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PubMed2006

Promoter prediction and annotation of microbial genomes based on DNA sequence and structural responses to superhelical stress.

BACKGROUND: In our previous studies, we found that the sites in prokaryotic genomes which are most susceptible to duplex destabilization under the negative superhelical stresses that occur in vivo are statistically highly significantly associated with intergenic regions that are known or inferred to contain promoters. In this report we investigate how this structural property, either alone or together with other structural and sequence attributes, may be used to search prokaryotic genomes for promoters. RESULTS: We show that the propensity for stress-induced DNA duplex destabilization (SIDD) is closely associated with specific promoter regions. The extent of destabilization in promoter-containing regions is found to be bimodally distributed. When compared with DNA curvature, deformability, thermostability or sequence motif scores within the -10 region, SIDD is found to be the most informative DNA property regarding promoter locations in the E. coli K12 genome. SIDD properties alone perform better at detecting promoter regions than other programs trained on this genome. Because this approach has a very low false positive rate, it can be used to predict with high confidence the subset of promoters that are strongly destabilized. When SIDD properties are combined with -10 motif scores in a linear classification function, they predict promoter regions with better than 80% accuracy. When these methods were tested with promoter and non-promoter sequences from Bacillus subtilis, they achieved similar or higher accuracies. We also present a strictly SIDD-based predictor for annotating promoter sequences in complete microbial genomes. CONCLUSION: In this report we show that the propensity to undergo stress-induced duplex destabilization (SIDD) is a distinctive structural attribute of many prokaryotic promoter sequences. We have developed methods to identify promoter sequences in prokaryotic genomes that use SIDD either as a sole predictor or in combination with other DNA structural and sequence properties. Although these methods cannot predict all the promoter-containing regions in a genome, they do find large sets of potential regions that have high probabilities of being true positives. This approach could be especially valuable for annotating those genomes about which there is limited experimental data.

Bacillus subtilis↗
PubMed2005

MDM2 negatively regulates the human telomerase RNA gene promoter.

BACKGROUND: We have previously demonstrated that NF-Y and Sp1 interact with the human telomerase RNA (hTR) promoter and play a central role in its regulation. We have also shown that pRB activates the hTR promoter, but the mechanism of pRb directed activation is unknown. It has recently been reported that pRB induces Sp1 activity by relieving inhibition mediated by mdm2. The aim was to investigate possible roles for mdm2 in hTR promoter regulation. METHODS: Chromatin immunoprecipitation was used to determine binding of mdm2 to the hTR promoter. Transfection and luciferase assays were used to investigate mdm2 repression of the promoter activity and interaction with known transcriptional modulators. RESULTS: Here we show using chromatin immunoprecipitation that mdm2 specifically binds the hTR promoter in vivo. Transient co-transfection experiments using an hTR promoter luciferase reporter construct show that hTR promoter activity is inhibited by over-expression of mdm2 in 5637 bladder carcinoma cells (p53 and pRB negative, low mdm2). Titration of mdm2 was able to antagonise activation of hTR promoter activity mediated by pRB or Sp1 over-expression, although in the presence of pRB, mdm2 could not repress promoter activity below basal levels. Using an Sp1 binding site mutation construct we showed that mdm2 repression did not absolutely require Sp1 binding sites in the hTR promoter, suggesting the possibility of pRB/Sp1 independent mechanisms of repression. Finally, we show that NF-Y mediated transactivation of the hTR promoter was also suppressed by mdm2 in a dose-dependent manner. CONCLUSIONS: These studies suggest that mdm2 may inhibit the hTR promoter by multiple mechanisms. Mdm2 may directly repress activation by both pRB and Sp1, or activation by NF-Y. Furthermore, the ability of mdm2 to interact and interfere with components of the general transcription machinery might partly explain the general repressive effect seen here. Elucidation of new regulators affecting hTR basal promoter activity in cancer cells provides a basis for future studies aimed at improving our understanding of the differential hTR expression between normal and cancer cells.

CCAAT-Binding Factor↗
PubMed2005

Viral promoters can initiate expression of toxin genes introduced into Escherichia coli.

