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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...

The atonal homolog 5 (ATH5) protein is central to the transcriptional network regulating the specification of retinal ganglion cells, and its expression comes under the spatiotemporal control of several basic helix-loop-helix (bHLH) proteins in the course of retina development. Monitoring the in vivo occupancy of the ATH5 promoter by the ATH5, Ngn2, and NeuroM proteins and analyzing the DNA motifs they bind, we show that three evolutionarily conserved E-boxes are required for the bHLH proteins to control the different phases of ATH5 expression. E-box 4 mediates the activity of Ngn2, ATH5, and NeuroM along the pathway leading to the conversion of progenitors into newborn neurons. E-box 1, by mediating the antagonistic effects of Ngn2 and HES1 in proliferating progenitors, controls the expansion of the ATH5 expression domain in early retina. E-box 2 is required for the positive feedback by ATH5 that underlies the up-regulation of ATH5 expression when progenitors are going through their last cell cycle. The combinatorial nature of the regulation of the ATH5 promoter suggests that the bHLH proteins involved have no assigned E-boxes but use a common set at which they either cooperate or compete to finely tune ATH5 expression as development proceeds.Retina development in vertebrates relies on regulatory proteins, most of which are widely expressed in the developing nervous system. However, the expression of the basic helixloop-helix protein atonal homolog 5 (ATH5) 3 appears to be restricted to retina ontogenesis. ATH5 activates neurogenesis and is required for the production of retinal ganglion cells (RGCs) (1-4). It is transiently expressed during the period of development when RGCs are produced and underlies the pathway leading to the conversion of proliferating progenitors into newborn RGCs (5, 6). Retinotopic differences in the timing of RGC production may reflect the wave-like expression of ATH5 (5, 7). In zebrafish, a signal from the optic stalk appears to induce the first patch of ATH5-expressing cells, and the continued spread of ATH5 expression beyond that patch may require a cascade of cell-to-cell interactions (7,8). It has been suggested that Sonic hedgehog derived from newborn RGCs drives a self-propagating wave of ATH5 expression and RGC production across the zebrafish retina (9, 10). In contrast, related experiments in zebrafish and chick highlight the importance of intrinsic factors for triggering ATH5 expression and neurogenesis (11-13).The presence of consensus E-box binding sites in the highly conserved upstream sequence of the ATH5 gene suggests that bHLH transcription factors are directly involved in the regulation of ATH5. This idea is supported by experiments showing the selective binding of neuronal bHLH proteins to the upstream sequence of ATH5 and the simultaneous changes in the expression level of ATH5 in response to several of these proteins (5, 6, 14 -16). These findings indicate a requirement for different combinations of bHLH proteins in regulating the different phases of ATH5 expression...

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