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PubMed1996

Transcriptional regulation of sertoli cell differentiation by follicle-stimulating hormone at the level of the c-fos and transferrin promoters.

One of the primary endocrine hormones that influence the onset of Sertoli cell differentiation at puberty and help maintain differentiation in the adult testis is FSH. FSH can modulate the majority of Sertoli cell differentiated functions, including stimulation of the iron-binding protein transferrin. Previous studies have shown that FSH alters the levels of cAMP and the immediate early gene c-fos. The current study was designed to investigate the transcriptional regulation of Sertoli cell differentiation by examining the actions of FSH on the promoter of the immediate early gene c-fos and the promoter of the downstream differentiated function gene transferrin. The regulation of c-fos by FSH was investigated with various chloramphenicol acetyltransferase (CAT) constructs containing segments of the c-fos promoter, such as the serum response element (SRE), cAMP response element (CRE), and AP1/phorbol ester/TPA response element (TRE), that were transfected into cultured Sertoli cells. Observations indicate that FSH can stimulate all three response elements, as well as a whole c-fos promoter construct. Interestingly, FSH was found to have a more dramatic effect on the SRE-CAT than a cAMP analog, suggesting a difference in the actions of the two agents. Gel mobility shift assays were performed to confirm the reporter gene results. Nuclear extracts of FSH-stimulated Sertoli cells caused a labeled AP1 oligonucleotide to form a DNA/protein complex (i.e., gel shift), indicating activation of the c-fos gene and binding of the c-fos/jun complex. Nuclear extracts from both FSH- and cAMP-stimulated Sertoli cells promoted similar gel shifts with SRE and CRE oligonucleotides. This observation supports the reporter gene data in indicating that FSH can influence both the SRE and CRE. A gel mobility shift assay was also performed with an oligonucleotide containing the 5'-flanking ETS domain of the SRE (ETS-SRE) that allows the formation of a ternary complex. FSH-stimulated Sertoli cell nuclear extracts were found to promote a unique ETS-SRE gel shift not present in cAMP-stimulated cells. The observations imply that FSH actions on the SRE are in part distinct from the actions of cAMP. Transferrin gene expression was examined to study the downstream regulation of Sertoli cell differentiation. CAT constructs containing deletion mutants of a 3-kb mouse transferrin promoter were used. When transfected into Sertoli cells, the 581-bp transferrin minimal promoter, previously shown to contain a CRE, had a significant response to cAMP and FSH. The 1.6-, 2.6-, and 3-kg transferrin promoter constructs also responded to FSH and cAMP to the same extent as, or to a lesser extent than, the 581-bp minimal promoter. Interestingly, the actions of FSH on the 581-bp minimal transferrin promoter were more dramatic than those of cAMP. The importance of FSH-induced c-fos in the regulation of transferrin expression was demonstrated in the current study when a c-fos antisense oligonucleotide was found to partially inhibit (50%) the ability of FSH to induce the expression of a transferrin promoter (CAT) construct. Therefore, FSH appears to act through multiple transcriptional activation pathways. The first involves cAMP and the CRE at both early-event genes (e.g., c-fos) and downstream genes (e.g., transferrin). It is likely that other pathways involve alternate signal transduction events (e.g., calcium mobilization) and promoter response elements (e.g., SRE). These multiple pathways may act in a compensatory manner to assure the ability of FSH to influence Sertoli cell differentiation and/or in a synergistic manner to amplify FSH actions.

Animals↗
PubMed2000

Critical nucleotides in the interaction of CatR with the pheBA promoter: conservation of the CatR-mediated regulation mechanisms between the pheBA and catBCA operons.

