1, the body wall type isoform, bound specifically to all three body wall type CeTNIs (CeTNI-1, -2, -3) (Fig. 7E, left panel, Fig. 7F) but that of pharynx type CeTNC-2 bound to all four CeTNIs (Fig. 7E, right panel, Fig. 7F).
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Discussion |
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Structural differences among four TnI genes and TNI isoforms of C. elegans
We have fully characterized the gene structure of the four TnI genes in C. elegans and the amino acid sequences of the TNI isoforms they produce. Results obtained on TnI gene structures (Fig. 1) and phylogenetic analysis from the sequence comparisons (Figs 2 and 3) allowed us to determine the evolutionary relationships of the four TnI genes. In particular, we found that the TnC-binding sites of three of the genes, tni-1, -2 and -3 are split by an intron while that of tni-4 is not. The actin/TnC-binding sites are split differently in the tni-3 and tni-4 genes, but the amino acid sequences are identical between them. Although the presence of N-terminal extensions of the four CeTNI are a common feature found in other invertebrates, the C-terminal extensions of CeTNI-1, -2 and -3 are unique. From these results we speculate that the tni-4 gene could be closer to the ancestral gene for the four TnI genes in C. elegans (Fig. 3A,B). This assumption was strongly supported by the following expression and functional analyses.
Functional differences in four CeTNI isoforms
Differences in expression patterns between the four TnI genes
Following gene duplication, it is proposed that new isoforms can evolve so that the organism can achieve new capabilities (MacLean et al. 1997), in this case by the evolution of different muscle types. A comparison of the tissue and stage-specific expression patterns should therefore be revealing about the evolution of this gene family. The TnI expression patterns obtained using the tni-1::rfp and tni-2::gfp or tni-2::gfp and tni-3::rfp transgenic animals were important in identifying major differences in expression of the three body wall muscles TnI isoforms (Fig. 4C). The three genes, tni-1, tni-2 and tni-3, were co-expressed in all the body wall muscles, but at different levels (Fig. 4C, panel a–l) under the control of the 5' upstream regulatory sequences (Fig. 5A).
The tni-4 gene is expressed solely in the pharyngeal muscles (Fig. 4B, panel d) which are formed at much earlier stages of the nematode development than the body wall muscles. The tni-3 gene could have duplicated from the original and formed a body wall isoform. Compared to the rhythmic contractions of the pharynx, the body wall muscle contractions are stimuli-dependent and calcium-regulated.
Phenotypic differences by RNAi treatment in four CeTNI isoforms
The effects of interfering with isoform function using isoform-specific RNAi constructs shows that the CeTnI genes have clearly evolved to achieve differences in functional importance. While RNAi for the tni-1 and -2 genes produce abnormal locomotion, only RNAi treatment interfering with tni-3 function led to abnormal muscle morphology, egg laying defects, and constipation, the latter two as a result of an absence of contractions of the vulva and anus, and associated vacuolization. This suggests that within the body wall-expressed genes, the tni-3 gene may be especially important. Inhibition of tni-4 gene expression produced large numbers of dead embryos from injected individuals, with survivors showing no pharyngeal pumping; RNAi treated embryos arrested at gastrulation long before pumping was initiated. Clearly, the multiple TnI genes have evolved to encode functionally different isoforms with different expression patterns.
Nongthomba et al. (2003, 2004) report on muscle hypercontraction phenotypes in Drosophila caused by the mutant alleles of the TnI genes. We observed uncoordinated phenotypes in tni-1 (RNAi) and tni-2 (RNAi) animals that showed weak backward movements. This suggests that defects in Ca2+ regulation can produce hypercontraction or Unc phenotypes in C. elegans as observed in Drosophila. Currently, no mutant has been isolated in the tni-3 gene, but the phenotypes seen with the tni-3 (RNAi) animals (Fig. 6C–F), from egg laying defect (Egl), protruding vulva (Pvl) and constipation (Con), it is possible to isolate some tni-3 mutants suggest tni-3 mutants might be recoverable by selecting for these phenotypes. Isolating mutants in the tni-3 gene would allow us to determine the sites responsible for the functional differences in this isoform compared to the others.
Interaction patterns between four CeTNI and two CeTNC isoforms
The interaction between TnI and TnC is a pivotal one in the function of the Tm-Tn complex. We have therefore explored TnC-TnI in vitro binding to determine whether there are isoform-specific differences that may indicate that the isoforms have functionally diverged during evolution. The results of the protein overlay assay (Fig. 7F) imply that the body wall CeTNI-2 bound to the body wall and pharynx types of CeTNC (Fig. 7C, left panel) whereas pharynx CeTNI-4 only bound to pharynx CeTNC-2 (Fig. 7C, right panel). In contrast, the body wall CeTNC-1 bound to three body wall CeTNI isoforms (Fig. 7D, left panel) but pharynx CeTNC-2 bound to both body wall and pharynx types of CeTNI isoforms (Fig. 7E, right panel).
