Isolation and sequence of cDNA clones coding for the precursor to the γ subunit of mouse muscle nicotinic acetylcholine receptor

Abstract

CDNA libraries have been constructed in plasmid (pBR322) and bacteriophage (λgt10) vectors with poly (A +) RNA isolated from the nonfusing mouse muscle cell line BC₃H‐1. The libraries were screened with a restriction fragment derived from a genomic clone coding for a human acetylcholine receptor γ subunit. Several clones were obtained whose cDNA inserts possessed nucleotide and deduced amino acid sequence homology with acetylcholine receptor γ subunits from Torpedo californica, chick, calf, and human. One isolate, λBMG419, has 88 nucleotides of 5′‐untranslated sequence, an open reading frame of 1,557 nucleotides coding for the precursor to the mouse acetylcholine receptor γ subunit, and 144 nucleotides of 3′‐untranslated sequence. Alignment of the λBMG419‐deduced amino acid sequence with homologs from other species predicts a precursor peptide of 519 amino acids and a mature protein of 497 amino acids, with nonglycosylated molecular weights of 58,744 and 56,424 daltons, respectively. Comparison of the deduced amino acid sequence of the mouse γ subunit with Torpedo, chick, calf, and human sequences showed overall homologies of 54%, 67%, 90%, and 90%, respectively; however, significantly higher homologies were found in several putative functional domains. Radiolabeled λBMG419 has been used to identify homologous RNA species, one of approximately 2 kb and one of about 3.5 kb, in poly (A +) RNA prepared from BC₃H‐1 cells and denervated mouse limb muscle. γ Subunit‐coding RNA species are considerably more abundant in denervated than in innervated muscle, suggesting that neural regulation of the abundance of theγ subunit is exerted through regulation of the amount of its mRNA.