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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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Map position and genome structure of the troponin C gene, pat-10. (A) Genomic map of the central part of chromosome I of the worm is shown. (B) Contig map of the region surrounding pat-10. Three YAC clones, Y37B3, Y55F5, and Y72D6 hit with cDNA fragment of the troponin C. Cosmid clones of B0490, W03G9, F54C1, C15C10 K04H3, and T21E10 were hybridized with cDNA fragment, and F54C1 and C15C10 contained the genomic fragment of pat-10. (C and D) Restriction map and intron/exon structure from sequence data of pat-10. It should be noted that only one transcript from the plasmid clone pTNC1 was matched to 674 bp of cDNA encoding 161 amino acid residues. H, HindIII; B, BamHI; E, EcoRI; P, PstI. The nucleotide sequence of pat-10 is in Fig. 2.
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Figure 1: Map position and genome structure of the troponin C gene, pat-10. (A) Genomic map of the central part of chromosome I of the worm is shown. (B) Contig map of the region surrounding pat-10. Three YAC clones, Y37B3, Y55F5, and Y72D6 hit with cDNA fragment of the troponin C. Cosmid clones of B0490, W03G9, F54C1, C15C10 K04H3, and T21E10 were hybridized with cDNA fragment, and F54C1 and C15C10 contained the genomic fragment of pat-10. (C and D) Restriction map and intron/exon structure from sequence data of pat-10. It should be noted that only one transcript from the plasmid clone pTNC1 was matched to 674 bp of cDNA encoding 161 amino acid residues. H, HindIII; B, BamHI; E, EcoRI; P, PstI. The nucleotide sequence of pat-10 is in Fig. 2.

Mentions: Worm culture and handling were done by established methods (Brenner 1974; Sulston and Hodgkin 1988). The nematode C. elegans Bristol N2 was used for DNA and protein analysis. The pat-10 mutant strains RW3608: pat-10(st575)/dpy-5(e61)unc-29(e1072), and RW3613: pat-10(st568)/unc-11(e47)dpy-5(e61) were used for analysis of mutation site and muscle structure by using segregated homozygous worms (Williams and Waterston 1994; see Fig. 7 I). Exon expression cloning of the troponin gene was essentially the same as was described (Kagawa et al. 1989) except using a cDNA library. A bacteriophage ZapII library of cDNA provided by R. Barstead (Oklahoma Medical Research Foundation, Oklahoma City, OK; Barstead and Waterston 1989) was screened to obtain positive clones with anti–troponin C against Ascaris protein (Nakae and Obinata 1993). Positive clones were obtained by screening four to eight plates (∼10,000 plaques per plate). Standard DNA recombinant techniques were followed (Sambrook et al. 1989). A 3.5-kb PstI fragment from C15C10 or F54C1 was subcloned into the PstI site of pUC119 to generate pTNC1 (see Fig. 1C and Fig. D). DNA and protein sequence data analyses were done by using the programs of HITACHI DNASIS and GENETYX-MAC. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession numbers D45895 and D45896 for genomic and cDNA sequences, respectively.


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)

Map position and genome structure of the troponin C gene, pat-10. (A) Genomic map of the central part of chromosome I of the worm is shown. (B) Contig map of the region surrounding pat-10. Three YAC clones, Y37B3, Y55F5, and Y72D6 hit with cDNA fragment of the troponin C. Cosmid clones of B0490, W03G9, F54C1, C15C10 K04H3, and T21E10 were hybridized with cDNA fragment, and F54C1 and C15C10 contained the genomic fragment of pat-10. (C and D) Restriction map and intron/exon structure from sequence data of pat-10. It should be noted that only one transcript from the plasmid clone pTNC1 was matched to 674 bp of cDNA encoding 161 amino acid residues. H, HindIII; B, BamHI; E, EcoRI; P, PstI. The nucleotide sequence of pat-10 is in Fig. 2.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Map position and genome structure of the troponin C gene, pat-10. (A) Genomic map of the central part of chromosome I of the worm is shown. (B) Contig map of the region surrounding pat-10. Three YAC clones, Y37B3, Y55F5, and Y72D6 hit with cDNA fragment of the troponin C. Cosmid clones of B0490, W03G9, F54C1, C15C10 K04H3, and T21E10 were hybridized with cDNA fragment, and F54C1 and C15C10 contained the genomic fragment of pat-10. (C and D) Restriction map and intron/exon structure from sequence data of pat-10. It should be noted that only one transcript from the plasmid clone pTNC1 was matched to 674 bp of cDNA encoding 161 amino acid residues. H, HindIII; B, BamHI; E, EcoRI; P, PstI. The nucleotide sequence of pat-10 is in Fig. 2.
Mentions: Worm culture and handling were done by established methods (Brenner 1974; Sulston and Hodgkin 1988). The nematode C. elegans Bristol N2 was used for DNA and protein analysis. The pat-10 mutant strains RW3608: pat-10(st575)/dpy-5(e61)unc-29(e1072), and RW3613: pat-10(st568)/unc-11(e47)dpy-5(e61) were used for analysis of mutation site and muscle structure by using segregated homozygous worms (Williams and Waterston 1994; see Fig. 7 I). Exon expression cloning of the troponin gene was essentially the same as was described (Kagawa et al. 1989) except using a cDNA library. A bacteriophage ZapII library of cDNA provided by R. Barstead (Oklahoma Medical Research Foundation, Oklahoma City, OK; Barstead and Waterston 1989) was screened to obtain positive clones with anti–troponin C against Ascaris protein (Nakae and Obinata 1993). Positive clones were obtained by screening four to eight plates (∼10,000 plaques per plate). Standard DNA recombinant techniques were followed (Sambrook et al. 1989). A 3.5-kb PstI fragment from C15C10 or F54C1 was subcloned into the PstI site of pUC119 to generate pTNC1 (see Fig. 1C and Fig. D). DNA and protein sequence data analyses were done by using the programs of HITACHI DNASIS and GENETYX-MAC. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession numbers D45895 and D45896 for genomic and cDNA sequences, respectively.

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