Recently, the crystal structure of the core Tn complex has been determined. The authors stated that the interaction sites between Tn and Tm/actin could be unique in each molecule of the complex in different tissues and animals (Takeda et al. 2003). Using our system, a reverse genetic approach in which a designed gene can be introduced in the animal should be possible and would allow investigation of which parts of the molecule, especially the isoform-specific parts, function to produce muscle-specific changes in function that subsequently contribute to animal behavior.
It is known that vertebrate cardiac TnC and TnI interact with each other more weakly than the corresponding components of skeletal muscle (Liao et al. 1994). At present, neither the site of interaction between CeTNI and CeTNC, nor the location of antibody detection sites of either antibodies are known. Molecular dissection techniques with epitope mapping of CeTNI will help to determine which part of the molecule interacts with other molecules (Hamada et al. 2002). If a difference can be found in the binding site the results will suggest coevolution between the CeTNIs and CeTNCs.
Functional relations to other animal TnIs
The C-terminal region of TnI is essential for Tm–actin and TnC interaction in the case of rabbit and chicken (Van Eyk & Hodges 1988). It is not known why the C-terminal extensions, which are extremely rich in glutamate, are needed for the function of the three body wall TnI isoforms in C. elegans (Figs 2 and 3A) and also in C. briggsae (not shown). In fact, there is, as yet, no direct evidence that these extensions are important for TnI functions. However, the C-termini of TnTs in Drosophila and crayfish Astacus show conserved glutamate-rich extensions (Fyrberg et al. 1990; Benoist et al. 1998; Domingo et al. 1998) that it has been proposed might enhance cooperation of Tn-Tm complexes within thin filaments (White et al. 1987). The CeTNI may have a homologous function. Glutamate has a negative charge, which may contribute to protein–protein interactions under different Ca2+ concentrations. The C-terminal hydrophilic regions of the three body wall type CeTNIs could be important for interactions with CeTNC-1 in body wall muscles.
The evolutionary relationships of the nematode TnI genes
The pattern that emerges from our studies of gene structure, protein sequence, expression patterns and biochemical function (CeTNI-CeTNC binding) is that the CeTnI genes fall into two distinct groups: the body wall-expressed genes (tni-1, -2 and -3) and the pharyngeal expressed gene, tni-4. Of these, the tni-4 gene appears most likely to be close to the ancestral gene from which this protein family evolved.
The data suggest that tni-3 may be the original body wall-expressed gene. It is expressed widely in the body wall and the RNAi studies show that it is required and has a greater effect on a number of body wall muscle functions — movement, vulval and anal contractions, while tni-1 and tni-2 gene functions seem restricted to locomotion. We propose that the tni-2 gene, that encoded CeTNI-2 for locomotion, arose from tni-3, and that tni-1 and tni-2 are close to each other in three points, represent the most recent gene duplication. This is supported by our observation that they are (i) located on the same chromosome (Fig. 1A), (ii) show a high homology of amino acid sequences, especially at the actin/TnC-binding site (Figs 2 and 3A), and (iii) are expressed in the same cells (Fig. 4C, panel c). Although these genes are functionally related to each other, tni-1 expression was weaker (Fig. 4) and occurred in fewer body wall muscles. We propose that tni-1 is a ‘spare’ gene; that is, it is the one of a duplicated pair of genes that has acquired the potential to function in a newly appeared tissue-specific manner in evolution. The relation between tni-1 and tni-2 readily fulfills these criteria. Currently, the C. elegans tni-2 gene encodes a body wall muscle isoform necessary for locomotion as seen from the RNAi treatments. tni-1 can be ‘a spare gene’ if it is sufficiently required for new function in a new functional tissue or in a different pattern of tissues. As more complete genome sequence data accumulate, this kind of gene, ‘a spare gene’, should be reported from other organisms. The relation between gene duplication and functional divergence to produce new or modified function tissues, changes in morphology and development processes is one of the key subjects in evolution. To know how a spare gene can get a function in newly appeared tissues is an interesting subject for the future.
Why has gene duplication been important in the evolution of protein families? Our studies have demonstrated with the TnI family in C. elegans not only that these genes likely share a common genetic origin but that they have acquired functional differences. Some of the amino acid differences between CeTNI isoforms must be important, suggesting that one TNI isoform could not readily substitute for another. To test for functional differences, it is necessary to introduce amino acid substitutions in different isoforms in those regions that interact with other members of troponin complex. As the actin/TnC-binding site between CeTNI-1 and CeTNI-2 or CeTNI-3 and CeTNI-4 are identical, this approach is hard to design. Introducing substitution into TnC-binding sites in four TnI genes could be possible but more efficient approaches could be done by injecting the chimera construct of the promoter/coding regions into the mutant animals.
