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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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Tissue-specific expression of the pat-10/lacZ genes. A fusion gene containing 5′ UTS of pat-10 and lacZ was used to study cellular expression. (A) Staining of body wall muscles, pTNCZ647; (B) pTNCZ292; (C) Expression of anterior part. It was noted that pharyngeal muscle was not stained. (D and E) pTNCZ647, side and top views of the vulva of young adult, respectively. Bar, 50 μm. (F and G) Summary of 5′ UTS of pat-10 and body wall–specific expressions. The position of regulatory sequences were shown: D, 1330/hlh-1 recognition; G, GC box; M, MEF2-binding sites, respectively.
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Figure 6: Tissue-specific expression of the pat-10/lacZ genes. A fusion gene containing 5′ UTS of pat-10 and lacZ was used to study cellular expression. (A) Staining of body wall muscles, pTNCZ647; (B) pTNCZ292; (C) Expression of anterior part. It was noted that pharyngeal muscle was not stained. (D and E) pTNCZ647, side and top views of the vulva of young adult, respectively. Bar, 50 μm. (F and G) Summary of 5′ UTS of pat-10 and body wall–specific expressions. The position of regulatory sequences were shown: D, 1330/hlh-1 recognition; G, GC box; M, MEF2-binding sites, respectively.

Mentions: Various upstream and internal regions of the troponin C gene, pat-10 of C. elegans were inserted into pPD transformation vectors (Fire et al. 1990) in-frame with the E. coli lacZ reporter gene. DNA fragments from 7.6 kb of BamHI containing 7,600 bp upstream of the first ATG at 1,146 and to 108 bp of the second exon were cloned into the BamHI site of pPD22.11. Series of another fragments deleting the 5′ upstream end of pat-10; 1,248 bp of PstI-BamHI, 647 bp of ApaLI-BamHI, and 292 bp of EcoRV-BamHI were also ligated to the processed vector. The number of constructed plasmid indicate the numbers of nucleotide from the first ATG at 1,146 (see Fig. 6). Preparation of plasmid DNAs for injection and transformation of C. elegans were carried out as was described (Mello et al. 1991).


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)

Tissue-specific expression of the pat-10/lacZ genes. A fusion gene containing 5′ UTS of pat-10 and lacZ was used to study cellular expression. (A) Staining of body wall muscles, pTNCZ647; (B) pTNCZ292; (C) Expression of anterior part. It was noted that pharyngeal muscle was not stained. (D and E) pTNCZ647, side and top views of the vulva of young adult, respectively. Bar, 50 μm. (F and G) Summary of 5′ UTS of pat-10 and body wall–specific expressions. The position of regulatory sequences were shown: D, 1330/hlh-1 recognition; G, GC box; M, MEF2-binding sites, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199735&req=5

Figure 6: Tissue-specific expression of the pat-10/lacZ genes. A fusion gene containing 5′ UTS of pat-10 and lacZ was used to study cellular expression. (A) Staining of body wall muscles, pTNCZ647; (B) pTNCZ292; (C) Expression of anterior part. It was noted that pharyngeal muscle was not stained. (D and E) pTNCZ647, side and top views of the vulva of young adult, respectively. Bar, 50 μm. (F and G) Summary of 5′ UTS of pat-10 and body wall–specific expressions. The position of regulatory sequences were shown: D, 1330/hlh-1 recognition; G, GC box; M, MEF2-binding sites, respectively.
Mentions: Various upstream and internal regions of the troponin C gene, pat-10 of C. elegans were inserted into pPD transformation vectors (Fire et al. 1990) in-frame with the E. coli lacZ reporter gene. DNA fragments from 7.6 kb of BamHI containing 7,600 bp upstream of the first ATG at 1,146 and to 108 bp of the second exon were cloned into the BamHI site of pPD22.11. Series of another fragments deleting the 5′ upstream end of pat-10; 1,248 bp of PstI-BamHI, 647 bp of ApaLI-BamHI, and 292 bp of EcoRV-BamHI were also ligated to the processed vector. The number of constructed plasmid indicate the numbers of nucleotide from the first ATG at 1,146 (see Fig. 6). Preparation of plasmid DNAs for injection and transformation of C. elegans were carried out as was described (Mello et al. 1991).

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