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PubMed2005

A novel method for prokaryotic promoter prediction based on DNA stability.

BACKGROUND: In the post-genomic era, correct gene prediction has become one of the biggest challenges in genome annotation. Improved promoter prediction methods can be one step towards developing more reliable ab initio gene prediction methods. This work presents a novel prokaryotic promoter prediction method based on DNA stability. RESULTS: The promoter region is less stable and hence more prone to melting as compared to other genomic regions. Our analysis shows that a method of promoter prediction based on the differences in the stability of DNA sequences in the promoter and non-promoter region works much better compared to existing prokaryotic promoter prediction programs, which are based on sequence motif searches. At present the method works optimally for genomes such as that of Escherichia coli, which have near 50 % G+C composition and also performs satisfactorily in case of other prokaryotic promoters. CONCLUSIONS: Our analysis clearly shows that the change in stability of DNA seems to provide a much better clue than usual sequence motifs, such as Pribnow box and -35 sequence, for differentiating promoter region from non-promoter regions. To a certain extent, it is more general and is likely to be applicable across organisms. Hence incorporation of such features in addition to the signature motifs can greatly improve the presently available promoter prediction programs.

Amino Acid Motifs↗

PubMed2005

Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1.

BACKGROUND: The promoter of the keratin 18 (K18) gene is 5- to 10-fold more active in tumorigenic (T-type) cell clones derived from the SW613-S human colon carcinoma cell line than in non-tumorigenic (NT-type) clones. We have reported previously that the mechanism responsible for this differential activity is acting on the minimal K18 promoter (TATA box and initiation site). This mechanism does not require the binding of a factor to a specific site on the DNA but involves the acetylation of a non-histone substrate. To get further insight into this mechanism, we investigated the effect of the adenovirus E1A protein on the activity of the K18 promoter, both in T and NT cells. RESULTS: Wild type adenovirus E1A protein and C-terminal deletion mutants inhibit the K18 promoter, specifically in T-type cells. The domain responsible for this inhibitory effect is located in the 12-25 region of the viral protein. E1A mutants that have lost this region but retain the PLDLS motif (the C-terminal binding site for CtBP1) stimulate the K18 promoter, specifically in NT cells. The inhibitory or stimulatory effects of the different E1A mutants are not dependent on a particular sequence of the promoter. An E1A N-terminal deletion mutant carrying point mutations in the PLDLS motif cannot stimulate the K18 promoter. CtBP1 interacts with CtIP, which is a known partner of BRCA1, itself a component of the RNA polymerase II holoenzyme. The stimulatory effect of two BRCA1 mutants, specifically in NT cells, implicates a tripartite BRCA1-CtIP-CtBP1 complex in the regulation of the K18 promoter. CONCLUSION: Since we have shown previously that the K18 promoter is stimulated by deacetylase inhibitors, specifically in NT cells, we conclude that the activity of the promoter is repressed in NT cells by a mechanism involving the recruitment, by a BRCA1/CtIP complex, of CtBP1 and associated deacetylases to the preinitiation complex. We propose a model depicting the mechanism responsible for the differential activity of the K18 promoter between T and NT cells of the SW613-S cell line.

Adenovirus E1A Proteins↗

PubMed2002

PRESTA: associating promoter sequences with information on gene expression.

BACKGROUND: Large sets of well-characterized promoter sequences are required to facilitate the understanding of promoter architecture. The major sequence databases are a prospective source of upstream regulatory regions, but suffer from inaccurate annotation. The software tool PRESTA (PRomoter EST Association) presented in this study is designed for efficient recovery of characterized and partially verified promoters from GenBank and EMBL libraries. RESULTS: The PRESTA algorithm examines the putative GenBank/EMBL promoters and automatically removes most of the poorly annotated entries. The remaining records are connected to expressed sequence tags (ESTs) through a high-stringency BLAST search. The frequency and source of recovered ESTs provide an estimate of the activity and expression pattern of the promoter, and the ESTs' 5' ends assist in transcription start-site verification. The PRESTA database provides easy access to non-redundant upstream regulatory regions recently extracted by the PRESTA algorithm. The current size of this resource is 552 human and 241 mouse promoters. Surprisingly, no overlap between the PRESTA database and the Eukaryotic Promoter Database (EPD) was detected by sequence comparison. CONCLUSIONS: The PRESTA algorithm demonstrates the principle of promoter verification by mapping EST 5' ends. The publicly available PRESTA database collects hundreds of characterized and partially verified promoter sequences and is complementary to other promoter databases.

