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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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Western blot identification of troponin C from a worm extract and bacterial expression. Protein fractions were separated on 10–20% gels and either stained with Coomassie brilliant blue (A) or transferred to nitrocellulose for indirect probing with anti–Ascaris-troponin C antiserum (B). Protein fractions are the immunotransfers contained in a total protein extract from the wild-type N2 (lane 1), bacterial protein from the troponin I clone, pCTNI-1 (lane 2), bacterial protein from the troponin C clone, pCTNC-1 (lane 3), bacterial protein from bluescript (lane 4), respectively. Molecular size marker (M).
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Figure 4: Western blot identification of troponin C from a worm extract and bacterial expression. Protein fractions were separated on 10–20% gels and either stained with Coomassie brilliant blue (A) or transferred to nitrocellulose for indirect probing with anti–Ascaris-troponin C antiserum (B). Protein fractions are the immunotransfers contained in a total protein extract from the wild-type N2 (lane 1), bacterial protein from the troponin I clone, pCTNI-1 (lane 2), bacterial protein from the troponin C clone, pCTNC-1 (lane 3), bacterial protein from bluescript (lane 4), respectively. Molecular size marker (M).

Mentions: Muscle expression of troponin C was confirmed by indirect immunofluorescence staining of wild-type animals (Fig. 8 and Fig. 9). An antibody was generated to nematode troponin C expressed in bacteria (see Materials and Methods), and in nematode protein extracts a band of the expected size (∼18 kD) was detected on SDS-PAGE gels as were larger bands (Fig. 4 B, lanes 1 and 3). These larger bands may be complexes of troponin C with troponin I and troponin I plus troponin T. It is known that troponins strongly bind to each other and can form troponin complexes (Fig. 4 B, lane 1; Grabarek et al. 1990). Using this antisera, we confirmed the expression pattern detected by the reporter constructs. Body wall muscle expression can be seen in embryos and carries on throughout the life of the animal (Fig. 8 and Fig. 9). In older animals vulva and anal muscle expression can also be observed (Fig. 8C and Fig. D). Some staining of pharyngeal muscle may come from cross-reaction with the second troponin C isoform, as suggested by the high degree of sequence homology between these proteins (data not shown).


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)

Western blot identification of troponin C from a worm extract and bacterial expression. Protein fractions were separated on 10–20% gels and either stained with Coomassie brilliant blue (A) or transferred to nitrocellulose for indirect probing with anti–Ascaris-troponin C antiserum (B). Protein fractions are the immunotransfers contained in a total protein extract from the wild-type N2 (lane 1), bacterial protein from the troponin I clone, pCTNI-1 (lane 2), bacterial protein from the troponin C clone, pCTNC-1 (lane 3), bacterial protein from bluescript (lane 4), respectively. Molecular size marker (M).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199735&req=5

Figure 4: Western blot identification of troponin C from a worm extract and bacterial expression. Protein fractions were separated on 10–20% gels and either stained with Coomassie brilliant blue (A) or transferred to nitrocellulose for indirect probing with anti–Ascaris-troponin C antiserum (B). Protein fractions are the immunotransfers contained in a total protein extract from the wild-type N2 (lane 1), bacterial protein from the troponin I clone, pCTNI-1 (lane 2), bacterial protein from the troponin C clone, pCTNC-1 (lane 3), bacterial protein from bluescript (lane 4), respectively. Molecular size marker (M).
Mentions: Muscle expression of troponin C was confirmed by indirect immunofluorescence staining of wild-type animals (Fig. 8 and Fig. 9). An antibody was generated to nematode troponin C expressed in bacteria (see Materials and Methods), and in nematode protein extracts a band of the expected size (∼18 kD) was detected on SDS-PAGE gels as were larger bands (Fig. 4 B, lanes 1 and 3). These larger bands may be complexes of troponin C with troponin I and troponin I plus troponin T. It is known that troponins strongly bind to each other and can form troponin complexes (Fig. 4 B, lane 1; Grabarek et al. 1990). Using this antisera, we confirmed the expression pattern detected by the reporter constructs. Body wall muscle expression can be seen in embryos and carries on throughout the life of the animal (Fig. 8 and Fig. 9). In older animals vulva and anal muscle expression can also be observed (Fig. 8C and Fig. D). Some staining of pharyngeal muscle may come from cross-reaction with the second troponin C isoform, as suggested by the high degree of sequence homology between these proteins (data not shown).

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