The promoter of the plasmid-borne pheBA genes encoding enzymes for phenol degradation resembles the catBCA promoter and is activated by CatR, the regulator of the chromosomally encoded catechol-degradative catBCA genes in Pseudomonas putida. In this study, site-directed mutagenesis of the pheBA promoter region was performed. The interrupted inverted repeat sequence of the CatR recognition binding site (RBS) of the pheBA promoter is highly homologous to that of the catBCA promoter. However, the RBS was shown not to be the sole important feature for high-affinity binding of CatR to this site. Mutagenesis of the activation binding site (ABS) of CatR, which overlaps the -35 hexamer sequence TTGGAT of the promoter, revealed that the two G nucleotides in this sequence are important for promoter activity but not for CatR binding. All other substitutions made in the ABS negatively affected both the promoter activity and CatR binding. The spacer sequence of the pheBA and catBCA promoters between the -10 and -35 hexamers is 19 bp, which is longer than optimal. However, reducing the spacer region of the pheBA promoter was not sufficient for CatR-independent promoter activation. An internal binding site (IBS) for CatR is located downstream of the transcriptional start site of the catBCA genes and it negatively regulates the operon. A similar IBS was identified in the case of the pheBA operon and tested for its functionality. The results indicate a conservation of CatR-mediated regulation mechanisms between the pheBA promoter and the catBCA promoter. This universal mechanism of CatR-mediated transcriptional activation could be of great importance in enabling catechol-degrading bacteria to expand their substrate range via horizontal transfer of the phenol degradative genes.

Bacterial Proteins↗
PubMed2005

In vivo analyses of constitutive and regulated promoters in halophilic archaea.

The two gvpA promoters P(cA) and P(pA) of Halobacterium salinarum, and the P(mcA) promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with beta-galactosidase activity, as reporter. For comparison, the P(fdx) promoter of the ferredoxin gene of Hbt. salinarum and the P(bgaH) promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed. P(fdx), driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas P(bgaH) and the three gvpA promoters yielded the largest activities during the stationary growth phase. Compared to P(fdx), the basal promoter activities of P(pA) and P(mcA) were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The P(cA) promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the P(cA)-TATA box and the BRE element were the reason for the lack of the basal P(cA) activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger P(pA) promoter and investigated in Hfx. volcanii transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the P(pA)-BRE element was substituted for the P(cA)-BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring P(pA)-BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of P(mcA)-BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of P(mcA).

Archaeal Proteins↗
PubMed2006

Promoter prediction in the rhizobia.

The ability to recognize and predict non-sigma54 promoters in the alphaproteobacteria is not well developed. In this study, 25 experimentally verified Sinorhizobium meliloti promoter sequences were compiled and used to predict the location of other related promoters in the S. meliloti genome. Fourteen candidate predictions were targeted for verification and of these at least 12 proved to be genuine promoters. As a result, the experimental identification of 12 novel promoters linked to genes rpoD, topA, rpmJ, trpS, ropB1, metC, rpsT, secE, trkH and three tRNA genes is reported. In all, 99 predicted and verified promoters are reported, including those linked with 13 tRNA genes, eight ribosomal protein genes and a number of other physiologically important or essential genes. On the basis of sequence conservation and a mutational analysis of promoter activity, the -35 and -10 consensus for these promoters is 5-CTTGAC-N17-CTATAT. This promoter structure, which seems to be widely conserved amongst several other genera in the alphaproteobacteria, shares significant similarity with, but is skewed by a 1 nt step from, the canonical Escherichia coli sigma70 promoter. Perhaps this difference is responsible for the observation that S. meliloti promoters are often poorly expressed in E. coli. In this regard, expression data from plasmid-borne gfp-reporter fusions to eight of the S. meliloti promoters verified in this work revealed that while these promoters were very active in S. meliloti and Agrobacterium tumefaciens only very low, near-background activity was detected in E. coli.

Bacterial Proteins↗
PubMed1995

Differential DNA bending introduced by the Pseudomonas putida LysR-type regulator, CatR, at the plasmid-borne pheBA and chromosomal catBC promoters.