It is well established that different protein classes undergo molecular evolution at different rates, presumably reflecting different functional constraints. However, it is also the case that different isoforms of the ‘same’ protein, encoded by a multigene family, may evolve at different rates. Excitation-contraction coupling in body wall for locomotion, vulva for egg laying and anal for defecation show functionally different patterns and these are likely to be reflected at molecular level. This may explain why three CeTNI isoforms function within the body wall muscles although we cannot identify each of the three functions. Alternatively, the presence of more than one isoform may be necessary to modulate muscle function. As we have established TnI studies at the molecular, tissue and whole animal levels in the nematode, we can use this system to investigate in more detail the functional differences of CeTNI isoforms at the molecular and physiological levels. It is also possible to develop the worm model to study TnI mutations in human myopathies, including cardiomyopathy.
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The C. elegans strains; wild-type Bristol N2, mutant unc-27 (e155), microinjected with the reporter genes and RNAi treated animals used in this study were grown under standard conditions (Brenner 1974).
DNA handling and sequencing
Conventional methods were used for all DNA manipulations except where otherwise stated. The purified pBluescript SK (–) plasmids with the cloned RACE fragments were sequenced by Dye Terminator cycle sequencing kits with a 373 A DNA sequencer (Perkin Elmer, Applied Biosystems Division). DNASIS‘ and GENETYX-MAC were used for sequence data analysis.
Cloning strategy and detection of mutation
A polyclonal antibody against Ascaris TnI (Nakae & Obinata 1993) used for immunoscreening of lZAPII cDNA library was provided by Dr Barstead (Oklahoma Medical Research Foundation, Oklahoma City, OK). The pCTnI-2 plasmid contained the cDNA insert at the EcoRI site of pBluescript SK (–). The 6.5 kb EcoRI fragment of tni-2 and 5 kb HindIII genomic fragments of tni-1 were obtained from a genomic DNA library constructed with size-fractionated genomic fragments cloned into a processed plasmid (pUC118). All positive colonies were identified by ECL System (Amersham Corp).
Double-stranded DNA was purified using a single worm PCR (polymerase chain reaction) method (Barstead & Waterston 1991). The Taq polymerase used for PCR was from TaKaRa Biochem. Amplified product was sequenced for localization of mutation site of unc-27 (e155).
RNA isolation and 5¢ RACE
Total RNA was isolated from wild-type nematodes using the TRIzol LS reagent (Gibco BRL), following the manufacturer's instructions. The isolated RNA was stored in three times its volume at 99% (v/v) at -80 C.
Reverse transcription (RT)-PCR and 5¢ RACE reactions were performed using RACE system (Gibco BRL), following the manufacturer's instructions. Primers used were TNI1–2a (5¢-CGAAGCTTCTTCTTACGCTC-3¢) for tni-1, TNI2–2a (5¢-CCGAGCTCAGCAGCCTTGTTC-3¢) for tni-2, Ti3Ex3A2 (5¢-AAGTCGACATTTCATCGTAGACTGCCTCG-3¢) for tni-3 and Ti4Ex3A2 (5¢-AAACTAGTATAGTACGCTGCTGAAGGGTCT-3¢) for tni-4. These cDNA products were tail-tagged with terminal deoxynucleotidyl transferase (TdT, Gibco BRL) and used to tag the cDNA products and PCR amplification for second strand DNA syntheses that were performed with SL1 primer as sense and TNI1–1a (5¢-TCTTGGCCTTCTTCTTGTTTCCGG-3¢) for tni-1, TNI2–1a (5¢-CTTCTTTCTTTCTGGAGTC-3¢) for tni-2, Ti3Ex3A1 (5¢-GACGAACGATGTAGCTGA-3¢) for tni-3 and Ti4Ex3A1 (5¢-GGGATAGTACGCTGCTGAAGGG-3¢) for tni-4 as anti-sense. Southern hybridization was used to confirm the presence and size of fragments of RACE products which were then cloned into pBluescript SK(–) for sequencing.
Construction of reporter gene fusions
We prepared tni-1::lacZ, tni-2::lacZ, tni-3::lacZ, tni-4::lacZ, tni-1::rfp, tni-2::gfp and tni-3::rfp fusions that accurately reflected the wild-type expression patterns. All constructs summarized in Fig. 4A are available upon request. Several transgenic C. elegans lines bearing extra chromosomal arrays containing the promoter::repoter fusion plasmids were obtained by microinjection together with the plasmid pRF4 as a marker. Strains with the extra chromosomal arrays were stained to detect b-galactosidase activity (Fire 1992). All constructs were assayed in more than two independent transgenic lines.
