Accessory Sites Research Articles

Giant Pleomorphic Adenoma of Infratemporal Fossa: A Rare Case Report.

Of all the head and neck tumors, salivary gland tumors account to 3%. Pleomorphic adenomas are one of the most common benign tumors arising from major salivary glands, although it could also develop from minor salivary glands situated at accessory sites like nasal cavity, pharynx, parapharyngeal space, lacrimal glands etc. Tumors of infratemporal fossa are quite unusual, mainly because of its hidden location in retromaxillary region. We report an unusual case of 65 years old male presenting with complaint of progressive left cheek swelling for 4 years. FNAC revealed pleomorphic adenoma of minor salivary gland tumor. Intraoperatively a giant lobulated tumor was seen occupying almost whole space of infratemporal fossa, which was removed in-toto via open approach. Patient was kept on regular follow up with no evidence of recurrence reported till date.

Modulating Ligand Dissociation through Methyl Isomerism in Accessory Sites: Binding of Retinol to Cellular Carriers.

Due to the poor aqueous solubility of retinoids, evolution has tuned their binding to cellular proteins to address specialized physiological roles by modulating uptake, storage, and delivery to specific targets. With the aim to disentangle the structure-function relationships in these proteins and disclose clues for engineering selective carriers, the binding mechanism of the two most abundant retinol-binding isoforms was explored by using enhanced sampling molecular dynamics simulations and surface plasmon resonance. The distinctive dynamics of the entry portal site in the holo species was crucial to modulate retinol dissociation. Remarkably, this process is controlled to a large extent by the replacement of Ile by Leu in the two isoforms, thus suggesting that fine control of ligand release can be achieved through a rigorous selection of conservative mutations in accessory sites.

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.

Selection of COMBO-FISH Probes for Multi-Purpose Applications

The common method of fluorescence in situ hybridisation (FISH) is based on labelling DNA by the use of single stranded DNA-probes with appropriate markers binding to the counterpart within the genome (6). Originally FISH was performed with DNA biochemical amplified either in yeast (Yeast Artificial Chromosome, YAC) or in bacteria (Bacterial Artificial Chromosome, BAC). To achieve DNA hybridisation using these probes is requiring thermal and chemical treatment for denaturation after fixation of the specimen (7,8). These treatments together with the relatively large amount of probe DNA added, lead to several changes of the three dimensional nuclear structure resulting in a poor reproduction of the chromosomal and chromatin arrangement (9). Furthermore the creation of those probes does not require any knowledge about the exact sequence of the desired genomic region but only about the specific enzymatic cutting locations within the genome. Thus the length of the labelling probes is determined by this enzymatic and chemical processes rather than by the target sequence specificity of the markers. Nevertheless, FISH is still a standard method (6) used with great success in various fields in research and diagnostics and has fundamental impact on nowadays medical care. As mentioned, the conventional FISH method has at least two major disadvantages, on the one hand the destruction of the three dimensional structure due to denaturation treatment, which is a general problem for research in the field of genomic alterations and evaluation of the 3D-chromatin structure, and on the other hand the fully or correctly targeting labelling which is already a problem for labelling probe production itself. Both drawbacks can be overcome by the combinatorial design and usage of short oligonucleotide sequences which bind specifically to the desired genomic sequence. The design of such probes has become possible since the human genome has totally been sequenced and since there are methods of automated synthesising pre-determined DNA sequences industrially. The problem of conserving the original chromosomal structure can be solved by using triplex forming oligonucleotides (TFOs) of a length between 15 and 30 bases, that bind as a third strand into the major groove of the double stranded DNA within the nucleus. The usage of TFOs avoids denaturation of the specimen and in this way conserves the native structure of the chromatin. On the other hand such short probes lack uniqueness within the genome. This problem is solved by the usage of combinatorial sets of short oligonucleotides which only cover the selected region but do not co-localise anywhere else in the genome. This method is called Combinatorial Oligo Fluorescence In Situ Hybridisation (COMBO-FISH) (10). It can be used as well with the described TFOs as with common double-strand forming probes (Watson-Crick binding probes) both composed of DNA or peptide nucleic acids (PNAs) (10,11,12). Schmitt et al. have described the algorithms for designing such TFO-probes in ref. (12). By now this algorithm can also be used to identify and design Watson-Crick-binding probes (13). In the course of these algorithms one has to find the first and the last nucleotide of the genetic region of interest (ROI) first. With this numbers a set of oligonucleotides binding to this genomic ROI with a minimum of accessory binding sites and no other co-localisation site within the genome is computed. As parameters for this search a minimum and a maximum length of the oligonucleotides, the number of probes determined and the maximum cluster size of probes which are allowed to bind within a sequence of selectable length outside the labelling site are to be defined. The algorithms Schmitt et al. (12) created can be used as well on the genomic sequence database of the human genome as provided by the NCBI (14) as on a self-created database including the

