No abstract

The alpha-chain of the nicotinic acetylcholine receptor carries the binding sites both for cholinergic ligands and for most experimentally induced or naturally occurring antibodies to the native receptor. By means of expression cloning in Escherichia coli, fusion proteins were derived from specific fragments of a complementary DNA encoding the mouse alpha-chain, allowing the mapping of the toxin-binding site to residues 160-216 and the main immunogenic region to residues 6-85. This approach permits the independent study of different functional domains of a complex receptor molecule and should be generally applicable to other proteins for which complementary DNA clones are available.

Four genes encode the related protein subunits that assemble to form the nicotinic acetylcholine receptor (nAChR) at the motor endplate of vertebrates. We have isolated from the chicken genome four additional members of the same gene family whose protein products, termed alpha 2, alpha 3, alpha 4 and n alpha (non‐alpha) probably define three distinct neuronal nAChR subtypes. The neuronal nAChR genes have identical structures consisting of six protein‐coding exons and specify proteins that are best aligned with the chicken endplate alpha subunit, whose gene we have also characterized. mRNA transcripts encoding alpha 4 and n alpha are abundant in embryonic and in adult avian brain, whereas alpha 2 and alpha 3 transcripts are much scarcer. The same set of neuronal genes probably exists in all vertebrates since their counterparts have also been identified in the rat genome.

The characterisation of interspecies differences in gene regulation is crucial to understanding the molecular basis of phenotypic diversity and evolution. The atonal homologue Atoh7 participates in the ontogenesis of the vertebrate retina. Our study reveals how evolutionarily conserved, non-coding DNA sequences mediate both the conserved and the species-specific transcriptional features of the Atoh7 gene. In the mouse and chick retina, species-related variations in the chromatin-binding profiles of bHLH transcription factors correlate with distinct features of the Atoh7 promoters and underlie variations in the transcriptional rates of the Atoh7 genes. The different expression kinetics of the Atoh7 genes generate differences in the expression patterns of a set of genes that are regulated by Atoh7 in a dose-dependent manner, including those involved in neurite outgrowth and growth cone migration. In summary, we show how highly conserved regulatory elements are put to use in mediating non-conserved functions and creating interspecies neuronal diversity.

We have cloned and sequenced a fragment of the chicken genome approximately 9 kilobases in length that comprises the genes encoding the 6 and 'y subunits of the nicotinic acetyicholine receptor. The two genes are homologous and have identical structures: both consist of 12 exons, some of which precisely correspond to predicted structural domains of the receptor subunits. The 8 and y subunit gehes are encoded by the same DNA strand and are very closely linked, there being only 740 base pairs between the last codon of 8 and the initiator codon of y. Blot analysis demonstrates that the genes we describe are unique in the genome. Comparison of the predicted protein sequence for the correspodding subunits of chicken and of the elasmobranch Torpedo reveals a high degree of conservation in Aome but not all of the protein domains.The nicotinic acetylcholine receptor (AcChok) mediates synaptic transmission at the vertebrate neuromuscular junction. Located in the folds of the postsynaptic membrane, the AcChoR is a cation channel whose opening is triggered by acetylcholine. AcChoR activation normally results in an influx of Na' that depolarizes the postsynaptic membrane and leads to muscle contraction. The AcChoR is the best understood of all ligand-gated ion channels due to its relative abundance in the electrocytes of Torpedo or Electrophorus, from which workable quantities of receptor can be purified. The receptor is a pentamer of four different subunits in the stoichiometty a2f8W. All subunits are glycosylated and span the membrane. They assemble into a barrel-shaped structure whose central cavity is thought to be the gatpd ion channel (for recent reviews see refs. 1-3). That the four subunits participate in the assembly of a single functional AcChoR molecule was elegantly verified by showing that all four mRNA species must be injected in Xenopus oocytes to allow detection of physiologically active receptor (4). Microsequencing of the amino ends of the four purified Torpedo subunits demonstrated that they were related and suggested that they had evolved from an ancestral gene by a series of duplications (5).Several laboratories recently succeeded in cloning and sequencing some (6-9) or all (10-12) of the cDNAs encoding the four subunits of Torpedo AcChoR. It was found that there is significantly more homology between the deduced protein sequences of the a-,8 and y-8 pairs than between any other combinations of subunits, suggesting that an initial duplication of the ancestral gene had given rise to two protogenes, one of which later yielded the y and 8 genes while the other gave rise to the a and p genes (12). Moreover, all subunits were shown to have four very hydrophobic stretches three of them closely grouped-of sufficient length to span the membrane. This finding suggested a model for the transmembrane insertion of the receptor whereby each subunit weaves four times through the membrane to yield an assembly of twenty transmembrane a helices, a subset of which constitutes the channel per se (8,9,12). A relate...