BACKGROUND: The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. RESULTS: We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. CONCLUSION: According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

Bacterial Proteins↗
PubMed1994

Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter.

Insulin-like growth factor-binding protein-1 (IGFBP-1) is produced by the liver and regulated by glucocorticoids and insulin at the level of gene transcription. To identify DNA sequences mediating the effects of glucocorticoids and insulin on IGFBP-1 promoter activity we created luciferase reporter gene constructs and performed transfection studies in H4IIE hepatoma cells. Initial studies confirmed that the IGFBP-1 promoter is functional when inserted in the correct orientation, but not in the reverse orientation. Dexamethasone (DEX) increased promoter activity 10-fold, and insulin reversed this effect of DEX by 85% at 8 h. The effects of DEX were abolished when constructs were truncated to 89 bases from the RNA cap site, and DNase footprinting with the DNA-binding domain of the human glucocorticoid receptor identified an imperfect palindrome containing two receptor-binding sites separated by three nucleotides typical of a glucocorticoid response element (GRE) at this location. Mutation of either binding site (or half-site) disrupted the effects of DEX, confirming that this sequence functions as a GRE. Two other regions of the promoter also footprinted with the glucocorticoid receptor protein and contained sequences consistent with glucocorticoid receptor-binding sites; however, neither of these footprints contained the full structure expected of a functional GRE, and neither mutation nor deletion of these other sequences altered the effects of DEX on promoter activity. To identify the DNA sequences required for the effects of insulin on glucocorticoid-stimulated promoter activity, we created internal deletions throughout the IGFBP-1 promoter region. Deletion of the 22-basepair (bp) sequence immediately 5' from the GRE disrupted the effect of insulin and appeared to increase basal promoter activity at least 2-fold in each of eight experiments (P < 0.001 vs. intact promoter). This region of the IGFBP-1 promoter contains a 19-bp palindrome (CAAAACAAACTTATTTTG) that overlaps the 5'-end of the GRE and is fully conserved in the human IGFBP-1 promoter. Each half of this palindrome resembles previously identified insulin response sequences, and deletion/mutation analysis suggests that each half may contribute to the effects of insulin on promoter activity. Gel shift studies confirmed that this palindrome binds H4IIE nuclear proteins. In summary, we have identified a GRE in the 5'-promoter region of the rat IGFBP-1 gene approximately 90 bp up-stream from the RNA cap site as well as a contiguous 22-bp region that plays a critical role in mediating the effects of insulin on glucocorticoid-stimulated promoter activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

Basal and vitamin D-responsive activity of the rat osteocalcin promoter in stably transfected osteosarcoma cells: requirement of upstream sequences for control by the proximal regulatory domain.

Osteocalcin (OC) is a bone-specific vitamin D- responsive protein that is developmentally expressed during osteoblast differentiation. In transient transfection assays, as little as approximately 0.1 kilobase (kb) of the OC proximal promoter is sufficient for basal expression. Because eukaryotic genes are packaged as nucleosomes that contribute to both chromatin organization and transcriptional control, we functionally examined the activity of OC promoter constructs within a chromatin context. ROS 17/2.8 osteosarcoma cells were stably transfected with a series of rat OC promoter-reporter constructs, containing progressive 5'-deletions. The results demonstrate that in contrast to transient transfection assays, the proximal 0.11-kb promoter is no longer active when integrated in the genome. Progressive gain of basal expression with 0.35-, 0.53-, and 0.72-kb promoters suggests that upstream sequences facilitate the formation of an appropriate higher order nuclear structure, thereby potentiating the activity of the chromosomally integrated proximal promoter elements. This is consistent with location of both deoxyribonuclease I (DNase I)-hypersensitive sites and nuclear matrix protein-DNA interaction sites in the osteocalcin promoter. Vitamin D responsiveness in the stably transfected cells is obtained with the inclusion of 0.53 kb or additional upstream promoter sequences. Therefore, these sequences satisfy the requirements for binding of basal and enhancer transcription factors as well as interactions between them within a chromatin context. Both maximal basal expression and maximal vitamin D responsiveness are obtained with cells carrying either the 0.72-kb or the 1.1-kb promoter fragment. Cells carrying the 1.1-kb promoter show DNase I hypersensitivity at both the basal promoter and the vitamin D response element-containing domains, locations that also exhibit DNase I hypersensitivity in the endogenous OC promoter. In addition, we have documented changes in the basal activity and vitamin D responsiveness of the stably integrated 1.1 kb promoter as a function of cell density-mediated growth inhibition, which is accompanied by up-regulation of bone phenotypic genes. Thus, important aspects of OC gene transcriptional regulation that cannot be investigated in transient transfection assays can be addressed using ROS 17/2.8 cells stably transfected with OC promoter-reporter constructs.

