The alpha4beta2 nicotinic acetylcholine receptors (nAChRs), a major subtype in the brain, have been shown to be modulated by chronic treatment with nicotine. In this study, the regulation of recombinant human alpha4beta2 nAChR subtype by (-)-nicotine and other cholinergic channel modulators was studied using human embryonic kidney 293 cells stably expressing this subunit combination. The treatment of transfected cells with (-)-nicotine and other activator ligands, including (-)-cytisine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (+/-)-epibatidine, resulted in concentration-dependent increases in the levels of alpha4beta2 nAChRs. The increase in [3H]cytisine binding sites was initiated by low concentrations of (-)-nicotine (<100 nM); was maximal at 10 microM (15-fold), rapid (t0.5 = 4.0 +/- 0.5 hr), and totally reversible (t0.5 = 11.7 +/- 0.1 hr); and occurred with no change in ligand binding affinity. Antagonists, including dihydro-beta-erythroidine, d-tubocurarine, and methyllycaconitine, also elicited significant increases in receptor levels. A good correlation was observed between the Ki values for binding inhibition and the EC50 values for receptor up-regulation. Treatment of cells with mecamylamine, a noncompetitive antagonist, did not change receptor levels or alter (-)-nicotine-evoked up-regulation. (-)-Nicotine-evoked up-regulation was blocked by cycloheximide, suggesting a role for protein synthesis. Treatment of cells with (-)-nicotine or dihydro-beta-erythroidine differentially modulated the efficacy of acetylcholine to activate cation efflux. Both 6-beta-[beta'(piperidino)propionyl]forskolin and phorbol-12-myristate-13-acetate increased [3H]cytisine binding sites and nAChR function and enhanced the effects of chronic (-)-nicotine treatment in a synergistic manner. These results collectively demonstrate that human alpha4beta2 nAChRs can be differentially up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.

To study the functions of the 13 gyp genes, gvpMLKJIHGFEDACN, on plasmid pNRC100 of Halobacterium halobium in gas vesicle formation, we carried out linker scanning mutagenesis of the gene cluster. We constructed a 24.5-kb Escherichia coli-H. halobium shuttle plasmid, pFL2, containing the gyp gene cluster and introduced a kanamycin resistance (K) cassette into each gene (except for gvpA). Transformation of H. halobium SD109, which had the entire gyp gene cluster deleted, with pFL2 and mutated pFL2 derivatives showed that while the unmutated gene cluster successfully programmed gas vesicle formation, derivatives with insertion of the c cassette in any of the gyp genes, except gvpM, did not lead to production of normal gas vesicles. Insertions in gvpL, -K, -J, -I, and -F resulted in a complete block in gas vesicle synthesis, while insertions in gvpH, -G, -E, -D, -C, and -N resulted in greatly reduced gas vesicle synthesis. In most cases, the block in gas vesicle synthesis did not result from polar effects, since similar results were obtained for derivatives of the insertion mutants in which most of the internal portion of the K cassette was deleted and only small (15 to 54-bp) insertions remained. The only exceptions were for gvpH and gvpD, where deletion of the internal portion of the K insertions resulted in phenotypic reversion. Electron microscopic analysis of the K mutants revealed that interruptions of gvpC and gvpN result in the formation of smaller gas vesicles than in the wild type, while interruptions ofgvpF, -G, -H, -I, -J, -K, and -L produce no discernible vesicle intermediates. These results indicate that gvpA, -C, and -N, which have the rightward transcriptional orientation, encode structural proteins, with gvpC and gvpN necessary for late stages of vesicle formation, and gvpL,

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The bop gene of wild-type Halobacterium halobium NRC-1 is transcriptionally induced more than 20-fold under microaerobic conditions. bop transcription is inhibited by novobiocin, a DNA gyrase inhibitor, at concentrations subinhibitory for growth. The exposure of NRC-1 cultures to novobiocin concentrations inhibiting bop transcription was found to partially relax plasmid DNA supercoiling, indicating the requirement of high DNA supercoiling for bop transcription. Next, the bop promoter region was cloned on an H. halobium plasmid vector and introduced into NRC-1 and S9, a bop overproducer strain. The cloned promoter was active in both H. halobium strains, but at a higher level in the overproducer than in the wild type. Transcription from the bop promoter on the plasmid was found to be inhibited by novobiocin to a similar extent as was transcription from the chromosome. When the cloned promoter was introduced into S9 mutant strains with insertions in either of two putative regulatory genes, brp and bat, no transcription was detectable, indicating that these genes serve to activate transcription from the bop promoter in trans. Deletion analysis of the cloned bop promoter from a site ϳ480 bp upstream of bop showed that a 53-bp region 5 to the transcription start site is sufficient for transcription, but a 28-bp region is not. An 11-bp alternating purine-pyrimidine sequence within the functional promoter region, centered 23 bp 5 to the transcription start point, was found to display DNA supercoiling-dependent sensitivity to S1 nuclease and OsO 4 , which is consistent with a non-B-DNA conformation similar to that of left-handed Z-DNA and suggests the involvement of unusual DNA structure in supercoiling-stimulated bop gene transcription.The bop gene, encoding bacterio-opsin, the purple membrane protein of Halobacterium halobium, was one of the first archaeal genes to be cloned and sequenced (11). After cloning, several studies focused on the transcription of the bop gene and its regulation by oxygen and light. The start site for transcription was shown to map just 2 nucleotides upstream of the coding region by purification of the message, capping of its 5Ј end, and sequencing (6). Although this established bop mRNA as a primary transcript, no easily identifiable promoter could be found. Only a weak similarity to the TATA-like (box A) element was present (17). Interestingly, an 11-bp-long alternating purine-pyrimidine sequence centered 23 bp 5Ј to the transcription start site was observed and hypothesized to influence transcription by adopting left-handed Z-DNA conformation, which would be stabilized by high salt concentration and DNA supercoiling in H. halobium.The bop gene was shown to be transcriptionally induced at least 20-fold under microaerobic conditions in wild-type H. halobium strains (36, 43). Interestingly, bop gene transcription was inhibited by novobiocin, suggesting the involvement of DNA supercoiling in the modulation of promoter activity (42). Moreover, the involvement of two upstream genes in bop gene tr...