RNA mediated interference
The four TnI genes were amplified separately from their cDNAs by PCR using M13 (-21) and M13RV primers (Gibco). The cDNAs corresponding to their full coding regions were inserted in the pBluescript SK(–) vector. RNA was synthesized in vitro from the DNA template with Ambion MAXIscript‘ using T3 and T7 polymerases. DNA removal, RNA extraction, suspension and annealing were performed as described (Montgomery et al. 1998). The dsRNA was injected into the gonads of wild-type worms. For triple RNAi, dsRNAs of three genes were mixed together and injected into wild-type worms. After recovery, worms were transferred to fresh NGM plates and further transferred to another fresh culture plates every 12 h. The injected worms are referred to as P0, and subsequent transfers as P1, P2, etc. The embryos for observation were washed from the plate by the hypochlorite method as described (Lewis & Fleming 1995) at 300 and 500 min; 12, 18, 24, 36, 60 and 72 h and kept for 5 h at 20 C in M9 buffer for any developmental change that might occur (Anyanful et al. 2001). The approximate timing of tni-4 (RNAi) eggs were determined so as to allow comparison to control, buffer injected embryos. A Nomarski interference microscope (Zeiss Axioplan 2) at 400¥ was used for observing embryos and worms.
Troponin C and troponin I expression vectors
A 650 bp EcoRI fragment of cDNA clone, pCTNC1 (Terami et al. 1999), was inserted into the EcoRI site of pET28 (+) (Novagen). A 650 bp KpnI-EcoRI fragment of tnc-2 was cloned into the KpnI and EcoRI sites of pUC119 (STRATAGENE) from yk366f10 cDNA clone. A SalI-EcoRI fragment from this plasmid was recloned into the SalI and EcoRI sites of pET28b (+).
A 1 kb EcoRI to KpnI fragment of tni-1 was cloned into the EcoRI and SmaI sites of pGEX-4T2 from yk103h4 cDNA clone. A 1 kb EcoRI fragment of tni-2 was cloned into the EcoRI site of pGEX-4T3 (Pharmacia Biotech.) from cDNA clone, cm15e4 of tni-2 ligated into pBluescript SK(–). The 1 kb EcoRI to KpnI fragment of tni-3 was cloned into the EcoRI and SmaI sites of pGEX-4T1 from yk147e9 cDNA clone. PCR product of tni-4 encoding the tni-4 cDNA without its own stop codon was generated and subcloned in frame at the BamHI and SmaI sites of pGEX-4T2 from yk328a10 cDNA clone.
Antibody preparation, immunostaining, Western analysis and protein overlay assay
Antisera used in this study were prepared by immunizing rabbits with gel homogenates containing 1 mg of the His-tag fusion peptide which was isolated by preparative SDS-PAGE and the corresponding band was excised from the Coomassie Blue stained gel (Hamada et al. 2002). Briefly, the immunization procedure was as follows: antigens were injected three times into rabbits at 2-week intervals. Antiserum was collected 1 week after the last injection. Immunostaining employed b-mercaptoethanol-collagenase treatment (Williams & Waterston 1994) for worms. Antibody was affinity-purified from the antiserum by adsorption on to a membrane to which the band of the corresponding fusion peptide had been transferred. The antibody concentration was estimated from the density of the IgG band using BSA as a standard. The amount of protein loaded in each lane was approximately 0.002 mg. Western analysis was performed using the ECL detection system (Amersham International Plc.). GST-troponin I fusion proteins were over-expressed in the host strain JM109 and BL21 (DE3) and reacted with bacterial protein including TNC. Using bacterially expressed proteins, we characterized the interactions between CeTNI and CeTNC by overlay assay (Terami et al. 1999).
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Acknowledgements |
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ZAPII cDNA expression library, to T. Obinata for the anti-TNI antibody against Ascaris and to Y. Kohara for the cDNA clones. We acknowledge John C. Sparrow for his generous help in critically reading the manuscript. We are thankful to F. Anokye-Danso, T. Hamada and S. Mahmood for their technical support and advice. Some of the strains used in this study were obtained from the Caenorhabditis Genetics Center, which is funded by the National Institute of Health and the National Center for Research Resources. A part of this work was supported by the ‘Japan-Korea Basic Scientific Cooperation Program.’ R. R. is a foreign student receiving a student scholarship from the Ministry of Education, Culture, Sports, Science and Technology of Japan. |
Footnotes |
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* Correspondence: E-mail: hkagawa{at}cc.okayama-u.ac.jp
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Received: 31 August 2004
Accepted: 2 December 2004
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