Animals↗
PubMed1992

Cyclic adenosine 3',5'-monophosphate activation of the rat prolactin promoter is restricted to the pituitary-specific cell type.

Pituitary lactotroph cell function and PRL gene expression are highly regulated by the cAMP-protein kinase-A (PKA) pathway. To further our understanding of the molecular mechanisms by which cAMP/PKA regulates rat (r) PRL promoter activity and to determine whether cAMP regulation is cell type specific, we 1) transected intact (-425), internal and 5'-deletion, and site-specific mutants of the rPRL promoter ligated to the firefly luciferase reporter gene into both pituitary and nonpituitary cell lines; and 2) assessed the role of the cAMP-cAMP response element-binding protein (CREB) pathway in GH4 rat pituitary cells. The data show that deleting the rPRL promoter from -425 to -116 did not abolish cAMP regulation, implying that proximal elements, such as the basal transcription element (-112/-80) or the pituitary-specific footprint (FP) I (-67/-45), mediate the cAMP response. However, nucleotide changes within FP I or FP II (-130/-120) did not alter the rPRL promoter response to 1 microM forskolin (FSK), despite the 77% and 26% reductions in basal rPRL promoter activity caused by these mutations, respectively. Furthermore, internal deletion of either the basal transcription element of FP I element also failed to affect cAMP regulation of the rPRL promoter, again despite the 90% and 93% reductions in basal promoter activity by these deletions, respectively. Since these internal deletion constructs otherwise contain rPRL promoter sequences from -425 to +73, including the up-stream pituitary-specific FPs III and IV, the data suggest that any one of these cell-specific elements is capable of imparting cAMP regulation to the proximal rPRL promoter. To directly test the implication that the cAMP response of the rPRL promoter is restricted to the pituitary-specific cell type, we took advantage of a 5'-deletion mutant truncated at position -116 and a FP II site-specific mutant, since constructs containing these rPRL promoters are active in nonpituitary cells. Despite the 6.6- and 18.5-fold stimulations over wild-type rPRL promoter activity in nonpituitary cells, respectively, these mutations remained completely unresponsive to FSK treatment. To document that the cAMP-CREB pathway was functional in GC/GH4 rat pituitary cells, CREB was affinity purified from GC rat pituitary cells, and DNase-I protection studies showed that it does not bind to the proximal rPRL promoter. Also, the human glycoprotein alpha-subunit promoter was induced 10-fold by FSK in GH4 rat pituitary cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Cloning and characterization of the 4.2 kb region of the rat thyrotropin receptor promoter.

We previously identified an approximately 200 bp "minimal promoter" of the rat TSH receptor (TSHR) gene which is essential for the promoter activity. In the present study, we have cloned and characterized an upstream region of the TSHR promoter to disclose additional functional element(s). We screened a rat genomic library and obtained a DNA fragment which contained a 4.2 kb 5'-flanking region. This fragment was 2.5 kb longer than that we previously studied (1.7 kb). To assess the promoter activity, chimeric plasmids containing the 4.2 kb promoter and its 5'-deletions ligated to a chloramphenicol acetyltransferase gene were transfected into thyroid and non-thyroid cells. These plasmids expressed significant promoter activity in FRTL-5 and FRT thyroid cells, but not in BRL liver cells. The strongest promoter activity was expressed by the -199 bp promoter, and the longer promoter expressed rather decreased activity. Co-expression of thyroid transcription factor-1 (TTF-1) increased the activity of the promoter region from -3187 to -199 bp, which encompassed one or two TTF-1 binding sites we previously identified, but not the -4206 bp promoter. In addition, FRTL-5 stable transfectants each having a chimeric construct were cultured in the presence or absence of TSH. All transfectants expressed higher promoter activity in the absence of TSH than in the presence of TSH, in particular, the -3187 bp plasmid expressed significantly higher activity by comparison to the -2617 and -4206 bp constructs. This result indicates that the region between -3187 and -2617 bp may contribute to TSH/cAMP-induced suppression and also suggests that the region between -4206 and -3187 bp involves the element(s) for constitutive suppression of the promoter activity. These results not only suggest that the 4.2 kb upstream region of the TSHR gene possibly contains some elements for the regulation of the gene expression, but also emphasize the importance of the minimal promoter region which we previously identified for the efficient expression of the gene.