Numerical and functional responses to the presence of a competitor – the case ofAggregatasp. (Apicomplexa: Aggregatidae) andOctopicola superba(Copepoda: Octopicolidae)

Evidence of interference competition between the eimeriorin coccidian Aggregata sp. and the octopicolid copepod Octopicola superba at the level of the gills of naturally infected Octopus vulgaris is evaluated. Numerical and functional responses are considered for analysis, and the fundamental and realized spatial niches (FSNs and RSNs) are measured as part of the study. While it was not possible to measure the FSN of Aggregata sp., the analysis of the infection levels of O. superba recorded for non-concomitantly and concomitantly infected hosts suggests that the gills and body skin constitute, respectively, the main and accessory sites of infection of the parasite. According to the evidence found, the gills function mainly as an accessory site of infection of Aggregata sp., in specimens in which the caecum and intestine are massively infected. Evidence for a negative interaction between Aggregata sp. and O. superba has been found while controlling for a potential confounding effect of host size. Furthermore, the presence of O. superba on gill lamellae appears to have been negatively affected by the presence of Aggregata sp., while this latter remained mostly undisturbed. The mean number of oocysts of Aggregata sp. in the gills was higher in spring and summer, which were also the seasons presenting the broadest RSN for O. superba.

Characterization of the tyrosine recombinase MrpA encoded by the Streptomyces coelicolor A3(2) plasmid SCP2*

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to significantly increase the stability of SCP2*-derived plasmids in Streptomyces lividans. The present report gives a functional characterization of MrpA. A sequence alignment revealed that MrpA shares highly conserved residues with members of the tyrosine recombinase family. After overexpression and Strep-tag purification, a DNase I footprint analysis and a gel mobility shift assay allowed for the identification of the 36-bp MrpA binding site mrpS. The mrpS site shows the configuration typical for tyrosine recombinases and contains two MrpA binding sites. The activity of MrpA was explored in vivo in E. coli cells and in vitro using purified MrpA. Depending on the position and orientation of the mrpS sites, three activities were detected: integration, resolution, and inversion. No accessory sites or proteins were required. Substitution of the conserved tyrosine (Y354F) by site-directed mutagenesis resulted in a complete loss of recombination activity but it still allowed the binding of MrpA to mrpS. The results define MrpA as a new site-specific tyrosine recombinase that acts with mrpS. In addition, we suggest that Y354 provides the nucleophile for DNA cleavage during recombination.

Unexplained Extensive Skull Base and Atlas Pneumatization

Pneumatization of the mastoid and petrous portions of the temporal bones begins late in fetal life, accelerates near and immediately after birth, and continues through infancy, early childhood, and, occasionally, early adult life. This pneumatization can vary greatly, and in cases of well-pneumatized temporal bones, accessory occipital air cells may occur and communicate with the mastoid air cells and middle ear. Other accessory sites of air cell pneumatization include the zygomatic process, squamosal region, and styloid process of the temporal bone. 1 Pneumatization of the occipital bone, including the craniocervical junction, is rare and, to our knowledge, has previously been reported only 10 times. 2-10 In 4 of those cases, 2,7,8 there was a history of trauma.

