The promoter of the mouse odorant receptor gene M71.

From a repertoire of approximately 2000 odorant receptor (OR) alleles in the mouse genome, a mature olfactory sensory neuron (OSN) is thought to choose only one functional allele of one OR gene for expression. OSNs that express a given OR gene are scattered throughout an epithelial region that is gene specific. The DNA sequences that enable OR gene choice and specify the epithelial pattern are not known. Within the upstream regions of several mouse, rat, and human OR genes, we have previously recognized putative homeodomain and O/E-like binding sites in proximity to each other. Here, we define a minimal promoter region for expression of the mouse OR gene M71 with small transgenes. This region contains a homeodomain and an O/E-like binding site. Combined mutations in both sites abolish transgene expression. When identical mutations are introduced at the endogenous M71 locus by gene targeting, the number of M71-expressing OSNs is reduced by a factor of three and the epithelial pattern is ventralized. The stronger impact observed with the mutant transgenes compared to the targeted mutations may reflect a multiplicity of regulatory sites within the OR gene cluster. We propose that these homeodomain and O/E sites regulate the probability of M71 gene choice differentially across the olfactory epithelium.

Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb.

An olfactory sensory neuron (OSN) expresses selectively one member from a repertoire of approximately 1000 odorant receptor (OR) genes and projects its axon to a specific glomerulus in the olfactory bulb. Both processes are here recapitulated by MOR23 and M71 OR minigenes, introduced into mice. Minigenes of 9 kb and as short as 2.2 kb are selectively expressed by neurons that do not coexpress the endogenous gene but coproject their axons to the same glomeruli. Deletion of a 395 bp upstream region in the MOR23 minigene abolishes expression. In this region we recognize sequence motifs conserved in many OR genes. Transgenic lines expressing the OR in ectopic epithelial zones form ectopic glomeruli, which also receive input from OSNs expressing the cognate endogenous receptor. This suggests a recruitment through homotypic interactions between OSNs expressing the same OR.

Multiple new and isolated families within the mouse superfamily of V1r vomeronasal receptors.

Seven-transmembrane-domain proteins encoded by the vomeronasal receptor V1r and V2r gene superfamilies, and expressed by vomeronasal sensory neurons, are believed to be pheromone receptors in rodents. Four V1r gene families have been described in the mouse (V1ra, V1rb, V1rc and V3r). Here we have screened near-complete mouse genomic databases to obtain a first global draft of the mouse V1r repertoire, including 104 new V1r genes. It comprises eight new and extremely isolated families in addition to the four families previously identified. Members of these new families were expressed in vomeronasal sensory neurons. The genome-wide view revealed great sequence diversity within the V1r superfamily. Phylogenetic analyses suggested an ancient original radiation, followed by the isolation, divergence and expansion of families by extensive gene duplications and frequent gene loss. The isolated nature of these gene families probably reflects a specialization of different receptor classes in the detection of specific types of chemicals.

Trans membrane domain IV is involved in ion transport activity and pH regulation of the NhaA-Na(+)/H(+) antiporter of Escherichia coli.

We have previously shown that the activity of NhaA is regulated by pH and found mutations that affect dramatically the pH dependence of the rate but not the K(m) (for Na(+) and Li(+)) of NhaA. In the present work, we found that helix IV is involved both in ion translocation as well as in pH regulation of NhaA. Two novel types of NhaA mutants were found clustered in trans membrane segment (TMS) IV: One type (D133C, T132C, and P129L) affects the apparent K(m) of NhaA to the cations with no significant effect on the pH profile of the antiporter; no shift of the pH profile was found when the activity of these mutants was measured at saturating Na(+) concentration. In contrast, the other type of mutations (A127V and A127T) was found to affect both the K(m) and the pH dependence of the rate of NhaA whether tested at saturating Na(+) concentration or not. These results imply that residues involved in the ion translocation of NhaA may (A127) or may not (D133, T132, and P129) overlap with those affecting the pH response of the antiporter. All mutants cluster in the N-terminal half of the putative alpha-helix IV, one type on one face, the other on the opposite. Cys accessibility test demonstrated that although D133C is located in the middle of TMS IV, it is inhibited by N-ethylmaleimide and is exposed to the cytoplasm.

Studies on the effect of ethanol on the Na+, and the Na+,K+ATPase activities of plasma membranes of rat kidney proximal tubular cells

In the present work it was investigated the effect of 2 percent ethanol on the Na+ and on the Na+, K(+)-ATPase activities. The differential effect of the alcohol on the two ATPases (approximately 40 percent inhibition of the Na(+)-ATPase and approximately 10 percent inhibition of the Na+, K(+)-ATPase), is not due to a higher degree of denaturalization of the enzyme, nor to a faster effect of ethanol on the Na(+)-than on the Na+, K(+)-ATPase. Our results show that ethanol affects the selectivity of the Na+, K(+)-ATPase for Na+ and/or for K+, enhancing the Na+ affinity for the K+ sites, and/or reducing the K+ affinity for its own sites. This effect was not seen for the Na(+)-ATPase, indicating that 2 percent ethanol inhibits the two ATPases in a totally different way