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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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Mobility-shift assay of the wild-type troponin C and characterization of mutant troponin C by using bacterially expressed proteins. (A) SDS-PAGE and Coomassie brilliant blue staining; (B) Western analysis using affinity-purified anti–troponin C antibody. Lane B, Total protein of E. coli JM109; lanes 1 and 2, bacterial protein from cDNA clone, pCTNC-1; lanes 3 and 4, mutant troponin C clone, pCPAT-10-m1; lanes 5 and 6, mutant troponin C clone, pCPAT-10-m2; lane N, total protein of the wild-type N2. Sample solution contained Ca2+ + (1 mM CaCl2, lanes 1, 3, and 5) and Ca2+ − (5 mM EGTA, lanes 2, 4, and 6), respectively. (C) Blotted sheet incubated with bacterially produced troponin I followed by detection with affinity-purified anti–TNI-1. For C, samples and conditions were the same as in A and B. It was noted that PAT-10-m1 did not show a band shift with and without calcium (B, lanes 3 and 4) and PAT-10-m2 did not bind troponin I (C, lanes 5 and 6). For details see the text.
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Figure 5: Mobility-shift assay of the wild-type troponin C and characterization of mutant troponin C by using bacterially expressed proteins. (A) SDS-PAGE and Coomassie brilliant blue staining; (B) Western analysis using affinity-purified anti–troponin C antibody. Lane B, Total protein of E. coli JM109; lanes 1 and 2, bacterial protein from cDNA clone, pCTNC-1; lanes 3 and 4, mutant troponin C clone, pCPAT-10-m1; lanes 5 and 6, mutant troponin C clone, pCPAT-10-m2; lane N, total protein of the wild-type N2. Sample solution contained Ca2+ + (1 mM CaCl2, lanes 1, 3, and 5) and Ca2+ − (5 mM EGTA, lanes 2, 4, and 6), respectively. (C) Blotted sheet incubated with bacterially produced troponin I followed by detection with affinity-purified anti–TNI-1. For C, samples and conditions were the same as in A and B. It was noted that PAT-10-m1 did not show a band shift with and without calcium (B, lanes 3 and 4) and PAT-10-m2 did not bind troponin I (C, lanes 5 and 6). For details see the text.

Mentions: A mutant clone having one of each mutation was constructed by two-step procedures as follows. Two fragments, one having a mutation sequence at the mutation site and another having a mutation at restriction site, were synthesized by PCR. Second PCR was performed by using two annealed fragments as a template. After digestion with restriction enzymes, only a fragment having a mutation site was ligated into vector. Constructed mutant clones were named pat-10-m1 for mutation at the second calcium-binding site and pat-10-m2 for mutation missing COOH-terminal helix, respectively (Fig. 5).


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)

Mobility-shift assay of the wild-type troponin C and characterization of mutant troponin C by using bacterially expressed proteins. (A) SDS-PAGE and Coomassie brilliant blue staining; (B) Western analysis using affinity-purified anti–troponin C antibody. Lane B, Total protein of E. coli JM109; lanes 1 and 2, bacterial protein from cDNA clone, pCTNC-1; lanes 3 and 4, mutant troponin C clone, pCPAT-10-m1; lanes 5 and 6, mutant troponin C clone, pCPAT-10-m2; lane N, total protein of the wild-type N2. Sample solution contained Ca2+ + (1 mM CaCl2, lanes 1, 3, and 5) and Ca2+ − (5 mM EGTA, lanes 2, 4, and 6), respectively. (C) Blotted sheet incubated with bacterially produced troponin I followed by detection with affinity-purified anti–TNI-1. For C, samples and conditions were the same as in A and B. It was noted that PAT-10-m1 did not show a band shift with and without calcium (B, lanes 3 and 4) and PAT-10-m2 did not bind troponin I (C, lanes 5 and 6). For details see the text.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Mobility-shift assay of the wild-type troponin C and characterization of mutant troponin C by using bacterially expressed proteins. (A) SDS-PAGE and Coomassie brilliant blue staining; (B) Western analysis using affinity-purified anti–troponin C antibody. Lane B, Total protein of E. coli JM109; lanes 1 and 2, bacterial protein from cDNA clone, pCTNC-1; lanes 3 and 4, mutant troponin C clone, pCPAT-10-m1; lanes 5 and 6, mutant troponin C clone, pCPAT-10-m2; lane N, total protein of the wild-type N2. Sample solution contained Ca2+ + (1 mM CaCl2, lanes 1, 3, and 5) and Ca2+ − (5 mM EGTA, lanes 2, 4, and 6), respectively. (C) Blotted sheet incubated with bacterially produced troponin I followed by detection with affinity-purified anti–TNI-1. For C, samples and conditions were the same as in A and B. It was noted that PAT-10-m1 did not show a band shift with and without calcium (B, lanes 3 and 4) and PAT-10-m2 did not bind troponin I (C, lanes 5 and 6). For details see the text.
Mentions: A mutant clone having one of each mutation was constructed by two-step procedures as follows. Two fragments, one having a mutation sequence at the mutation site and another having a mutation at restriction site, were synthesized by PCR. Second PCR was performed by using two annealed fragments as a template. After digestion with restriction enzymes, only a fragment having a mutation site was ligated into vector. Constructed mutant clones were named pat-10-m1 for mutation at the second calcium-binding site and pat-10-m2 for mutation missing COOH-terminal helix, respectively (Fig. 5).

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