Glucocorticoids Regulate Transcription of the Gene for Phosphoenolpyruvate Carboxykinase in the Liver via an Extended Glucocorticoid Regulatory Unit

The hepatic transcriptional regulation by glucocorticoids of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) gene is coordinated by interactions of specific transcription factors at the glucocorticoid regulatory unit (GRU). We propose an extended GRU that consists of four accessory sites, two proximal AF1 and AF2 sites and their distal counterpart dAF1 (-993) and a new site, dAF2 (-1365); together, these four sites form a palindrome. Sequencing and gel shift binding assays of hepatic nuclear proteins interacting with these sites indicated similarity of dAF1 and dAF2 sites to the GRU proximal AF1 and AF2 sites. Chromatin immunoprecipitation assays demonstrated that glucocorticoids enhanced the binding of FOXO1 and peroxisome proliferator-activated receptor-alpha to AF2 and dAF2 sites and not to dAF1 site but enhanced the binding of hepatic nuclear transcription factor-4alpha only to the dAF1 site. Insulin inhibited the binding of these factors to their respective sites but intensified the binding of phosphorylated FOXO1. Transient transfections in HepG2 human hepatoma cells showed that glucocorticoid receptor interacts with several non-steroid nuclear receptors, yielding a synergistic response of the PEPCK-C gene promoter to glucocorticoids. The synergistic stimulation by glucocorticoid receptor together with peroxisome proliferator-activated receptor-alpha or hepatic nuclear transcription factor-4alpha requires all four accessory sites, i.e. a mutation of each of these markedly affects the synergistic response. Mice with a targeted mutation of the dAF1 site confirmed this requirement. This mutation inhibited the full response of hepatic PEPCK-C gene to diabetes by reducing PEPCK-C mRNA level by 3.5-fold and the level of circulating glucose by 25%.

Allosteric modulation of myristate and Mn(III)heme binding to human serum albumin

Human serum albumin (HSA) is best known for its extraordinary ligand binding capacity. HSA has a high affinity for heme and is responsible for the transport of medium and long chain fatty acids. Here, we report myristate binding to the N and B conformational states of Mn(III)heme-HSA (i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower affinity than Fe(III)heme, and displays a water molecule coordinated to the metal. Myristate binding to a secondary site FAx, allosterically coupled to the heme site, not only increases optical absorbance of Mn(III)heme-bound HSA by a factor of approximately three, but also increases the Mn(III)heme affinity for the fatty acid binding site FA1 by 10-500-fold. Cooperative binding appears to occur at FAx and accessory myristate binding sites. The conformational changes of the Mn(III)heme-HSA tertiary structure allosterically induced by myristate are associated with a noticeable change in both optical absorbance and NMR spectroscopic properties of Mn(III)heme-HSA, allowing the Mn(III)-coordinated water molecule to exchange with the solvent bulk. At pH = 10.0 both myristate affinity for FAx and allosteric modulation of FA1 are reduced, whereas cooperation of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme binds to HSA with higher affinity than at pH 7.0 even in the absence of myristate, and the metal-coordinated water molecule is displaced. As a whole, these results suggest that FA binding promotes conformational changes reminiscent of N to B state HSA transition, and appear of general significance for a deeper understanding of the allosteric modulation of ligand binding properties of HSA.

Synapsis in Phage Bxb1 Integration: Selection Mechanism for the Correct Pair of Recombination Sites

Recombination by site-specific recombinases is a highly concerted process that requires synapsis of the correct pair of DNA substrates. Phage-encoded serine-integrases are unusual among the serine-recombinase family, which includes transposon resolvases and DNA invertases, in that they utilize two simple but different DNA substrates (attB and attP) and do not require accessory sites, additional proteins, or DNA supercoiling. Synapsis must therefore be directed solely by integrase-DNA interactions. We show here that the Bxb1 serine-integrase binds as a dimer to its two DNA substrates (attB, attP) and recombinant products (attL, attR) with similar affinities. However, synapsis occurs only between attP and attB, and not between any of the other nine possible site combinations. The Bxb1 integrase domain structure, the unusual DNA-binding properties of the integrase, and the characterization of a mutant protein with altered site-discrimination, are consistent with synaptic selectivity being derived from DNA sequence-induced changes in the conformations of integrase-DNA complexes.

