Worm Breeder's Gazette 13(5): 27 (February 1, 1995)
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.
Department of Biology, Faculty of Science, Okayama University, Okayama 700 JAPAN c5191 8@jpnkudpc.bitnet, hkagawa@sc.okayama-u.ac.jp
In C. elegans, there are two different types of muscles; the pharyngeal muscles and body wall muscles. We cloned and sequenced the tropomyosin gene, tmy-1 of the worm which encodes more than three isoforms; two of body muscles and one of pharyngeal muscle. Tmy-1 gene expression was controlled by two promoters and alternative splicing. We have already known the place of the tmy-1 gene expression by injecting series of tmy-1 gene-lacZ fusion plasmids into the oocyte. The expression of promoter activity was detected by histochemical procedure of beta- galactosidase activity. The first promoter controlling two isoforms expressed in the body wall, vulva and male tail muscles. The second promoter specifically expressed in the pharyngeal muscles (Imadzu et al. WBG 13#3p56). To know the results of promoter/lacZ expression were consistent with the results of protein levels, we raised antibody against bacterial produced tropomyosin followed by immunostaining. Fusion protein of b-Gal-CeTMIII was isolated from sonicated bacterial extract by ammonium sulfate precipitation and ion exchange column chromatography. Affinity purification of antibody was prepared by the protocol of Macef and Koch (J. Cell Sci. 92, 61-70 1988), using the proteins immobilized on nitrocellulose membrane. Worms processed with 2X Laemmli buffer run on SDS PAGE. Blotted membrane was stained with 0.01percent Ponseau red in 5percent acetic acid and corresponding band was cut for affinity purificaffon of antibody. Purified protein from SDS-PAGE sometimes is not good antigen for raising antibody for the purpose of histochemistry. We should mention the effect of SDS treatment on the difference between "before" and "after" immunization. Affinity purified antibody by using worm protein was better than that by using bacterial produced protein. Affinity purified anti-CeTMIII antibody stained pharyngeal muscle conversely anti CeTMI/CeTMII antibody stained body wall muscles by indirect immunofluorescence microscopy. Affinity purified antibody also detected a low molecular mass of CeTMII (256 amino acids) on Western blot analysis. These results confirm that promoter-lacZ micro injection experiments were really the result of the transcription and translation of the correct gene. In our micro injection experiments on troponin I (Kuroda et al), troponin C (Matsumoto et al) and ryanodine receptor genes (Sakube et al., WBG 13#2p58), these genes expressed in the body wall muscles. This suggest that muscle contraction in the body wall could initiate with calcium signal of thin filament linked; troponins-tropomyosin. This might suggest that pharyngeal muscle contract with thick filament linked; CaMd-myosin light chain pathway. Upstream carrier of calcium in the pharyngeal may be inositol 1,4,5 triphosphate. Yuji Kohara et al and Baylis et al. isolated the cDNA clones of InsP3 which might express in the pharyngeal (WBG 13#4p18, WBG 13#4p68) .