The plasmid-borne pheBA operon of Pseudomonas putida strain PaW85 allows growth of the host cells on phenol. The promoter of this operon is activated by the chromosomally encoded LysR-type regulator CatR, in the presence of the inducer cis,cis-muconate. cis,cis-muconate is an intermediate of catechol degradation by the chromosomally encoded ortho or beta-ketoadipate pathway. The catBC operon encodes two enzymes of the beta-ketoadipate pathway and also requires CatR and cis,cis-muconate for its expression. The promoters of the pheBA and catBC operons are highly homologous, and since both respond to CatR, it is likely that the pheBA promoter was recruited from the ancestral catBC promoter. Gel shift assays and DNase I footprinting have shown that the pheBA promoter has a higher binding affinity for CatR than the catBC promoter. Like the catBC promoter, the pheBA promoter forms two complexes (C1 and C2) with CatR in the absence of cis,cis-muconate, but only forms a single complex (C2) in the presence of cis,cis-muconate. Like the catBC promoter CatR repression binding site (RBS) and activation binding site (ABS) arrangement, the pheBA promoter demonstrates the presence of a 26 bp segment highly homologous to the RBS that is protected by CatR from DNase I digestion in the absence of the inducer. An additional 16 bp sequence, similar to the catBC promoter ABS, is protected only when the inducer cis-cis-muconate is present. The binding of CatR in absence of cis,cis-muconate bends the catBC and pheBA promoter regions to significantly different degrees, but CatR binding in the presence of cis,cis-muconate results in a similar degree of DNA bending. The evolutionary implications of the interactions of CatR with these two promoters are discussed.

Adipates↗
PubMed1990

Characterisation of tissue-specific trans-acting factor binding to a proximal element in the rat growth hormone gene promoter.

Using an exonuclease III protection assay, tissue-specific binding of rat pituitary tumour cell (GH3 cell) nuclear factors to a proximal region (-68 to -138) of the rat growth hormone gene promoter has been detected. The binding is particularly strong between the borders -68 to -102. The binding is eliminated in the presence of excess unlabelled rat growth hormone gene promoter sequences but also by proximal (-423 to +38) or distal (-1960 to -1260) rat prolactin gene promoter sequences and simian virus 40 enhancer/promoter sequences. Extracts of rat pituitaries showed identical binding characteristics. Methylation interference analysis indicated that the contact points between the pituitary-specific factor and the proximal rat growth hormone gene promoter-binding element (-65 to -95) are over a conserved sequence which occurs twice in the rat growth hormone gene promoter and at least eight times in the rat prolactin gene 5'-flanking sequences. This sequence has previously been proposed to constitute the binding site for the somatotroph/lactotroph tissue-specific transcription factor. Gel-retardation and exonuclease III competition analysis showed that three of the rat prolactin gene promoter elements (-46 to -71, -156 to -180 and -174 to -204) share the ability to bind the pituitary-specific factor. The binding to the most proximal rat prolactin gene promoter element (-46 to -71) was clearly more avid than to the rat growth hormone gene promoter (-65 to -95) proximal element. However, both these elements displayed the formation of two gel-retarded complexes while the more distal rat prolactin gene binding elements (-156 to -180 and -174 to -204) formed only the smaller of the two complexes. Finally, we demonstrated by co-transfection competition analysis that plasmids containing the most proximal rat prolactin gene promoter binding element completely inhibited transcription from the rat growth hormone gene promoter while rat growth hormone gene promoter sequences only partially inhibited transcription from the rat prolactin gene promoter. This suggests that the higher affinity for factor binding displayed by the proximal rat prolactin gene promoter binding site in vitro is reflected in factor binding activity in vivo.

Animals↗
PubMed2006

Health promotion in the practice setting: findings from a review of clinical issues.