Two Mechanisms for Mannose-binding Protein Modulation of the Activity of Its Associated Serine Proteases

Serum mannose-binding protein (MBP) neutralizes invading microorganisms by binding to cell surface carbohydrates and activating MBP-associated serine proteases-1, -2, and -3 (MASPs). MASP-2 subsequently cleaves complement components C2 and C4 to activate the complement cascade. To analyze the mechanisms of activation and substrate recognition by MASP-2, zymogen and activated forms have been produced, and MBP.MASP-2 complexes have been created. These preparations have been used to show that MBP modulates MASP-2 activity in two ways. First, MBP stimulates MASP-2 autoactivation by increasing the rate of autocatalysis when MBP.MASP-2 complexes bind to a glycan-coated surface. Second, MBP occludes accessory C4-binding sites on MASP-2 until activation occurs. Once these sites become exposed, MASP-2 binds to C4 while separate structural changes create a functional catalytic site able to cleave C4. Only activated MASP-2 binds to C2, suggesting that this substrate interacts only near the catalytic site and not at accessory sites. MASP-1 cleaves C2 almost as efficiently as MASP-2 does, but it does not cleave C4. Thus MASP-1 probably enhances complement activation triggered by MBP.MASP-2 complexes, but it cannot initiate activation itself.

Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation.

Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid. Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.

Épidémiologie descriptive des amyloses diagnostiquées au CHU de Rennes de 1995 à 1999

Introduction. – The frequency of amyloidosis is not well known in France. We compiled a register of amyloidosis diagnosed from 1995 to 1999 in the University Hospital of Rennes. Patients and methods. – This retrospective study was performed between 01 January 1995 and 31 december 1999. Diagnosis was assessed on positivity of red Congo by anatomopathology. Immunohistochemistry allowed the definition the type of amyloidosis. Clinical data, staging and outcome of patients were analysed. Results. – Forty-three amyloidosis were diagnosed (27 women, 16 men) with an incidence of 8,6 new cases per year. Mean age was 63.7 years. Five diagnosis were realised in 1995, six in 1996, six in 1997, 12 in 1998, 14 in 1999. Twenty amyloidosis were AL type (46.5%), seven AA (16.3%), 1 β2 microglobulin type, 15 (35%) remained of undetermined type. Thirty-three amyloidosis (77%) were systemic, 10 were localized to one organ (23%). When diagnosis was made, biopsies concerned affected organs in 86% of the cases, accessory sites (labial salivary glands, bone marrow) in only 14% of the cases. Twenty-five patients died (58%). Two deaths were treatment-related, 16 to amyloidois, seven patients died of another complaint. Conclusion. – Increased incidence of amyloidosis needs to be confirmed. We emphasize the importance of immunohistochemical typing on frozen samples, the value of accessory biopsies and the need for complete extension staging.

AML3/CBFα1 Is Required for Androgen-specific Activation of the Enhancer of the Mouse Sex-limited Protein (Slp) Gene

A complex 120-base pair enhancer, derived from the mouse sex-limited protein (Slp) gene, is activated solely by the androgen receptor (AR) in specific tissues, although it contains a hormone response element recognized by several steroid receptors. The generation of this transcriptional specificity has been ascribed to the interactions of the receptor with tissue-specific nonreceptor factors bound to accessory sites within the enhancer. Protein-DNA interaction assays revealed two factors binding the 5' part of the enhancer that differ widely in abundance between cells showing AR-specific activation of the Slp element compared with those that also permit activation by glucocorticoid receptor (GR). The factor designated B formed a complex centered on the sequence TGTGGT, a core motif recognized by members of the AML/CBFalpha transcription factor family. This complex was competed by a high affinity binding site specific for AML/CBFalpha and was specifically supershifted by an antibody to AML3/CBFalpha1, placing factor B within the AML3/CBFalpha1 subclass. Interestingly, this factor was shown to bind to a second site in the 3' part of the enhancer, positioned between the two critical AR binding sites. Transfection studies revealed that AML1-ETO, a dominant-negative AML/CBFalpha construct, abrogated AR induction of the enhancer, but not of simple hormone response elements. Furthermore, overexpression of AML3/CBFalpha1 could rescue the AML1-ETO repression. Finally, glutathione S-transferase-AML/CBFalpha fusion proteins demonstrated direct interaction between AML/CBFalpha and steroid receptors. Although this interaction was equivalent between AML1/CBFalpha2 and AR or GR, AML3/CBFalpha1 showed stronger interaction with AR than with GR. These data demonstrate that AML3/CBFalpha1 is functionally required for hormonal induction of the Slp enhancer and that direct, preferential protein-protein interactions may contribute to AR-specific activation. These results demonstrate an intriguing role of AML3/CBFalpha1 in steroid- as well as tissue-specific activation of target genes.