Animals↗
PubMed1999

Regulation of the laminin C1 promoter in cultured mesangial cells.

Expression of the genes encoding several matrix proteins, including the laminin gamma1 and beta1 subunits, is increased in glomeruli or renal cortex from diabetic animals or in mesangial cells cultured in high concentrations of glucose. Transforming growth factor (TGF)-beta1 and IGF-1 have been implicated as mediators of this response. In the present study, we assessed the influence of high glucose concentrations and the roles of TGF-beta1 and IGF-1 in the regulation of laminin C1 gene expression in cultured mesangial cells. Culture of normal rat mesangial cells (RMC) or SV40-transformed mouse mesangial (MES-13) cells in 500 mg/dl D-glucose for 2 days to 3 weeks significantly increased laminin C1 mRNA abundance compared with cells cultured in 100 mg/dl D-glucose. IGF-1 also increased laminin C1 mRNA abundance in RMC or MES-13 cells, whereas TGF-beta1 was without effect. The influence of raising the medium glucose concentration on laminin C1 promoter activity was further studied in MES-13 cells that had been stably transfected with a reporter gene containing the promoter linked to luciferase. Culture in 500 mg/dl D-glucose for 4 h to at least 1 week increased laminin C1 promoter activity compared with cells maintained in 100 mg/dl glucose. In contrast, culture of cells in medium that contained 400 mg/dl mannitol or 400 mg/dl L-glucose in addition to 100 mg/dl D-glucose did not increase laminin C1 promoter activity. The ability of high glucose to increase laminin C1 promoter activity was absolutely dependent on the presence of serum. Consistent with results obtained with mRNA, TGF-beta1 had no influence on promoter activity in stable integrants. Whereas IGF-1 transiently increased promoter activity in stable integrants, the increase was not sustained (6 h). Moreover, neutralizing antibody to TGF-beta or to IGF-1 receptor did not suppress increases in laminin C1 promoter activity induced by culture of stable integrants in high glucose. Several inhibitors of protein kinase C, including bisindolylmaleimide (GFX), myristoylated PKC inhibitor peptide, and LY333531, were also without effect on increases in laminin C1 promoter activity induced by culture in high glucose. Exposure to the NO donor (+/-)-s-nitroso-n-acetylpenicillamine (SNAP) blocked increases in laminin C1 promoter activity induced by serum and by culture in high glucose without influencing promoter activity in cells cultured in the absence of serum and in 100 mg/dl glucose. The ability of high glucose concentrations and IGF-1 to increase laminin C1 promoter activity in cultured mesangial cells, and the suppression of glucose actions by the NO donor SNAP, provide potential mechanisms whereby the synthesis of the laminin gamma1 chain may be regulated in the glomerulus in diabetes. Of note, the mechanism by which high glucose increases laminin C1 promoter activity appears to differ from mechanisms previously described for some other glucose actions on matrix protein synthesis. In this regard, TGF-beta and protein kinase C were not implicated as mediators of the effect of high glucose on laminin C1 promoter activity.

Animals↗
PubMed2003

Cloning and characterization of the promoters of temperate mycobacteriophage L1.

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the beta-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 min of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli sigma70-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early Pleft promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the Pleft equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the -10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus -10 and -35 hexamers of the E. coli sigma70 promoters.

Amino Acid Sequence↗
PubMed2001

Malignant breast epithelial cells stimulate aromatase expression via promoter II in human adipose fibroblasts: an epithelial-stromal interaction in breast tumors mediated by CCAAT/enhancer binding protein beta.

