Worm Breeder's Gazette 16(2): 34
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Biology, Faculty of Science, Okayama University, 3-1-1 Tsushima naka, Okayama 700-8530, Japan|
The single tropomyosin gene tmy-1 (lev-11) of Caenorhabditis elegans, which spans 13 kilobases and includes 14 exons, encodes three isoforms by alternative splicing. Isoforms I and II designated as CeTMI and CeTMII respectively, expresses mainly in body wall muscles with promoter region 660 - 800bp upstream. CeTMIII expresses exclusively in the pharynx utilizing an internal promoter. (Kagawa et. al., 1995) To identify the functional region and tissue expression of the genome of a fourth isoform, we employed Rapid Amplification of cDNA Ends (5', 3' RACE) to determine the cDNA and microinjection with lacZ fusion plasmid techniques for tissue expression. We elucidated CeTMIV, a 256 amino acid isoform generated by different alternative splicing and similar to CeTMIII in exon patterning with the exception of exons 5c (which was exclusive for CeTMIV) and 9b. The tmy::lacZ fusion gene with 3.3kb upstream promoter region and 1.1kb CeTMIV specific exons expressed in the pharynx and posterior gut. Further deletion of upstream region located the primary promoter region to 846bp upstream the initial codon. We also show within the fusion gene the presence of both the myo-2 enhancer like and the ges-1 like sequences needed for pharynx and gut expression respectively. Just like CeTMIV, we also located the primary promoter region of CeTMIII to 846bp upstream the initial codon. Finally to demonstrate that tropomyosin is essential for survival, we inactivated the gene by RNA-mediated interference and the worms arrested early in larval development. The stage of arrest was similar to lev-11(st557) mutation observed in CeTMI and CeTMII, where the mutation of G to A in exon 1 raises a stop codon resulting in translation termination. (Kagawa et. al., 1997) These results complete the study of the four tropomyosin isoforms and illustrates the complexities of the tissue specific differences unique to tropomyosins. The results also show and confirm the necessity of tropomyosin requirement for assembling body wall, pharynx and other muscles in C. elegans. Kagawa et. al., J Mol. Biol. (1995) 251, 603 - 613 Kagawa et. al., Cell Struct. & Funct. (1997) 22, 213 - 218 Okkema & Fire, Development (1994) 120, 2175 - 2186.,