BACKGROUND: Many agencies over the last few decades have called for the explicit inclusion and application of health promotion in all aspects of nursing practice. This article reviews the body of empirical (research-based) and theoretical (conceptual) literature addressing health promotion in nursing clinical practice, to uncover its nature, intention, and impact. APPROACH: The aim of this review was to identify from the nursing literature what is reported on in relation to health promotion activity, both theoretical and clinical in the practice setting. An extensive review identified that literature pertaining to these issues clustered around the categories of: theorising and conceptualising the meaning and purpose of health promotion in nursing practice, the development and testing of tools and models for practice, and the exploration of attitudes, behaviours, roles and functions of nurses and health professionals in practice. FINDINGS: Against more recent trends in health promotion, the literature shows that broader and wide-ranging health promotion reform has yet to have universal impact, whereas conventional health education activities remain the mainstay for the majority of "health promotion" activities in nursing practice. Where nurses purport to be conducting "health promotion" they are often conducting behavioural, lifestyle, and risk-orientated health education. The findings of this review therefore support the notion that much of nursing-related health practice is firmly located in reactive ill health and disease risk-specific preventive locations, instead of incorporating and instilling broader societal, economic, ecological, and political dimensions of health promotion. Recommendations for wider reform, developed from personal opinion and supported by the literature, are presented. CONCLUSIONS: It is argued that nursing bodies, as well as individual nurses themselves, should seek current health promotion locations in nursing practice, and find effective ways to implement health promotion strategies and, subsequently, equip practitioners with the necessary resources and skills to engage with other health promotion agencies. Future research should be aimed at implementing and evaluating broad health promotion processes.

Clinical Competence↗
PubMed2000

In vitro and in vivo analyses of constitutive and in vivo-induced promoters in attenuated vaccine and vector strains of Vibrio cholerae.

The optimal promoter for in vivo expression of heterologous antigens by live, attenuated vaccine vector strains of Vibrio cholerae is unclear; in vitro analyses of promoter activity may not accurately predict expression of antigens in vivo. We therefore introduced plasmids expressing the B subunit of cholera toxin (CtxB) under the control of a number of promoters into V. cholerae vaccine strain Peru2. We evaluated the tac promoter, which is constitutively expressed in V. cholerae, as well as the in vivo-induced V. cholerae heat shock htpG promoter and the in vivo-induced V. cholerae iron-regulated irgA promoter. The functionality of all promoters was confirmed in vitro. In vitro antigenic expression was highest in vaccine strains expressing CtxB under the control of the tac promoter (2 to 5 microgram/ml/unit of optical density at 600 nm [OD(600)]) and, under low-iron conditions, in strains containing the irgA promoter (5 microgram/ml/OD(600)). We orally inoculated mice with the various vaccine strains and used anti-CtxB immune responses as a marker for in vivo expression of CtxB. The vaccine strain expressing CtxB under the control of the tac promoter elicited the most prominent specific anti-CtxB responses in vivo (serum immunoglobulin G [IgG], P </= 0.05; serum IgA, P </= 0.05; stool IgA, P </= 0.05; bile IgA, P </= 0.05), despite the finding that the tac and irgA promoters expressed equivalent amounts of CtxB in vitro. Vibriocidal antibody titers were equivalent in all groups of animals. Our results indicate that in vitro assessment of antigen expression by vaccine and vector strains of V. cholerae may correlate poorly with immune responses in vivo and that of the promoters examined, the tac promoter may be best suited for expression from plasmids of at least certain heterologous antigens in such strains.

Animals↗
PubMed1995

Activation of transcription initiation from the nac promoter of Klebsiella aerogenes.