Expression of aromatase P450 (P450arom), which catalyzes the formation of estrogens, is aberrantly increased in adipose fibroblasts surrounding breast carcinomas, giving rise to proliferation of malignant cells. Aromatase in human adipose tissue is primarily expressed in undifferentiated fibroblasts under the control of several distinct and alternatively used P450arom promoters. In tumor-free breast adipose tissue, P450arom is usually expressed at low levels via a distal promoter (I.4), whereas in the breast adipose tissue bearing a tumor, P450arom is increased through the activation of two proximal promoters, II and I.3. Because the in vivo activation of P450arom promoter II is a key event responsible for aberrantly high P450arom expression in breast tumors, we studied the molecular basis for the enhancement of P450arom promoter II using human adipose fibroblasts (HAFs) in primary culture treated with T47D breast cancer cell-conditioned medium (TCM) as a model system. Upon treatment with TCM, HAFs displayed a striking induction of P450arom mRNA levels via promoter II usage. This effect appeared to be specific for malignant breast epithelial cells, because conditioned media from breast cancer cell lines T47D and MCF-7 induced promoter II activity, whereas normal breast epithelial cells or liver or prostate cancer cell lines did not produce such an effect. Although treatment with a cyclic AMP analogue also caused a switch in the promoter use from I.4 to II in cultured HAFs, TCM-induced promoter II use was found to be mediated via a cyclic AMP-independent pathway. Use of serial deletion mutants of the promoter II 5'-flanking sequence revealed the presence of critical cis-acting elements in the -517/-278 bp region, which regulate the baseline activity. TCM caused a 5.7-fold induction of the -517-bp promoter II construct, whereas site-directed mutagenesis of a CCAAT/enhancer binding protein (C/EBP) binding site (-317/-304 bp) abolished both baseline and TCM-induced activities. Ectopic expressions of C/EBPalpha and C/EBPbeta, but not C/EBPdelta, significantly induced promoter II activity. Moreover, we demonstrated the presence of both C/EBPbeta and C/EBPdelta but not C/EBPalpha in a DNA-protein complex formed by the nuclear extract from TCM-treated HAFs and a probe containing this critical C/EBP binding element (-317/-304 bp). Finally, treatment of HAFs with TCM strikingly induced C/EBPbeta expression, whereas this did not affect the levels of C/EBPalpha or C/EBPdelta transcripts. In conclusion, malignant breast epithelial cells secrete factors, which induce aromatase expression in adipose fibroblasts via promoter II. This is, at least in part, mediated by a TCM-induced up-regulation and enhanced binding of C/EBPbeta to a promoter II regulatory element.

Adipose Tissue↗
PubMed2001

[Differences in human and rat FSH receptors promote activity as a result of the transcriptional factors: E2F1, E2F4 and E2F5 overexpression].

UNLABELLED: FSH-R expression in granulosa cells varies during the course of ovarian ontogenesis, as well as, during each menstrual cycle. Expression of follicle-stimulating hormone receptors (FSH-R) on Sertoli cells of the testis and ovarian granulosa cells depend on many paracrine and autocrine factors. The modulation of FSH-R synthesis is accomplished via a number of mechanisms including regulation of the promoter activity. Little is known about factors involved in control of FSH-R transcription in different species. The aim of the present study was to investigate differences in the regulation of human and rat FSH-R promoter activity by E2F transcriptional factors. MATERIAL AND METHODS: The 5'-flanking regions of human and rat FSH receptor gene subcloned in the pGL3 plasmid were transiently transfected into cultured CHO cells and rat granulosa cells. Rat granulosa cells were obtained by puncturing ovaries from DES primed immature Sprague-Dawley rats. Promoter activity was determined by measuring firefly luciferase luminescence of the cell lysate. Transfection efficiency was normalized by the renilla luciferase activity generated by co-transfected pRL-CMV vector. In order to determine the influence of E2F1, E2F4 and E2F5, on FSH-R promoter activity, cells were transfected either with promoter construct alone or with its mixture with selected expression vector. RESULTS: Rat FSH-R promoter construct (-1033/+6 bp) and human FSH-R promoter construct (-1485/-1 bp) were both active in transfected cells. Overexpression of E2F1 protein decreased both, human and rat wild type FSH-R promoter activity. Overexpression of E2F4 did not affect neither rat nor human FSH-R gene transcription. Expression vector for E2F5 increased both, human and rat, FSH-R promoter activity. Folds of increase were markedly higher in case of rat FSH-R construct transfection, comparing to human FSH-R promoter. CONCLUSIONS: Results suggest, that the E2F1 and E2F5 factors might play an opposite role in the regulation of FSH-R promoter activity. More pronounced stimulatory effect of E2F5 on the rat FSH-R can be explained by the presence of E2F site in the promoter. Since there is no E2F sensitive element in the human FSH-R promoter sequence, E2F1 and E2F5 can also indirectly influence FSH-R promoter activity.