Algorithms↗

PubMed2005

Stimulation of the novel estrogen receptor-alpha intronic TERP-1 promoter by estrogens, androgen, pituitary adenylate cyclase-activating peptide, and forskolin, and autoregulation by TERP-1 protein.

The estrogen receptor-alpha (ERalpha) pituitary-specific variant, TERP-1, is regulated dramatically by physiological status. We examined hormonal regulation of the TERP-1 promoter in transient transfection assays in GH3 somatolactotrope cells. We found that 17beta-estradiol (E2), genistein, androgen, pituitary adenylate cyclase-activating peptide, and forskolin (FSK) all stimulated TERP-1 promoter activity, whereas progesterone had no effect. ERalpha bound to a palindromic estrogen response element (ERE) and two half-site EREs; mutation of any of these sites decreased basal expression and completely obliterated E2 stimulation. In contrast, mutation of an activator protein-1 site decreased basal and FSK-stimulated promoter activity, but not E2 or androgen stimulation. The pure antiestrogen ICI 182,780 suppressed E2 and genistein, but not FSK or androgen, stimulation. Similarly, mutation of the ERE palindrome or half-site EREs suppressed promoter stimulation by E2 and genistein, but not by androgen or FSK. Because TERP-1 levels regulate ERalpha function on model promoters, we tested TERP-1 modulation of its own and other physiological promoters. TERP-1 suppressed basal and E2-stimulated expression of its own promoter. TERP-1 suppression required the ERE regions of the promoter, and the dimerization domain of TERP-1. TERP-1 overexpression also suppressed E2 stimulation of the progesterone receptor and prolactin promoters. Thus, estrogens, androgen, and FSK can stimulate TERP-1 promoter activity, and increased TERP-1 expression modulates E2 stimulation of physiological promoters. These data suggest that TERP-1 regulation may play a significant role in modifying pituitary ERalpha responses.

Androgens↗

PubMed1991

In vivo regulation of the activity of the two promoters of the rat acetyl coenzyme-A carboxylase gene.

The acetyl-coenzyme-A carboxylase (ACC) gene contains two promoters: promoter I (PI) and promoter II (PII). Depending upon which promoter is active, two classes of ACC mRNA are formed. The physiological significance of the presence of two promoters in the gene is not clear at this time. However, this question can be indirectly approached by examination of their expression patterns under different physiological conditions. We have examined the activities of these two promoters under different physiological conditions by means of primer extension analysis. Under normal conditions, the Wistar rat, fed standard chow ad libitum, expresses only basal levels of PI in white adipose tissue and PII in the liver. Starvation leads to the virtual disappearance of transcriptional products from these promoters. When fatty acid synthesis is stimulated by refeeding a fat-free diet to starved rats, both PI and PII are activated in the liver; however, in white adipose tissue, only PI, not PII, is responsive to this nutritional induction. On the other hand, in streptozotocin-diabetic rats, in which the activity of both promoters in both tissues is depressed, the administration of insulin quickly induces PI in adipose tissues, but has no significant effect on either of the promoters in the liver. During the weaning transition, the increase in hepatic lipogenesis is accompanied by activation of PI and PII when the pups are weaned onto a fat-free diet. Weaning onto a standard chow causes only a slight increase in PII. During the lactation period, profound alterations occur in the metabolism of the lipogenic tissues. In the lactating rat mammary gland only PII is active, and its activity is increased throughout lactation, reaching a plateau by day 7. Concomitantly, all ACC gene activity is completely shut off in the adipose tissue, while in the liver, PII, the only promoter active, is affected only minimally. The fat accumulation of the genetically obese Zucker rats is largely due to an abnormally high hepatic lipogenesis. In the obese Zucker rat (fa/fa), the level of expression of PII is similar to that in its lean siblings; however, PI is constitutively expressed at high levels, comparable to those in the Wistar rat that has been subjected to the starvation/refeeding induction. These studies demonstrate that the in vivo transcriptional control of the dual promoter rat ACC gene is a highly regulated and tissue-specific process.

