The minimal promoter of the human alpha 3 nicotinic receptor subunit gene. Molecular and functional characterization.

The minimal promoter of the human alpha(3) nicotinic receptor subunit gene has been mapped to a region of 60 base pairs and found to contain two Sp1 sites, one of which is essential for promoter activity. DNase footprinting has revealed the presence of another region of interaction with nuclear factors (named F2) immediately downstream of the Sp1 sites. This region has been found to be functional since it is capable of stimulating the minimal promoter. The F2 protection is completely and specifically competed by an AP2 consensus oligonucleotide that has been proved to bind AP2alpha exclusively. However, the AP2alpha recombinant protein was unable to bind the F2 region directly, thus suggesting that AP2alpha may participate in F2 protection by protein-protein interactions with other nuclear factors. The minimal promoter has been shown to be stimulated by two additional regions, one located downstream of F2 and the other upstream of the minimal promoter itself. In neuronal cells, the combined stimulatory activities of these three regions have synergistic effects, whereas in non-neuronal cells, there is a negative interference between the upstream and downstream regions. These opposite transcriptional effects may account for at least part of the neuro-specific expression profile of the alpha(3) gene.

Neuronal and extraneuronal expression and regulation of the human alpha5 nicotinic receptor subunit gene.

The mRNA encoding the human alpha5 nicotinic subunit was detected in several structures of the nervous system but appeared to be mainly expressed in cerebellum, thalamus, and the autonomic ganglia. For the first time, the alpha5 transcript was also detected in several non-neuronal tissues, with maximal expressions being found throughout the gastrointestinal tract, thymus, and testis. Many other extraneuronal sites expressed alpha5, but there were also nonexpressing organs, such as the liver, spleen, and kidney. To understand the transcriptional mechanisms controlling such a diversified expression of alpha5 in neuronal and nonneuronal cells, we isolated the 5'-regulatory region of the human gene and characterized its properties. Here we identify the alpha5 core promoter and demonstrate that the DNA regions surrounding it contain elements (with positive or negative activities) that work in a tissue-specific fashion. In particular, the segment specifying the 5'-untranslated region in neuronal cells has most of the properties of an enhancer because it activates a heterologous promoter in a position- and orientation-independent fashion. We therefore conclude that the expression of alpha5 relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs.

Transcriptional regulation of the human alpha5 nicotinic receptor subunit gene in neuronal and non-neuronal tissues.

The human alpha5 nicotinic receptor subunit gene appears to be expressed in several structures of the nervous system, but also in a number of non-neuronal tissues, with maximal expressions occurring in the entire gastrointestinal tract, thymus and testis. To understand whether specific transcriptional mechanisms are involved in the tissue-specific expression of the alpha5 subunit in neuronal and non-neuronal cells, we isolated the 5'-regulatory region of the human gene and characterized its functional properties. We demonstrate that specific DNA elements, with positive or negative activities depending on the cell type, are responsible for the diversified expression of the alpha5 subunit in different tissues. We therefore conclude that the expression of the alpha5 subunit relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs.

Expression of the alpha3 nicotinic receptor subunit mRNA in aging and Alzheimer's disease.

Changes in the number of high-affinity nicotine binding sites have been widely reported in specific regions of the human brain during aging and in degenerative neurological diseases associated with aging, such as Alzheimer's disease. Nicotinic receptors are highly diverse and a description of the molecular subtypes affected in such conditions has not been achieved to date. To investigate the status of the alpha3 subunit-containing subtypes in such conditions, we assessed by in situ hybridisation the alpha3 mRNA density in the hippocampus, entorhinal cortex and thalamus of Alzheimer's patients and age-matched controls. No significant difference in the expression of the alpha3 mRNA, either qualitative or quantitative, was found between Alzheimer's individuals and controls in any of the analysed areas. This result suggests that the nicotine binding changes occurring in these areas in Alzheimer's patients are not correlated to a variation of the alpha3 mRNA in the same regions. Nevertheless, a negative correlation between the alpha3 mRNA density and the age was observed in the entorhinal cortex of both the Alzheimer's and the normal subjects, suggesting a potentially extensive decay of the alpha3-expressing neurons or loss of alpha3-containing receptors in intact neurons of the entorhinal cortex in the late elderly.

Expression and transcriptional regulation of the human alpha3 neuronal nicotinic receptor subunit in T lymphocyte cell lines.