Animals↗
PubMed2002

Evidence-based health promotion: applying it in practice.

INTRODUCTION: In health promotion, we should use interventions established by evidence to be effective in improving the health of the community. This paper reviews the concepts, evaluation and use of evidence in health promotion. METHODS: A literature search of evidence-based health promotion and evaluation of health promotion was conducted using Medline, Social Science Citation Index (SSCI), PsycLIT and evidence-based web sites on health promotion, health education and community preventive services. Recent issues of key journals on health promotion, health education and public health were also hand-searched. RESULTS: The concept of evidence in health promotion interventions is complex due to its multidimensional nature. Evidence of effectiveness in health promotion is assessed by combining quantitative data on effect change in outcome measures and qualitative data on process evaluation of health promotion activities. Limitations to the use of randomised trials in community-based health promotion interventions include ethical and logistic problems in maintaining randomisation of subjects over long periods, absence of experimental conditions in the real-world setting, contamination of control subjects and the multidimensional nature of health promotion interventions. CONCLUSION: Randomised controlled trials should be used to evaluate the effectiveness of most health education and behavioural interventions in clinical settings. When such trials are not feasible as in community-based health promotion interventions, quasi-experimental designs provide strong evidence. Multiple methods are needed to assess evidence of effectiveness of health promotion programmes. Appropriate practice of evidence-based health promotion requires consideration of quality of available evidence, local values and prevailing resources.

Evidence-Based Medicine↗
PubMed1992

Human IL-1 receptor antagonist promoter. Cell type-specific activity and identification of regulatory regions.

To study the molecular mechanisms involved in transcriptional regulation of the human IL-1R antagonist (IL-1ra) we have isolated 1680-bp of 5'-flanking region DNA from the IL-1ra gene. This region of DNA was sequenced and cloned into the luciferase expression vector pA3Luc (pRA-1680.Luc) for use in gene transfer studies aimed at determining the cis-acting DNA elements required for IL-1ra expression. Sequence analysis of the IL-1ra promoter revealed a TATAA box at -26, with consensus sequences for possible NF-kB-, NFIL-1 beta A-, AP-1-, and CRE-binding sites located further upstream. When transfected into a variety of human and murine cell lines, the cloned IL-1ra promoter was preferentially active in those cell lines in which expression of the endogenous IL-1ra gene could be detected. The cloned promoter and the endogenous IL-1ra promoter utilized the same transcriptional start site. This promoter activity was LPS-inducible in the RAW 264.7 murine macrophage cell line. In the human monocytic cell line U937, IL-1ra promoter activity was inducible by LPS or PMA treatment, but the combination of LPS and PMA led to the greatest increase in promoter activity, identical to the pattern of expression of endogenous IL-1ra mRNA as detected by polymerase chain reaction analysis. A series of 5'-truncated promoter constructs having a common 3'-end at +27 were created to map potential cis-acting transcriptional elements important for full IL-1ra promoter activity. Removal of sequences between -294 and -148 led to a greater than 90% decrease in both unstimulated and LPS-induced promoter activity; further deletion to -85 led to an almost complete abrogation of promoter activity. These studies demonstrate that the cloned IL-1ra promoter behaves in a manner consistent with that of the endogenous gene. Two regions within the IL-1ra promoter are identified which are required for full promoter activity.

