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Genomic organization, expression, and analysis of the troponin C gene pat-10 of Caenorhabditis elegans.

Terami H, Williams BD, Kitamura Si, Sakube Y, Matsumoto S, Doi S, Obinata T, Kagawa H - J. Cell Biol. (1999)

Bottom Line: We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site.The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I.These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Faculty of Science, Okayama University, Okayama, 700-8530 Japan.

ABSTRACT
We have cloned and characterized the troponin C gene, pat-10 of the nematode Caenorhabditis elegans. At the amino acid level nematode troponin C is most similar to troponin C of Drosophila (45% identity) and cardiac troponin C of vertebrates. Expression studies demonstrate that this troponin is expressed in body wall muscle throughout the life of the animal. Later, vulval muscles and anal muscles also express this troponin C isoform. The structural gene for this troponin is pat-10 and mutations in this gene lead to animals that arrest as twofold paralyzed embryos late in development. We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site. The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I. These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.

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Comparison of amino acid sequences of troponin C-1 of C. elegans with a second troponin C of C. elegans and those of Drosophila, crayfish, and rabbits. Alignment of C. elegans troponin C, CeTNC-1 (pat-10), CeTNC-2 (cosmid ZK673.7), Drosophila troponin C Dm73F (Fyrberg et al. 1994), crayfish type (Kobayashi et al. 1989), rabbits, cardiac (Wilkinson 1980) and skeletal (Zot et al. 1987). Four calcium-binding sites are shown (I–IV). Mutation sites in the pat-10(st575) mutant are shown at 64 D to N and 153 W to termination (*), respectively. Shading indicates the residues that are identical in these six troponin C proteins.
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Figure 3: Comparison of amino acid sequences of troponin C-1 of C. elegans with a second troponin C of C. elegans and those of Drosophila, crayfish, and rabbits. Alignment of C. elegans troponin C, CeTNC-1 (pat-10), CeTNC-2 (cosmid ZK673.7), Drosophila troponin C Dm73F (Fyrberg et al. 1994), crayfish type (Kobayashi et al. 1989), rabbits, cardiac (Wilkinson 1980) and skeletal (Zot et al. 1987). Four calcium-binding sites are shown (I–IV). Mutation sites in the pat-10(st575) mutant are shown at 64 D to N and 153 W to termination (*), respectively. Shading indicates the residues that are identical in these six troponin C proteins.

Mentions: Initially, we screened a C. elegans cDNA expression library with a polyclonal antiserum raised against purified Ascaris troponin C (Nakae and Obinata 1993), and recovered a 0.7-kb cDNA encoding a 161 residue polypeptide with extensive homology to troponin C from other organisms (see below, Fig. 3). To isolate the corresponding genomic sequence and identify its chromosomal location, we hybridized the cDNA to a filter containing a gridded set of yeast artificial chromosome (YAC) clones that span most of the nearly complete C. elegans genomic physical map (Coulson et al. 1988). We detected strong hybridization to a set of four overlapping YAC clones on chromosome I in the interval defined by the unc-38 and dpy-5 genes (see Fig. 1A and Fig. B). We further mapped the hybridizing region to cosmids F54C1 and C15C10 (Fig. 1 B), and ultimately to a 3.5-kb PstI fragment (Fig. 1 C) which contains the entire troponin C gene, pat-10 (see below, Fig. 2).


Genomic organization, expression, and analysis of the troponin C gene pat-10 of Caenorhabditis elegans.

Terami H, Williams BD, Kitamura Si, Sakube Y, Matsumoto S, Doi S, Obinata T, Kagawa H - J. Cell Biol. (1999)

Comparison of amino acid sequences of troponin C-1 of C. elegans with a second troponin C of C. elegans and those of Drosophila, crayfish, and rabbits. Alignment of C. elegans troponin C, CeTNC-1 (pat-10), CeTNC-2 (cosmid ZK673.7), Drosophila troponin C Dm73F (Fyrberg et al. 1994), crayfish type (Kobayashi et al. 1989), rabbits, cardiac (Wilkinson 1980) and skeletal (Zot et al. 1987). Four calcium-binding sites are shown (I–IV). Mutation sites in the pat-10(st575) mutant are shown at 64 D to N and 153 W to termination (*), respectively. Shading indicates the residues that are identical in these six troponin C proteins.
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Related In: Results  -  Collection

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Figure 3: Comparison of amino acid sequences of troponin C-1 of C. elegans with a second troponin C of C. elegans and those of Drosophila, crayfish, and rabbits. Alignment of C. elegans troponin C, CeTNC-1 (pat-10), CeTNC-2 (cosmid ZK673.7), Drosophila troponin C Dm73F (Fyrberg et al. 1994), crayfish type (Kobayashi et al. 1989), rabbits, cardiac (Wilkinson 1980) and skeletal (Zot et al. 1987). Four calcium-binding sites are shown (I–IV). Mutation sites in the pat-10(st575) mutant are shown at 64 D to N and 153 W to termination (*), respectively. Shading indicates the residues that are identical in these six troponin C proteins.
Mentions: Initially, we screened a C. elegans cDNA expression library with a polyclonal antiserum raised against purified Ascaris troponin C (Nakae and Obinata 1993), and recovered a 0.7-kb cDNA encoding a 161 residue polypeptide with extensive homology to troponin C from other organisms (see below, Fig. 3). To isolate the corresponding genomic sequence and identify its chromosomal location, we hybridized the cDNA to a filter containing a gridded set of yeast artificial chromosome (YAC) clones that span most of the nearly complete C. elegans genomic physical map (Coulson et al. 1988). We detected strong hybridization to a set of four overlapping YAC clones on chromosome I in the interval defined by the unc-38 and dpy-5 genes (see Fig. 1A and Fig. B). We further mapped the hybridizing region to cosmids F54C1 and C15C10 (Fig. 1 B), and ultimately to a 3.5-kb PstI fragment (Fig. 1 C) which contains the entire troponin C gene, pat-10 (see below, Fig. 2).

Bottom Line: We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site.The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I.These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Faculty of Science, Okayama University, Okayama, 700-8530 Japan.

ABSTRACT
We have cloned and characterized the troponin C gene, pat-10 of the nematode Caenorhabditis elegans. At the amino acid level nematode troponin C is most similar to troponin C of Drosophila (45% identity) and cardiac troponin C of vertebrates. Expression studies demonstrate that this troponin is expressed in body wall muscle throughout the life of the animal. Later, vulval muscles and anal muscles also express this troponin C isoform. The structural gene for this troponin is pat-10 and mutations in this gene lead to animals that arrest as twofold paralyzed embryos late in development. We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site. The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I. These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.

Show MeSH