The expression of neurotransmitter receptors on the surface of immunocompetent cells is generally accepted as evidence that the nervous system can influence immune responses, even though many aspects of these interactions remain to be elucidated. In this article, we analyzed the expression of the alpha3 nicotinic receptor subunit in human cell lines of myeloid and lymphoid origin and show that the alpha3 mRNA and the receptor molecules containing this subunit are specifically expressed in T lymphocyte cell lines. We have previously characterized the structural properties of the human alpha3 nicotinic subunit gene promoter and defined its functional profile in neuronal cells; in this study, we analyzed the activity of the alpha3 promoter in T lymphocytes and found that the same minimal promoter located in the 0.16-kb BglII-AccIII fragment is responsible for the expression of the alpha3 mRNA in both neuronal and T lymphocyte cell lines. However, the alpha3 transcription initiation patterns in the two cell types were both qualitatively and quantitatively different, and the minimal promoter was differentially modulated by downstream and upstream regulatory elements. These findings suggest that distinct transcriptional mechanisms allow the same promoter to be regulated in a tissue-specific fashion, according to the different functional needs of the two cell types.

Identification of two replicons in phage-plasmid P4.

DNA replication of phage-plasmid P4 proceeds bidirectionally from the ori1 site (previously named ori), but requires a second cis-acting region, crr. Replication depends on the product of the P4 alpha gene, a protein with primase and helicase activity, that binds both ori1 and crr. A negative regulator of P4 DNA replication, the Cnr protein, is required for copy number control of plasmid P4. Using a plasmid complementation test for replication, we found that two replicons, both dependent on the alpha gene product, coexist in P4. The first replicon is made by the cnr and alpha genes and the ori1 and crr sites. The second is limited to the alpha and crr region. Thus, in the absence of the ori1 region, replication can initiate at a different site. By deletion mapping, a cis-acting region, ori2, essential for replication of the alpha-crr replicon was mapped within a 270-bp fragment in the first half of the alpha gene. The ori2 site was found to be dispensable in a replicon that contains ori1. A construct that besides crr and alpha carries also the cnr gene was unable to replicate, suggesting that Cnr not only controls replication from ori1, but also silences ori2.

Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha.

Bacteriophage P4 DNA replication depends upon the phage-encoded alpha protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr). The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr. We searched for P4 mutants that suppressed this phenotype (Cnr resistant [alpha cr]). Eight independent mutants that grew in the presence of high levels of Cnr were obtained. None of these can establish the plasmid state. Each of these mutations lies in the DNA binding domain of gp alpha that occupies the C terminus of the protein. Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V. A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of alpha protein in vitro. The alpha cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein. We present evidence that Cnr protein interacts with the gp alpha domain that binds specifically to DNA and that gp(alpha)cr mutations impair this interaction. We hypothesize that gp alpha-Cnr interaction is essential for the control of P4 DNA replication.

Structural and functional characterization of the human alpha3 nicotinic subunit gene promoter.

We describe the structural and functional features of the human alpha3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3' region, specifying the 5' UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-kappaB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human alpha3 gene 5' regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.

Multiple regulatory mechanisms controlling phage-plasmid P4 propagation.

Bacteriophage P4 autonomous replication may result in the lytic cycle or in plasmid maintenance, depending, respectively, on the presence or absence of the helper phage P2 genome in the Escherichia coli host cell. Alternatively, P4 may lysogenize the bacterial host and be maintained in an immune-integrated condition. A key step in the choice between the lytic/plasmid vs. the lysogenic condition is the regulation of P4 alpha operon. This operon may be transcribed from two promoters, PLE and PLL, and encodes both immunity (promoter proximal) and replication (promoter distal) functions. PLE is a constitutive promoter and transcription of the downstream replication genes is regulated by transcription termination. The trans-acting immunity factor that controls premature transcription termination is a short RNA encoded in the PLE proximal part of the operon. Expression of the replication functions in the lytic/plasmid condition is achieved by activation of the PLL promoter. Transcription from PLL is insensitive to the termination mechanism that acts on transcription starting from PLE.PLL is also negatively regulated by P4 orf88, the first gene downstream of PLL. An additional control on P4 DNA replication is exerted by the P4 cnr gene product.

A new gene of bacteriophage P4 that controls DNA replication.

Bacteriophage P4 replication may result in either a lytic cycle or plasmid maintenance, depending on the presence or absence, respectively, of helper phase P2 genome. Bacteriophage P4 DNA replication depends on the product of gene alpha, which has origin recognition, primase, and helicase activities. An open reading frame with the coding capacity for a protein of 106 amino acids (orf106) is located upstream of the alpha gene. Genes orf106 and alpha are transcriptionally coregulated. Three amber mutations and an internal deletion (del51) were introduced into orf106. All of the amber mutations exhibited a polar effect on transcription of the downstream alpha gene. The P4 del51 mutant was slightly defective in lytic growth and could not be propagated in the plasmid state. In this latter condition, P4 DNA overreplication was observed. Overexpression of Orf106 severely inhibited P4 DNA replication, preventing P4 lytic growth and plasmid maintenance. The inhibitory effect of Orf106 on P4 replication was not observed when both orf106 and alpha were overexpressed. We suggest that orf106 is involved in P4 replication and that a balanced expression of orf106 relative to alpha may be necessary for proper P4 DNA replication. In particular, orf106 appears to be essential for the control of P4 genome replication in the plasmid state. We propose that orf106 be named cnr, for copy number regulation.