Acetyl-CoA Carboxylase↗

PubMed2002

Ligands for the peroxisomal proliferator-activated receptor gamma and the retinoid X receptor inhibit aromatase cytochrome P450 (CYP19) expression mediated by promoter II in human breast adipose.

Local estrogen biosynthesis in breast adipose tissue, catalyzed by P450 aromatase, contributes to the growth of breast carcinomas. Aromatase expression is regulated by a number of alternative promoters, and in normal adipose tissue it is primarily regulated via the distal promoter I.4. However, in breast adipose containing a tumor, aromatase expression is regulated by the proximal promoter II in response to tumor-derived factors. Previously we have shown that peroxisomal proliferator-activated receptor gamma (PPARgamma) ligands inhibit aromatase expression in normal breast adipose tissue mediated by promoter I.4. In the present study, we investigated the effects of the PPARgamma ligand troglitazone and the retinoid X receptor (RXR) ligand LG101305 on aromatase expression mediated by promoter II. In cultured human breast adipose stromal cells, troglitazone or LG101305 alone inhibited aromatase activity and expression stimulated by inducers of promoter II, in a concentration-dependent manner, and this inhibition was greater in the presence of both ligands. Reporter gene assays showed that troglitazone and LG101305 inhibit transcription from promoter II of the CYP19 gene. However, EMSAs showed that PPARgamma and RXRalpha do not bind to promoter II of the CYP19 gene, indicating that PPARgamma- and RXR-mediated inhibition of aromatase expression via promoter II occurs through an indirect mechanism of action. Because ligands for PPARgamma and RXR inhibit aromatase expression in healthy breast adipose (via promoter I.4), as well as expression induced by tumor-derived factors (via promoter II), such compounds could find utility in the treatment of estrogen-dependent breast cancers.

Adipose Tissue↗

PubMed2002

Overexpression of aromatase P450 in leiomyoma tissue is driven primarily through promoter I.4 of the aromatase P450 gene (CYP19).

The CYP19 gene encoding aromatase P450 (estrogen synthetase) is expressed in several extragonadal sites and regulated in a tissue-specific fashion, which is achieved by alternative use of the seven different promoters (and corresponding exons 1) of the CYP19 gene. Previously, we demonstrated that aromatase P450 is overexpressed in leiomyoma tissue and that in situ estrogen synthesized in leiomyoma tissues possibly plays a role in leiomyoma growth. To elucidate the mechanism of overexpression of aromatase P450, we determined the promoter use of aromatase P450 in leiomyomas. 5'-Rapid amplification of cDNA ends analysis revealed that of six leiomyoma nodules tested, four nodules contained I.4-specific transcript of aromatase P450 alone, one nodule contained PII-specific transcript alone, and the remaining nodule contained both I.4- and PII-specific transcripts simultaneously. The levels of aromatase transcripts were then quantified by competitive RT-PCR assay. Among 21 leiomyomas, I.4-specific transcript and PII-specific transcript were predominant in 18 and 2 leiomyomas, respectively, whereas the remaining leiomyoma was negative for aromatase P450 expression. We next compared the aromatase activity of leiomyoma cells stimulated by promoter-specific regulatory factors. A combination of IL-1beta and dexamethasone, known as a potent inducer of promoter I.4-driven transcription, effectively increased aromatase activity. A combination of (Bt)(2)cAMP, 3-isobutyl-1-myethylxanthine, and PGE(2), known as inducers of promoter II-driven transcription, also increased aromatase activity, but the increases found were smaller than that induced by dexamethasone and IL-1beta. The transcriptional ability of the promoter I.4 sequence was confirmed by transient transfection assay using primary cells released from leiomyomas and established cells from normal myometrium (KW cells). Luciferase vectors containing promoter I.4 sequence (-340/+14 or longer) showed a significant increase in luciferase activity in response to dexamethasone. Deletion or mutation of a putative glucocorticoid-responsive element in the promoter I.4 sequence eliminated promoter activity. These results indicate that promoter I.4 is the major promoter responsible for overexpression of aromatase P450 in leiomyomas and that a glucocorticoid-responsive element within it plays a substantial role in the expression of aromatase P450.

Adult↗

PubMed1989

Chicken calmodulin promoter activity in proliferating and differentiated cells.

