The third and fourth tropomyosin isoforms of Caenorhabditis elegans are expressed in the pharynx and intestines and are essential for development andmorphology

The tropomyosin gene tmy-1/lev-11 of Caenorhabditis elegans spans 14.5 kb S cience and Technology and encodes three isoforms by alternative splicing. T o identify, characterize and compare the genome and tissue expression of a fourth isoform, the technique of rapid amplification of cDNA ends and micro injection with lacZ and gfp fusion plasmids were employed. We elucidated Ce T-MIV, a fourth isoform of tmy-1, which encoded a 256 residue polypep tide. CeTMIV isoform had a similar promoter region to CeTMIII isoform, but was a lternatively spliced to generate a cDNA that differed in two, exons. The tm y-1::lacZ and tiny-1::gfp fusion genes, with 3.2 kb promoter sequence and 1 .1 kb of CeTMIV isoform specific exons, were expressed in the pharyngeal an d intestinal cells. Further unidirectional deletion of the sequence located the primary promoter region 853 by upstream from the initial codon. We sho w within the upstream region, the presence of B and C subelement-like seque nces of myo-2, which may be used to stimulate pharyngeal expression. Despit e the presence of a ges-1 like sequence, we were unable to locate the two G ATA sites required for intestinal expression. Reassessing tissue expression for CeTMIII isoform with newly constructed fusion plasmids, we showed furt her expression in germ-line tissue and intestinal cells in addition to phar yngeal expression. Finally, to demonstrate that tropomyosin is essential fo r development, we inactivated the body wall and pharynx-specific isoforms b y RNA-mediated interference. In addition to 50-75 % embryonic lethality in both cases, the worms that survived body wall interference had abnormal bod y morphology and uncoordinated movements, and those that survived pharynx i nterference had deformed pharynges and gut regions. These results show the function of tropomyosin in normal muscle filament assembly and embryonic de velopment, and illustrate the different expression patterns characteristic of tropomyosin isoforms in C. elegans. (C) 2001 Academic Press.