The third and fourth tropomyosin isoforms of Caenorhabditis elegans are expressed in the pharynx and intestines and are essential for development and morphology.
The tropomyosin gene tmy-1/lev-11 of Caenorhabditis elegans spans 14.5 kb and encodes three isoforms by alternative splicing. To identify, characterize and compare the genome and tissue expression of a fourth isoform, the technique of rapid amplification of cDNA ends and microinjection with lacZ and gfp fusion plasmids were employed. We elucidated CeTMIV, a fourth isoform of tmy-1, which encoded a 256 residue polypeptide. CeTMIV isoform had a similar promoter region to CeTMIII isoform, but was alternatively spliced to generate a cDNA that differed in two exons. The tmy-1::lacZ and tmy-1::gfp fusion genes, with 3.2 kb promoter sequence and 1.1 kb of CeTMIV isoform specific exons, were expressed in the pharyngeal and intestinal cells. Further unidirectional deletion of the sequence located the primary promoter region 853 bp upstream from the initial codon. We show within the upstream region, the presence of B and C subelement-like sequences of myo-2, which may be used to stimulate pharyngeal expression. Despite the presence of a ges-1 like sequence, we were unable to locate the two GATA sites required for intestinal expression. Reassessing tissue expression for CeTMIII isoform with newly constructed fusion plasmids, we showed further expression in germ-line tissue and intestinal cells in addition to pharyngeal expression. Finally, to demonstrate that tropomyosin is essential for development, we inactivated the body wall and pharynx-specific isoforms by RNA-mediated interference. In addition to 50-75 % embryonic lethality in both cases, the worms that survived body wall interference had abnormal body morphology and uncoordinated movements, and those that survived pharynx interference had deformed pharynges and gut regions. These results show the function of tropomyosin in normal muscle filament assembly and embryonic development, and illustrate the different expression patterns characteristic of tropomyosin isoforms in C. elegans.
Anyanful
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A
Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
Sakube
Y
Y
Takuwa
K
K
Kagawa
H
H
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019521:Body Patterning; D017173:Caenorhabditis elegans / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005091:Exons / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation; D017238:Genes, Helminth / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005854:Germ Cells / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology / Q000196:embryology; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D010614:Pharynx / Q000196:embryology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014335:Tropomyosin / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D020033:Protein Isoforms; D014335:Tropomyosin