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.

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The aim of this study is to investigate the function of the C-terminal extension of three troponin I isoforms, that are unique to the body wall muscles of Caenorhabditis elegans and to understand the molecular interactions within the TN complex between troponin I with troponin C/T, and tropomyosin. We constructed several expression vectors to generate recombinant proteins of three body wall and one pharyngeal troponin I isoforms in Escherichia coli. Protein overlay assays and Western blot analyses were performed using antibodies. We demonstrated that pharyngeal TNI-4 interacted with only the pha ryngeal isoforms of troponin C/T and tropomyosin. In contrast, the body wall TNI-2 bound both the body wall and pharyngeal isoforms of these components.Similar to other invertebrates, the N-terminus of troponin I contributes to interactions with troponin C. Full-length troponin I was essential for interactions with tropomyosin isoforms. Deletion of the C-terminal extension had no direct effect on the binding of the body wall troponin I to other muscle thin filament troponin C/T and tropomyosin isoforms.

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