H. Terami

Tissue expression of four troponin I genes and their molecular interactions with two troponin C isoforms in Caenorhabditis elegans.

Gene duplication is a major genetic event that can produce multiple protein isoforms. Comparative sequence and functional analysis of related gene products can provide insights into protein family evolution. To characterize the Caenorhabditis elegans troponin I family, we analyzed gene structures, tissue expression patterns and RNAi phenotypes of four troponin I isoforms. Tissue expression patterns were determined using lacZ/gfp/rfp reporter gene assays. The tni‐1, tni‐2/unc‐27 and tni‐3 genes, each encoding a troponin I isoform, are uniquely expressed in body wall, vulval and anal muscles but at different levels; tni‐4 was expressed solely in the pharynx. Expressing tni‐1 and ‐2 gene RNAi caused motility defects similar to unc‐27 (e155) mutant, a tni‐2 null allele. The tni‐3 RNAi expression produced egg laying defects while the tni‐4 RNAi caused arrest at gastrulation. Overlay analyses were used to assay interactions between the troponin I and two troponin C isoforms. The three body wall troponin I isoforms interacted with body wall and pharyngeal troponin C isoforms; TNI‐4 interacted only with pharyngeal troponin C. Our results suggest the body wall genes have evolved following duplication of the pharynx gene and provide important data about gene duplication and functional differentiation of nematode troponin I isoforms.

C. ELEGANS MODEL FOR STUDYING TROPOMYOSIN AND TROPONIN REGULATIONS OF MUSCLE CONTRACTION AND ANIMAL BEHAVIOR

There are two muscle tissues in the nematode Caenorhabditis elegans: the pharynx for feeding and the body wall for locomotion. These correspond to cardiac and skeletal muscles in vertebrates, respectively. Study of the muscle genes of C. elegans can be classified into three stages; first, mutant isolation and gene mapping, second, cloning and sequencing of the gene, and third, complete sequences of all genes. Many uncoordinated mutant animals have been isolated (Brenner, 1974; Waterston, 1988; Moerman and Fire, 1997) and the complete amino acid sequence of myosin heavy chain, twitchin, and paramyosin, (invertebrate specific core protein of thick filament), and were the first determined in any animals by analyzing the unc-54, unc-22, and unc-15 mutants, respectively (Karn et al., 1983; Benian et al., 1989; Kagawa et al., 1989). Tropomyosin and troponin components are also present but as with actin and myosin heavy chain in the worm, there are some differences in gene structure and sequence compared to those in other animals (Kagawa et al., 1995; Myers et al., 1996; Moerman and Fire, 1997). Deficiencies of body wall troponin C or tropomyosin in C. elegans cause the Pat (paralyzed arrest at embryonic two-fold stage) phenotype (Williams and Waterston, 1994; Terami et al., 1999) and those of troponin T cause Mup (muscle position abnormal) phenotype (Myers et al., 1996). After determining the complete genome sequences of the nematode (The C. elegans Sequence Consortium, 1998), we can find out how isoforms are related to each other. Only one troponin C gene, pat-10/tnc-1, is expressed in the body wall muscles and the gene defect causes a developmental arrest of the animals (Terami et al., 1999).

Calcium-bindings of wild type and mutant troponin Cs of Caenorhabditis elegans

Apparent Ca(2+)-binding constant (K(app)) of Caenorhabditis elegans troponin C (CeTnC) was determined by a fluorescence titration method. The K(app) of the N-domain Ca(2+)-binding site of CeTnC was 7.9+/-1.6 x 10(5) M(-1) and that of the C-domain site was 1.2+/-0.6 x 10(6) M(-1), respectively. Mg(2+)-dependence of the K(app) showed that both Ca(2+)-binding sites did not bind competitively Mg(2+). The Ca(2+) dissociation rate constant (k(off)) of CeTnC was determined by the fluorescence stopped-flow method. The k(off) of the N-domain Ca(2+)-binding site of CeTnC was 703+/-208 s(-1) and that of the C-domain site was 286+/-33 s(-1), respectively. From these values we could calculate the Ca(2+)-binding rate constant (k(on)) as to be 5.6+/-2.8 x 10(8) M(-1) s(-1) for the N-domain site and 3.4+/-2.1 x 10(8) M(-1) s(-1) for the C-domain site, respectively. These results mean that all Ca(2+)-binding sites of CeTnC are low affinity, fast dissociating and Ca(2+)-specific sites. Evolutional function of TnC between vertebrate and invertebrate and biological functions of wild type and mutant CeTnCs are discussed.