Animals↗
PubMed2003

[Health promotion benefits and obstacles as perceived by occupational medicine physicians in Poland].

This article presents the views of occupational medicine (OM) physicians concerning the following: (a) whether OM physicians should be involved in workplace health promotion: (b) whether they can benefit (if so in what way) from involvement in this activity; (c) whether they find the existing conditions favorable for launching health promotion projects; (d) what are the major obstacles? This paper is based on a survey carried out in the fall of 2002 in a group of 325 OM specialists who attend large groups of patients. Over 90% of the respondents believe that OM physicians should be involved in health promotion, however, one third of them do not plan any action in this field in the following year. Among the key benefits from health promotion, health gain is indicated by 80% of OM physicians. Only a few respondents perceive their involvement in health promotion as an opportunity to be more competitive in health service market, thereby to increase their income. None of those interviewed thought that involvement in health promotion win them greater respect among peers. Almost 80% of the respondents complained about unfavorable conditions for carrying out workplace health promotion. As the most significant obstacles they reported the lack of interest in health promotion among employers (86% of respondents), unsatisfactory gratification (76%), limited skill to raise funds for health promotion (64%), limited opportunities to advance education in this field (59%), and the lack of interest in health promotion among employees (57%). In general, OM physicians express their willingness to advance their knowledge of health promotion, although 64% of respondents are convinced that they are well trained in this area. According to the respondents, a proper way to promote health is not only to become very active, but to commit employers to assure the development of health promotion at workplace and to allocate more funds for health promotion projects.

Attitude of Health Personnel↗
PubMed2004

Limited specificity of promoter constructs for gene therapy in osteosarcoma.

Osteosarcoma (OS), a malignant bone neoplasia in childhood, has poor prognosis if metastases appear in the lung. A novel therapeutic approach could consist in a gene therapeutic treatment of OS metastases. However, if promiscuous viral vectors are applied for the delivery of potentially toxic transgenes, their misdelivery into normal tissues could cause severe complications. This problem could be circumvented by application of OS-specific promoters for transgene expression control. We analysed the function of promoters described to be tumour-, osteosarcoma- or osteoblast-specific. Expression rates driven by osteoblast- specific fragments from the collagen1A1-promoter, the human Osteocalcin-promoter, the bone-sialoprotein promoter and the beta-catenin promoter depending on vitamin supplementation were analysed in five OS cell lines, in normal lung fibroblasts and in a non-osteoblastic prostate cancer cell line (LNCaP) by dual luciferase assays. In addition, an unspecific but doxycyclin-repressible promoter construct (pAd.3r-luc) was examined. We found that all constructs were active in OS cell lines to varying extents. The complete human Osteocalcin promoter and the bone-sialoprotein promoter were partially induced by vitamin D3 or C respectively while the pAd.3r-luc activity could be shut down by doxycyclin. In contrast, the human Osteocalcin-promoter was not activated by vitamin D3 in LNCaP cells; its action remained relatively low. Interestingly, excepting the beta-catenin promoter, we measured strong activities of all promoters in lung fibroblast cells. Our study demonstrates that promoter activity should be evaluated not only for the target cells of the gene therapeutic approaches, but also for neighbouring normal tissues. Unspecific but repressible promoters could represent an alternative.

Cell Line, Tumor↗
PubMed1990

Characterization of transcriptional initiation from promoters P1 and P2 of the pyrBI operon of Escherichia coli K12.

Expression of the pyrBI operon of Escherichia coli K12, which encodes the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated by pyrimidine availability, primarily through an attenuation control mechanism, and also appears to be subject to stringent control. Previous in vitro transcription studies indicated that the pyrBI operon is transcribed from tandem promoters designated P1 and P2, which appeared to be of similar strength. In this study, we characterized these promoters in detail and examined their role in pyrBI expression. Our results show that although transcription is initiated at both promoters in vivo, greater than 99% of the pyrBI transcripts are initiated at promoter P2, indicating that this is the only physiologically significant promoter. The level of transcripts initiated at promoter P2 was found to be higher in cells grown under pyrimidine-limiting conditions compared to that in cells grown under conditions of pyrimidine excess, indicating pyrimidine-mediated regulation at the level of transcriptional initiation. In vitro characterization of transcription from the pyrBI promoter region showed that non-physiological reaction conditions used in the original identification of the two promoters greatly overestimated the strength of promoter P1. Further in vitro characterization of the two promoters showed that transcription from promoter P2, but not from promoter P1, is inhibited by guanosine tetraphosphate and exhibits salt and heparin sensitivity typical of a stringently controlled promoter. In addition, heparin-challenge experiments revealed a UTP-induced instability of transcriptional initiation complexes at promoter P2 which may be of regulatory significance.