A 1218-base pair (bp) portion of the chicken calmodulin promoter was sequenced and assayed for promoter activity. This portion of the promoter was found sufficient to produce accurate transcriptional initiation. The promoter sequence was GC rich, particularly in the 700 bp region 5' to the cap site. Eight plasmids were prepared containing the first calmodulin exon and 30-1218 bp of the promoter, ligated to the reporter gene chloramphenicol acetyl transferase. In chicken embryonic fibroblasts and proliferating BC3H-1 cells promoter activity increased progressively with increasing promoter length up to 617 bp. Extension of the promoter beyond 617 bp inhibited expression, as did sequences within the first calmodulin exon. In BC3H-1 cells differentiation was found to reduce calmodulin mRNA levels approximately 3-fold. Activity of the calmodulin promoter constructs also decreased by a similar extent with differentiation. Sequences up to 234 bp 5' to the calmodulin cap site were markedly less effective in elevating chloramphenicol acetyl transferase activity in differentiated BC3H-1 cells than in proliferating cells and may account for the lower overall activity of the calmodulin promoter in these cells. Within this region several sequences were identified, including an extensive homology to the rat calmodulin I gene promoter that could be significant in regulation of calmodulin expression.

Animals↗

PubMed1997

Identification of a novel transferable cis element in the promoter of an estrogen-responsive gene that modulates sensitivity to hormone and antihormone.

The estrogen receptor (ER) is a ligand-regulated transcription factor that acts at the promoters of estrogen-regulated genes to modulate their expression. In the present study, we examined three estrogen-regulated promoters, namely the rat progesterone receptor gene distal (PRD) and proximal (PRP) promoters and the human pS2 gene promoter, and observed marked differences in their sensitivity to stimulation by estrogen and repression of estrogen-stimulated transcription by antiestrogen (AE)-occupied ER. ER-containing MCF-7 human breast cancer cells were transfected with reporter gene constructs containing estrogen response elements upstream of the three gene promoters. In this system, PRP and PRD showed similar dose-response curves for stimulation by estradiol whereas pS2 was activated by even lower concentrations of estradiol. By contrast, PRD was much less sensitive to repression of estrogen-stimulated activity by all AEs studied, relative to the PRP and the pS2 promoters. Using deletion and mutational analysis, we have identified a transferable cis element at -131 to -94 bp in PRD that is involved in modulating the sensitivity of this promoter to both estrogens and AEs. The element reduced the magnitude of estrogen-stimulated activity, enhanced the ability of AEs to repress estrogen-stimulated activity, and elicited similiar effects when transferred to the promoter of another estrogen-responsive gene. Thus, removal of this region from PRD further accentuated the insensitivity of this promoter to AE while enhancing its sensitivity (both EC50 and fold induction) to estrogen. Gel mobility shift assays showed that proteins from nuclear extracts of MCF-7 cells interact with this element and that the binding of these proteins is inversely correlated with the transcriptional effectiveness of the ER. The findings demonstrate that a specific cis element from the promoter of an estrogen-responsive gene can alter the transcriptional activity of hormone and antihormone-occupied receptor bound at its response element near the promoter. Such ligand response modulatory elements, and changes in the levels and activity of factors that bind to such elements, may underlie the different sensitivities of steroid hormone-regulated genes to both hormones and antihormones.

Animals↗

PubMed1999

The equine luteinizing hormone beta-subunit promoter contains two functional steroidogenic factor-1 response elements.

The requirements for basal expression of the LH beta-subunit promoter in pituitary gonadotropes are largely unknown. We have used the equine (e) LHbeta subunit promoter as a model to unravel the combinatorial code required for gonadotrope expression. Through the use of 5'-deletion mutagenesis, a region between -185 and -100 of the eLHbeta promoter was shown to play a critical role in maintaining basal promoter activity in alphaT3-1 and LbetaT2 cells. This region encompasses the steroidogenic factor-1 (SF-1) binding site that has been reported to have a functional role in expression of the LHbeta promoter in other species. We have also identified an additional SF-1 site at -55 to -48. Binding of SF-1 to both sites was confirmed by electrophoretic mobility shift assays. Mutations within these sites, either individually or in combination, did not attenuate basal activity of the eLHbeta promoter in alphaT3-1 cells, but did diminish promoter activity in LbetaT2 cells. Interestingly, cotransfection with an expression vector encoding SF-1 induced eLHbeta promoter activity, and this induction was abrogated by mutations within the SF-1 sites in alphaT3-1 cells. Block replacement mutagenesis was performed on the -185/-100 region of the eLHbeta promoter to identify DNA response elements responsible for maintaining basal promoter activity. From this analysis, two regions emerged as being important: a distal 31-bp segment (-181 to -150) and an element located immediately 3' to the distal SF-1 site (-119 to -106). It is hypothesized that these two regions as well as the SF-1 sites represent regulatory elements that contribute to a combinatorial code involved in targeting expression of the eLHbeta promoter to gonadotropes.