The nac gene of Klebsiella aerogenes encodes a bifunctional transcription factor that activates or represses the expression of several operons under conditions of nitrogen limitation. In experiments with purified components, transcription from the nac promoter was initiated by sigma 54 RNA polymerase and was activated by the phosphorylated form of nitrogen regulator I (NRI) (NtrC). The activation of the nac promoter required a higher concentration of NRI approximately P than did the activation of the Escherichia coli glnAp2 promoter, and both the promoter and upstream enhancer element contributed to this difference. The nac promoter had a lower affinity for sigma 54 RNA polymerase than did glnAp2, and uninitiated competitor-resistant transcription complexes formed at the nac promoter decayed to competitor-sensitive complexes at a greater rate than did similar complexes formed at the glnAp2 promoter. The nac enhancer, consisting of a single high-affinity NRI-binding site and an adjacent site with low affinity for NRI, was less efficient in stimulating transcription than was the glnA enhancer, which consists of two adjacent high-affinity NRI-binding sites. When these binding sites were exchanged, transcription from the nac promoter was increased and transcription from the glnAp2 promoter was decreased at low concentrations of NRI approximately P. Another indication of the difference in the efficiency of these enhancers is that although activation of a nac promoter construct containing the glnA enhancer was relatively insensitive to subtle alterations in the position of these sites relative to the position of the promoter, activation of the natural nac promoter or a nac promoter construct containing only a single high-affinity NRI approximately P binding site was strongly affected by subtle alterations in the position of the NRI approximately P binding site(s), indicating a face-of-the-helix dependency for activation.

Bacterial Proteins↗
PubMed2001

Regulatory architecture of the iron-regulated fepD-ybdA bidirectional promoter region in Escherichia coli.

The overlapping and opposing promoter elements for the Escherichia coli fepDGC operon and the ybdA gene (encoding a 43-kDa cytoplasmic membrane protein) within the enterobactin gene cluster were investigated by measuring the effects of site-specific mutations on transcript levels and on expression of reporter genes in a bidirectional transcriptional fusion vector. Primary promoter structures for the opposing transcripts overlapped extensively such that their -10 sequences were almost directly opposed on the two strands of the DNA helix and their +1 transcription start sites were only 23 bp apart. Relative to the E. coli consensus sequence, both promoters were poorly conserved at the -35 position and mutations which strengthened the -35 element of either promoter significantly enhanced its transcription, decreased that of the opposing promoter, and dramatically altered iron-mediated regulation of expression. Both the fepD and ybdA primary promoters were shown to require a 5'-TGn-3' upstream extension of their -10 elements for optimal activities. Secondary promoters were identified for both fepD and ybdA, and their contributions to the overall expression levels were evaluated in these dual expression vector constructs. The data provided strong evidence that the architecture of the regulatory elements within the overlapping fepD and ybdA promoters is configured such that there is a direct competition for binding RNA polymerase and that the expression levels at these promoters are influenced not only by the activity of the opposing promoters but also by additional promoter sequence elements and perhaps accessory regulatory factors. Iron-mediated regulation of these promoters through the repressor protein Fur is a consequence of the relative promoter strengths and the position of an operator site that consists of two overlapping Fur-binding sequences in this compact regulatory region.

Bacterial Proteins↗
PubMed1990

Functional elements of the steroid hormone-responsive promoter of mouse mammary tumor virus.

Transcription from the promoter of mouse mammary tumor virus is subject to induction by several classes of steroid hormones as well as to repression by a negative regulatory element present in the long terminal repeats of proviral DNA. In order to characterize the functional elements of the promoter that in some way must respond to these regulatory signals, a number of promoter mutations were constructed, including a set of linker-scanning mutations across the entire promoter region. Analysis of these mutated promoters with a transient-transfection assay defined at least three mutation-sensitive promoter elements that are required for both basal and hormone-induced transcription. One mutation-sensitive region contains a TATA element located at approximately position -30 with respect to the start of transcription. A second mutation-sensitive region contains two 10-base-pair direct repeats located between positions -60 and -38, within which are embedded three copies of octamer-related sequences; complete disruption of this region of the promoter leads to a more severe decrease in transcription than do any of the linker-scanning mutations, suggesting that the repeated sequences may be at least partially functionally redundant. Gel electrophoresis mobility shift assays were used to demonstrate specific binding of a nuclear protein to this region of the promoter. A third mutation-sensitive region contains a binding site for nuclear factor 1 (NF-1) located between positions -77 and -63. Site-directed mutations in the NF-1-binding site which increase the apparent affinity of NF-1 for the promoter in vitro do not decrease the hormone dependence of transcription, suggesting that transcriptional activation mediated by steroid hormone-receptor complexes cannot be explained by facilitation or stabilization of the interaction of promoter sequences with NF-1 and consistent with the idea that binding of NF-1 is not rate determining in transcription from the mouse mammary tumor virus promoter. None of the promoter mutations functionally separates basal from glucocorticoid-induced transcription, suggesting that hormone induction does not make the promoter independent of any of the DNA-binding factors required for its basal activity.