Amino Acid Sequence↗
PubMed1990

Structure and autoregulation of the c-rel promoter.

Precise regulation of proto-oncogene expression appears to be essential for the proper growth and development of multi-cellular organisms. One aspect of this regulation is at the level of transcription from the proto-oncogene promoter(s). In order to characterize the promoter for the chicken c-rel proto-oncogene, we have isolated and sequenced genomic DNA containing the first exon of the chicken c-rel proto-oncogene. The c-rel promoter is structurally similar to the promoters of the so-called house-keeping genes, lacking the CAATT and TATA elements found in some cellular genes, but containing a G/C-rich box near the transcription start sites. There are multiple transcription start sites for the c-rel mRNA, which map near putative binding sites for the transcription factors HIP-1 and NF-kB. The c-rel promoter was functionally characterized by its ability to support expression of the firefly luciferase gene. The c-rel promoter is a relatively weak promoter, 100-times less active than the promoter within the spleen necrosis virus long terminal repeat (LTR). We have defined the c-rel promoter by analysis of a series of 5' deletion mutants of the c-rel promoter fused to the firefly luciferase gene. A DNA fragment containing 97 bp of 5' flanking sequence and 72 bp of 3' flanking sequence is sufficient for c-rel promoter function. Co-transfection of the c-rel promoter with a retroviral vector expressing the c-rel protein resulted in a decrease in expression from the minimal c-rel promoter. These results indicate that expression of the c-rel proto-oncogene is tightly regulated both at the level of basal promoter activity and by autoregulation of transcription by the c-rel protein.

Adenine↗
PubMed1995

Identification and characterization of the human myeloperoxidase promoter.

Myeloperoxidase (MPO) is a microbicidal protein present in the primary granules of myeloid cells. Transcription of the MPO gene is turned on only during the late myeloblast and promyelocyte stages of myeloid maturation. Identification of cis-regulatory elements and transcription factors which regulate the MPO gene should, therefore, shed light on myeloid maturation. We report transfection and in vitro transcription experiments which demonstrate promoter activity in the proximal 5'-flanking region of the human MPO gene. Using a chloramphenicol acetyl transferase (CAT) reporter vector system, and segments of the 5'-flanking MPO DNA, we constructed a series of MPO promoter-CAT expression vectors. By electroporation and lipofectin-mediated transient transfection assays, as well as by in vitro transcription studies, a 594-bp MPO DNA sequence (bp -583 to +11) showed promoter activity in a variety of MPO-expressing and non-MPO-expressing cell lines. Compared with the SV40 early promoter, the MPO promoter had greater relative activity in MPO-expressing than in non-MPO-expressing cell lines, suggesting slight tissue specificity. However, a CAT reporter plasmid containing 1099-bp of 5'-flanking MPO DNA showed greater specificity for MPO expressing cell lines. Analysis of a group of promoter deletion mutants showed that the minimal promoter was contained in a DNA fragment extending from bp-128 to +11. The remainder of the promoter region contained several segments which appeared to enhance the activity of the minimal promoter. One such enhancer sequence was homologous to an enhancer previously described in the human elastase promoter. Activity of the 594-bp MPO promoter in HL-60 was reduced by only approximately 30% following treatment of the cells with chemical inducers of maturation, but the 1099-bp MPO promoter showed 60% reduction in activity after DMSO treatment. A previously described enhancer region in intron 9 of the MPO gene had little or no effect on activity of the 594-bp MPO promoter. The availability of the MPO promoter will facilitate determination of other factors involved in the regulation of this myeloid-specific gene.

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