unc-94 encodes a tropomodulin in Caenorhabditis elegans |
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Authors: | Stevenson Tesheka O Mercer Kristina B Cox Elisabeth A Szewczyk Nathaniel J Conley Catharine A Hardin Jeffrey D Benian Guy M |
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Affiliation: | 1 Department of Pathology, Emory University, Atlanta, GA 30322, USA 2 Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322, USA 3 Department of Zoology, University of Wisconsin, Madison, WI 53706, USA 4 NASA Ames Research Center, M/S 239-11, Moffett Field, CA 94035, USA 5 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA |
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Abstract: | unc-94 is one of about 40 genes in Caenorhabditis elegans that, when mutant, displays an abnormal muscle phenotype. Two mutant alleles of unc-94, su177 and sf20, show reduced motility and brood size and disorganization of muscle structure. In unc-94 mutants, immunofluorescence microscopy shows that a number of known sarcomeric proteins are abnormal, but the most dramatic effect is in the localization of F-actin, with some abnormally accumulated near muscle cell-to-cell boundaries. Electron microscopy shows that unc-94(sf20) mutants have large accumulations of thin filaments near the boundaries of adjacent muscle cells. Multiple lines of evidence prove that unc-94 encodes a tropomodulin, a conserved protein known from other systems to bind to both actin and tropomyosin at the pointed ends of actin thin filaments. su177 is a splice site mutation in intron 1, which is specific to one of the two unc-94 isoforms, isoform a; sf20 has a stop codon in exon 5, which is shared by both isoform a and isoform b. The use of promoter-green fluorescent protein constructs in transgenic animals revealed that unc-94a is expressed in body wall, vulval and uterine muscles, whereas unc-94b is expressed in pharyngeal, anal depressor, vulval and uterine muscles and in spermatheca and intestinal epithelial cells. By Western blot, anti-UNC-94 antibodies detect polypeptides of expected size from wild type, wild-type-sized proteins of reduced abundance from unc-94(su177), and no detectable unc-94 products from unc-94(sf20). Using these same antibodies, UNC-94 localizes as two closely spaced parallel lines flanking the M-lines, consistent with localization to the pointed ends of thin filaments. In addition, UNC-94 is localized near muscle cell-to-cell boundaries. |
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Keywords: | GFP, green fluorescent protein RNAi, RNA-mediated interference MHC A, myosin heavy chain A SNP, single nucleotide polymorphism |
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