Topological selectivity of a hybrid site-specific recombination system with elements from Tn3 res/resolvase and bacteriophage P1 loxP/Cre

In order to investigate the functions of the parts of the Tn 3 recombination site res, we created hybrid recombination sites by placing the loxP site for Cre recombinase adjacent to the “accessory” resolvase-binding sites II and III of res. The efficiency and product topology of in vitro recombination by Cre between two of these hybrid sites were affected by the addition of Tn 3 resolvase. The effects of resolvase addition were dependent on the relative orientation and spacing of the elements of the hybrid sites. Substrates with sites II and III of res close to loxP gave specific catenated or knotted products (four-noded catenane, three-noded knot) when resolvase and Cre were added together. The product topological complexity increased when the length of the spacer DNA segment between loxP and res site II was increased. Similar resolvase-induced effects on Cre recombination product topology were observed in reactions of substrates with loxP sites adjacent to full res sites. The results demonstrate that the res accessory sites are sufficient to impose topological selectivity on recombination, and imply that intertwining of two sets of accessory sites defines the simple catenane product topology in normal resolvase-mediated recombination. They are also consistent with current models for the mechanism of catalysis by Cre.

Positive regulation of the rat CYP2B2 phenobarbital response unit by the nuclear receptor hexamer half-site·nuclear factor 1 complex

Abstract. A distal 163-bp fragment mediates phenobarbital responsiveness of the rat CYP2B2 gene. Multiple cis-acting elements in this fragment cooperate to form a phenobarbital response unit (PBRU). A nuclear factor 1 binding site and an associated nuclear receptor hexamer half-site are present in both the rat CYP2B2 PBRU and the homologous mouse Cyp2b10 sequence. Based on mutational analyses, the hexamer half-site has been reported to act positively in CYP2B2 and negatively in Cyp2b10. However, the specific mutations introduced into the rat and mouse hexamer half-sites were different, raising the possibility that the different roles attributed to the element may be a consequence of the different mutations used. We introduced into the rat CYP2B2 hexamer half-site the specific mutational change previously introduced into the Cyp2b10 sequence, where its effect was to increase the basal level of expression and to abolish phenobarbital responsiveness. In the rat context, this mutation reduced but did not abolish phenobarbital responsiveness and decreased, rather than increased, the basal level of expression. The residual phenobarbital responsiveness of the hexamer half-site mutant, as well as that of nuclear factor 1 mutants, indicates that these elements behave as positive accessory sites, suggesting that factors binding to them function as activators of phenobarbital-dependent transcription.

Hepatic nuclear factor 3 is an accessory factor required for the stimulation of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids.