Animals↗
PubMed1992

Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells.

Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA.

Activating Transcription Factors↗
PubMed1992

The Epstein-Barr virus immediate-early promoter BRLF1 can be activated by the cellular Sp1 transcription factor.

Disruption of viral latency in Epstein-Barr virus-infected cells is mediated through the activation of the BZLF1 (Z) immediate-early gene product. The Z protein can be derived from either of two promoters: the BZLF1 promoter, which directs transcription of a 1.0-kb mRNA encoding the Z gene product alone, or the upstream BRLF1 promoter, which directs transcription of a 2.8-kb bicistronic mRNA encoding the BRLF1 and BZLF1 immediate-early proteins. In this study we have examined the regulation of the BRLF1 promoter by viral and cellular factors. We found that the BRLF1 promoter is autoregulated by the BRLF1 transactivator through a nonbinding mechanism. We show that the BRLF1 (but not the BZLF1) promoter is highly responsive to the Sp1 transcription factor. Sp1 activation of the BRLF1 promoter is mediated through a consensus Sp1-binding site located from -39 to -44 (relative to the mRNA start site). We demonstrate that the BRLF1 promoter has high constitutive activity in C-33 cells (an epithelial cell line) and that the proximal Sp1-binding site is required for this activity. Despite the ubiquitous presence of Sp1 in many cell types, we found that the BRLF1 promoter has essentially no activity in lymphoid cell lines, suggesting that factors other than Sp1 may negatively regulate the BRLF1 promoter in these cells. Our findings demonstrate that the two potential promoters directing BZLF1 transcription are differentially regulated and that Sp1 can activate the BRLF1 promoter but not the BZLF1 promoter.

Animals↗
PubMed1992

Effect of genomic location on expression of beta-galactosidase mRNA controlled by the herpes simplex virus type 1 UL38 promoter.

To examine the effect of genomic location on the details of expression of selected herpes simplex virus promoters, we have constructed recombination vectors for placing such promoters controlling the beta-galactosidase reporter gene into two regions of the viral genome lacking any nearby promoter or regulatory elements. The first vector generates the promoter-beta-galactosidase reporter gene inverted within the locus of the gC (UL44) translational reading frame; the second replaces the LAT promoter and the first 600 bases of the primary transcript in both copies of the RL region. These locations were chosen to obviate any possible influence of upstream but noncontiguous heterologous or homologous DNA sequence elements upon promoter activity. When the reporter gene controlled by the strict late (gamma) UL38 promoter was placed in the gC location, it was significantly less active than in its normal location; in contrast, promoter activity was comparable to wild-type values when the promoter was recombined into the RL region. The low level of activity in the gC location could be partially alleviated by the incorporation of additional DNA sequences upstream of the UL38 promoter. Despite the effect of genomic location upon the level of expression, the kinetics of expression in either location mirrors the wild-type UL38 strict late kinetics of expression. Finally, we used deletional analysis to demonstrate that no more than 29 bases of DNA sequence 5' of the mRNA cap site are required for promoter activity in either location; this result is consistent with earlier results of transient-expression assays and indicates that the UL38 promoter shares general features with other strict late (gamma) herpes simplex virus promoters.

