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Acetylcholine receptor gene expression in Torpedo electric organ and skeletal muscle during embryonic development
$49 91 A c e t y l c h o l i n e r e c e p t o r gene e x p r e s s i o n in T o r p e d o electric organ and skeletal m u s c l e during e m b r y o n i c d e v e l o p m e n t . O. Asher 1, S. Fuchs 1, and M.C. Souroujonl, 2. Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel and 2The Open University of Israel, Tel Aviv 61392, Israel. Torpedo electric organ is a skeletal muscle homologue that forms during embryonic development. Ceils that give rise to the electric organ initially appear as myoblasts and differentiate during embryonic development into highly specialized electrocytes that express high amounts of acetylcholine receptor (AChR). In order to understand the regulatory mechanism that governs this high expression of AChR in the Torpedo electric organ, we compared the mRNA levels of the AChR subunits and myogenic factors in the electric organ and in the skeletal muscle during embryonic development. Northern blot analysis at different stages of development demonstrated that in the electric organ there was a large increase in the mRNA levels of all AChR subunits during development. The greatest change occurred concomitantly with synaptogenesis in embryo 4-6 cm long. On the other hand, in the muscle we observed only small changes in AChR transcripts during development. In order to find out whether myogenic factors are also responsible for the burst in the expression of AChR during differentiation of the electric organ of Torpedo we have compared the expression of MyoD and MRF4 in the electric organ and in the muscle during embryonic development. We found that the mRNA levels of MyoD and MRF4 changed only slightly during development and their amounts were quite comparable in the electric organ and muscle. These results demonstrate that in the electric organ the mRNA levels of the myogenic factors do not correlate with the expression of AChR subunits. It thus seems that these myogenic factors are not directly responsible for the large increase in the AChR mRNA levels in the electric organ during embryonic development and may implicate that another regulatory mechanism governs this unique burst in AChR expression.
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Cloning o f the m o n g o o s e acetylcholine receptor ~-subunit O. Asher and S. Fuchs Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. To elucidate the fine structure of the acetylcholine receptor (AChR) ligand binding site we have been studying the putative binding site from animal species that are resistant to ct-neurotoxins. Pervious work from our laboratory on the mongoose AChR binding site domain (Barchan et al, Prec. Natl. Acad. Sci. 89: 7717, 1992) demonstrated that there are five amino acid differences between the mongoose (ct-BTX resistant) and the mouse (ct-BTX sensitive) which cluster in the presumed ligand binding site, close to cysteines 192 & 193. Four of these differences are at positions 187, 189, 194 &197 which are conserved in animal species that are susceptible to ct-BTX. In order to find out whether other regions in the ct-subunit of the mongoose AChR may participate in determining its resistance to ¢t-BTX we have cloned by RT-PCR the entire AChR ¢t-subunit gene from the mongoose and compared it to the a-subunit gene of the mouse. Alignment of the mouse and mongoose ct-subunit sequences shows 89% homology in the nucleotides and 93% homology in the deduced amino acids. There are 29 amino acid differences between the mouse and the mongoose a-subunits, only six of which are at positions that are conserved in all animal species that bind aBTX. These six differences include the four amino acids substitutions in the binding site domain and two others at position 112 (Tyr to His) and 153 (Gly to Ser). It is possible that the latter two substitutions may also contribute in determining the resistance of the mongoose to ct-BTX. The other 23 amino acid differences are at positions which are not conserved among animal species that are susceptible to a-BTX and most of them represent minor conservative differences. Therefore, amino acids residues at these positions are probably not involved in conferring toxin resistance. The a-subunit specific cDNA from the mouse and the mongoose AChR were translated in vitro in a nuclease treated reticulocyte lysate system and were immunoprecipitated with polyclonal antibodies to the AChR. In both cases, distinct polypeptides of 41kDa were expressed, suggesting that both cDNA templates properly translated to yield their respective ct-subunit precursors.
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Cloning o f the m o n g o o s e acetylcholine receptor ~-subunit O. Asher and S. Fuchs Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. To elucidate the fine structure of the acetylcholine receptor (AChR) ligand binding site we have been studying the putative binding site from animal species that are resistant to ct-neurotoxins. Pervious work from our laboratory on the mongoose AChR binding site domain (Barchan et al, Prec. Natl. Acad. Sci. 89: 7717, 1992) demonstrated that there are five amino acid differences between the mongoose (ct-BTX resistant) and the mouse (ct-BTX sensitive) which cluster in the presumed ligand binding site, close to cysteines 192 & 193. Four of these differences are at positions 187, 189, 194 &197 which are conserved in animal species that are susceptible to ct-BTX. In order to find out whether other regions in the ct-subunit of the mongoose AChR may participate in determining its resistance to ¢t-BTX we have cloned by RT-PCR the entire AChR ¢t-subunit gene from the mongoose and compared it to the a-subunit gene of the mouse. Alignment of the mouse and mongoose ct-subunit sequences shows 89% homology in the nucleotides and 93% homology in the deduced amino acids. There are 29 amino acid differences between the mouse and the mongoose a-subunits, only six of which are at positions that are conserved in all animal species that bind aBTX. These six differences include the four amino acids substitutions in the binding site domain and two others at position 112 (Tyr to His) and 153 (Gly to Ser). It is possible that the latter two substitutions may also contribute in determining the resistance of the mongoose to ct-BTX. The other 23 amino acid differences are at positions which are not conserved among animal species that are susceptible to a-BTX and most of them represent minor conservative differences. Therefore, amino acids residues at these positions are probably not involved in conferring toxin resistance. The a-subunit specific cDNA from the mouse and the mongoose AChR were translated in vitro in a nuclease treated reticulocyte lysate system and were immunoprecipitated with polyclonal antibodies to the AChR. In both cases, distinct polypeptides of 41kDa were expressed, suggesting that both cDNA templates properly translated to yield their respective ct-subunit precursors.