Transcription of the hepatic phosphoenolpyruvate carboxykinase gene is stimulated by glucocorticoids and inhibited by insulin. The glucocorticoid response is mediated by a complex glucocorticoid response unit that consists of two glucocorticoid receptor (GR)-binding sites (GR1 and GR2) and two accessory factor-binding sites (AF1 and AF2). The complete unit is required for the full glucocorticoid response. The dominant insulin effect is mediated in part through an insulin response sequence that is coincident with the AF2 element. Members of the hepatic nuclear factor 3 (HNF3) and CCAAT enhancer binding protein (C/EBP) families bind to the AF2 element; however, there is no correlation between binding of these factors and the ability of the AF2 element to mediate an insulin response. We show here that binding of HNF3 does correlate with the stimulation of the glucocorticoid response by the AF2 element and that C/EBP is apparently not involved in this effect. This requirement for HNF3 is quite specific since the substitution of elements known to enhance the action of the GR in other promoters fails to recapitulate AF2 accessory factor activity. By contrast, an HNF3-binding site from the transthyretin gene is able to substitute for the wild type AF2 sequence and elicit a maximal glucocorticoid response. Based on current and previous observations, the glucocorticoid response unit consists of four DNA elements that bind four different proteins. These are: AF1 (hepatic nuclear factor 4/chicken ovalbumin upstream promoter transcription factor), AF2 (HNF3), GR1 (GR), and GR2 (GR).

Rate and Selectivity of Synapsis of resRecombination Sites by Tn 3Resolvase

Site-specific recombination catalysed by Tn 3resolvase requires the formation of an intermediate synaptic complex containing two resrecombination sites and several resolvase subunits. Synaptic complexes were observed directly by chemical crosslinking of resolvase subunits followed by agarose gel electrophoresis. The highest yield of synaptic complex was from a "standard" substrate, a supercoiled plasmid with ressites in direct repeat, but complexes were also made between sites in inverted repeat, or in nicked or linear molecules, or in separate molecules. The substrate selectivity for synapsis is less stringent than for recombination; thus recombination selectivity is dependent on steps after synapsis. The stability of the synapse after its formation might be a key factor, since unproductive synapses are less stable than productive ones. In a standard substrate, synapsis is fast relative to the rate of recombination. Crosslinking in active reaction mixtures yields synaptic complexes derived from both the substrate and the catenane recombination product. Although catalysis of strand exchange is at binding site I of res, a pair of isolated site I's do not synapse, whereas a synaptic complex is formed from a plasmid carrying two copies of resbinding sites II and III. Our data are consistent with a model in which the formation of the synaptic intermediate is driven, and its structure defined, by the initial interaction of these accessory sites.

Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium

CysB is a transcriptional activator for the cysteine regulon and negatively autoregulates its own gene, cysB. Transcription activation also requires an inducer, N-acetyl-L-serine. CysB is known to bind to activation sites just upstream of the -35 regions of the positively regulated cysJIH, cysK, and cysP promoters and to a repressor site centered at about +1 in the cysB promoter. Additional accessory sites have been found in positively regulated promoters. The hydroxyl radical footprinting experiments reported here indicate that the activation sites CBS-J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are composed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp. N-Acetyl-L-serine stimulates binding to these sites as well as to the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites. A second topology is found in the accessory site CBS-K2 and the repressor site CBS-B, which contain divergently oriented 19-bp half-sites separated by one or two helical turns. N-Acetyl-L-serine inhibits binding to these two sites. A third topology is present in the cysK and cysP promoters, where an additional half-site is oriented toward the activation site and separated from it by one helical turn. Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending. The marked dissimilarities of these half-site arrangements and of their responses to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to them with different combinations of subunits.

Dexamethasone stimulation of rat insulin-like growth factor binding protein-1 (IGFBP-1) promoter activity involves the interaction of multiple transcription factors

Using an improved procedure for transient transfection of H4-II-E rat hepatoma cells, we characterized the cis elements in the proximal promoter of the rat insulin-like growth factor binding protein-1 (rat IGFBP-1) gene that are required for basal (unstimulated) and dexamethasone-stimulated promoter activity. Three sites are required for optimal basal promoter activity: an AP-2 site (nt −286 to −293), the M4 region of the insulin response element (nt −108 to −99), and a hepatocyte nuclear factor-1 (HNF-1) site (nt −62 to −50). In addition to the glucocorticoid response element (nt −91 to −77), participation of two of three accessory sites is required for optimal stimulation by dexamethasone: the M4 and HNF-1 sites, and a third site located between nt −252 and −236. Further study will focus on how the interactions of tissue-specific and hormonally-responsive transcription factors are integrated.