Abstract
Sequences from a gene coding for mouse acetylcholine receptor alpha subunit have been inserted into a recombinant plasmid and cloned in Escherichia coli. mRNAs for acetylcholine receptors occur in low abundance in vertebrate muscle. To clone the mouse alpha-subunit cDNA, we made use of (i) a cell line, BC3H-1, that overproduces the alpha-subunit mRNA and (ii) a polysome fractionation procedure that results in enrichment of alpha-subunit mRNA. Polyadenylylated RNA was used to construct a cDNA library of 750 recombinant clones. Acetylcholine receptor-specific sequences were identified by hybrid-selected translation, followed by monoclonal antibody precipitation and peptide mapping of the translation product. One clone (pA59) that fit these criteria was found in the first 80 isolates. It had a 700-base-pair insert that was excised with Pst I. Blot-hybridization experiments with nick-translated pA59 DNA showed that BC3H-1 cells contain 100-1,000 times more alpha-subunit mRNA than does newborn or adult mouse muscle. Blot hybridization of restriction digests of mouse liver DNA revealed that pA59 is homologous to a very small number (probably one) of genomic sequences.
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