Animals↗
PubMed1993

cis-acting sequences in the Aleutian mink disease parvovirus late promoter important for transcription: comparison to the canine parvovirus and minute virus of mice.

We are currently investigating the regulation of transcription of the Aleutian mink disease parvovirus (ADV). ADV causes a chronic immune complex-mediated condition known as classical Aleutian disease, characterized by slow viral replication. This slow replication is an intrinsic property of ADV and distinguishes it from the more prototypic parvoviruses such as minute virus of mice (MVM) and canine parvovirus (CPV). We have previously suggested a role for the weak ADV promoters in the slow replication and thereby the absence of acute cytopathology and instead establishment of persistent ADV infection with progressive immune complex-mediated chronic lesions. In this study, we have mapped the cis-acting sequences around the ADV P36 promoter responsible for both constitutive transcription and transactivation mediated by the nonstructural protein 1. The mapping was performed by using endpoint deletions of the ADV P36 promoter and by making chimeras between the ADV P36 and MVM P38 promoters. We found the weak constitutive activity of the ADV P36 promoter to be caused by suboptimal promoter proximal sequences, while the low level of transactivation was caused mainly by an upstream region including sequences with homology to the transactivation responsive element (tar) of the H-1 parvovirus (M.-L. Gu, F.-X. Chen, and S. L. Rhode, Virology 187:10-17, 1992). We also found the corresponding regions in the MVM and CPV P38 promoters to be important for transactivation of these promoters by making 5' deletions of the promoter region. In addition, it was found that MVM tar-like and upstream sequences could transfer high nonstructural protein 1 responsiveness to the ADV promoter even though the distance between the tar-like element and the TATA box was significantly changed. On the basis of comparative data for ADV, MVM, CPV, and H-1, a new clustered motif (TTGGTT) is proposed to be the responsive cis-acting element for transactivation. Homology comparison of the specific transcriptional elements of the ADV P36, MVM P38, and CPV P38 promoters suggests that few, but crucial, changes in the ADV P36 promoter and upstream region are responsible for the weak constitutive activity and low level of transactivation of the ADV P36 promoter.

Aleutian Mink Disease↗
PubMed1994

Characterization of the internal promoter of simian foamy viruses.

Simian and human foamy viruses (HFV and SFV), genetically related members of the spumavirus genus of retroviruses, have complex genome structures which encode the gag, pol, and env genes for virion proteins as well as additional open reading frames. One of these open reading frames is a viral transactivator, encoded by genes designated taf for SFV and bel-1 for HFV, which augments transcription directed by the long terminal repeat (LTR) through cis-acting targets in the U3 domain of the LTR. Recently, an internal transcriptional promoter has been identified in sequences within the 3' end of the HFV env gene (M. Lochelt, W. Muranyi, and R. M. Flugel, Proc. Natl. Acad. Sci. 90:7317-7321, 1993). We have demonstrated by using transient expression assays in several tissue culture cell lines and by analyzing viral transcripts in infected cells that SFV-1 from a rhesus macaque and SFV-3 from an African green monkey also encode an internal promoter in the env gene. Transcription directed by the internal promoters of SFV-1 and SFV-3 is activated by the taf-1 and taf-3 gene products, respectively, in several cell types. The importance of a TATA box for the SFV-1 internal promoter was established by site-specific mutagenesis, and the 5' ends of transcripts initiating in the internal promoter have been determined. cis-acting sequences in the SFV-1 env gene required for the response to taf-1 are contained within a 121-bp element located 5' to the TATA box in the internal promoter. This taf-1-responsive element in the internal promoter functions in a position- and orientation-independent fashion in a heterologous promoter and thus has the properties of an enhancer which depends on taf-1 activity. Alignments reveal that the SFV-1 internal promoter and the SFV-1 LTR have little sequence relatedness. Cross-transactivation studies show that the transactivators of SFV-1 and HFV function on the internal promoter and LTR of the homologous virus but not on the heterologous virus. In summary, the genomes of simian and human foamy viruses direct viral transcription through both the promoter in the LTR and an internal promoter within the env gene, and each promoter contains unique enhancer-like elements regulated by the viral transactivator.

Animals↗
PubMed2000

A positive-strand RNA virus with three very different subgenomic RNA promoters.

Numerous RNA viruses generate subgenomic mRNAs (sgRNAs) for expression of their 3'-proximal genes. A major step in control of viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. We used barley yellow dwarf virus (BYDV) as a model system to study transcriptional control in a virus with multiple sgRNAs. BYDV generates three sgRNAs during infection. The sgRNA1 promoter has been mapped previously to a 98-nucleotide (nt) region which forms two stem-loop structures. It was determined that sgRNA1 is not required for BYDV RNA replication in oat protoplasts. In this study, we show that neither sgRNA2 nor sgRNA3 is required for BYDV RNA replication. The promoters for sgRNA2 and sgRNA3 synthesis were mapped by using deletion mutagenesis. The minimal sgRNA2 promoter is approximately 143 nt long (nt 4810 to 4952) and is located immediately downstream of the putative sgRNA2 start site (nt 4809). The minimal sgRNA3 core promoter is 44 nt long (nt 5345 to 5388), with most of the sequence located downstream of sgRNA3 start site (nt 5348). For both promoters, additional sequences upstream of the start site enhanced sgRNA promoter activity. These promoters contrast to the sgRNA1 promoter, in which almost all of the promoter is located upstream of the transcription initiation site. Comparison of RNA sequences and computer-predicted secondary structures revealed little or no homology between the three sgRNA promoter elements. Thus, a small RNA virus with multiple sgRNAs can have very different subgenomic promoters, which implies a complex system for promoter recognition and regulation of subgenomic RNA synthesis.

Base Sequence↗
PubMed2001

A novel silencer element in the bovine papillomavirus type 4 promoter represses the transcriptional response to papillomavirus E2 protein.

The long control regions (LCRs) of mucosal epitheliotropic papillomaviruses have similar organizations: a promoter region, an enhancer region, and a highly conserved distribution of E2 DNA binding sites (C. Desaintes and C. Demeret, Semin. Cancer Biol. 7:339--347, 1996). The enhancer of these viruses is epithelial cell specific, as it fails to activate transcription from heterologous promoters in nonepithelial cell types (B. Gloss, H. U. Bernard, K. Seedorf, and G. Klock, EMBO J. 6:3735--3743, 1987). Using the bovine papillomavirus type 4 (BPV-4) LCR and a bovine primary cell system, we have shown previously that a level of epithelial specificity resides in a papillomavirus promoter region. The BPV-4 promoter shows an enhanced response to transcriptional activators in epithelial cells compared with that of fibroblasts (K. W. Vance, M. S. Campo, and I. M. Morgan, J. Biol. Chem. 274:27839--27844, 1999). A chimeric lcr/tk promoter suggests that the upstream BPV-4 promoter region determines the cell-type-selective response of this promoter in fibroblasts and keratinocytes. Promoter deletion analysis identified two novel repressor elements that are, at least in part, responsible for mediating the differential response of this promoter to upstream activators in fibroblasts and keratinocytes. One of these elements, promoter repressor element 2 (PRE-2), is conserved in position and sequence in the related mucosal epitheliotropic papillomaviruses, BPV-3 and BPV-6. PRE-2 functions in cis to repress the basal activity of the simian virus 40 promoter and binds a specific protein complex. We identify the exact nucleotides necessary for binding and correlate loss of binding with loss of transcriptional repression. We also incorporate these mutations into the BPV-4 promoter and demonstrate an enhanced response of the mutated promoter to E2 in fibroblasts. The DNA binding protein in the detected complex is shown to have a molecular mass of approximately 50 kDa. The PRE-2 binding protein represents a novel transcriptional repressor and regulator of papillomavirus transcription.

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