Animals↗

PubMed1992

Role of the cyclic adenosine 3',5'-monophosphate response element in efficient expression of the rat thyrotropin receptor promoter.

The "minimal" promoter region of the TSH receptor gene, -195 to -39 basepairs (bp), exhibits basal promoter activity, thyroid specificity, and negative regulation by TSH via its cAMP signal. In FRT thyroid cells and by comparison to pTRCAT5'-199, 5'-deletion mutants of chloramphenicol acetyltransferase (CAT) constructs from -199 to -150 bp of the minimal promoter decrease basal CAT activity by 50%, whereas continued deletion to -146 bp increases activity more than 4-fold. Continued deletion to -131 bp results in basal activity less than that of the -199 bp construct. An octameric cAMP response element (CRE)-like sequence, TGAGGTCA, is within -146 to -131 bp and starts at -139 bp. Its mutation to a consensus CRE (TGACGTCA) or AP1 (TGAGTCA) site or mutation of several residues flanking its 3'-terminus can improve promoter activity as much as 8-fold compared to pTRCAT5'-199. A nonpalindromic mutation to CGAGGACA decreases basal promoter activity to the level of the 199-bp minimal promoter. The CRE-like sequence between -139 and -132 bp is a constitutive enhancer of promoter activity in FRT thyroid cells, since, ligated to a simian virus-40-promoter-driven CAT gene, it increases CAT activity in the absence of forskolin in proportion to copy number and independent of direction or position. It can, however, function as a cAMP-responsive CRE, as evidenced by the fact that forskolin increases the activity of the same simian virus-40-promoter-driven CAT gene constructs in Buffalo rat liver (BRL) cells. DNAase-I footprinting shows that the CRE region is protected by a purified binding region peptide of the CRE-binding protein, activating transcription factor-2, and recombinant AP1 (human c-jun) as well as by BRL, FRT, and FRTL-5 rat thyroid cell nuclear extracts. Gel mobility shift analyses show that multiple CRE-binding proteins in the BRL, FRT, and FRTL-5 cell nuclear extracts form complexes with the CRE-like site, that one of these is CRE-binding protein, and that all form complexes with mutant sequences of the CRE-like site in a manner that exactly parallels their effects on constitutive enhancer function in FRT thyroid cells. We show, therefore, that the CRE-like site in the minimal TSH receptor promoter functions as a constitutive enhancer of promoter activity in FRT thyroid cells yet is a cAMP-responsive CRE.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2001

Genomic context modulates insulator activity through promoter competition.

Chromatin insulators regulate gene expression by preventing inappropriate enhancer-promoter interactions. Our previous study showed that insulators do not merely function as rigid blockers, rather their activities are quantitative and selective. We have investigated the factors and mechanisms that determine the effectiveness of the suHw insulator in transgenic Drosophila. We show that the suHw-mediated blockage of the AE1 enhancer from a downstream promoter depends on the ability of the promoter to compete for AE1. Promoters that are highly competitive for the enhancer are blocked less effectively. Moreover, blockage of AE1 from its cognate ftz promoter can range from virtually complete to non-detectable, depending on the property of the neighboring upstream promoter. A highly competitive neighboring promoter enhances the suHw-mediated blockage, whereas a less competitive promoter reduces the insulator effectiveness. The influence on insulator effectiveness by both the interacting and the neighboring competing promoters correlates with their ability to compete for the enhancer, which was previously shown to depend on core promoter sequences. Our findings suggest a mechanism at the level of gene organization that modulates insulator effectiveness through promoter competition. The dependence of insulator function on its cis contexts may provide it with more regulatory flexibility while imposing organizational restraints on eukaryotic gene complexes.

Animals↗

PubMed2000

Transcriptional control of Ca(2+)-activated K(+) channel expression: identification of a second, evolutionarily conserved, neuronal promoter.

Neuronal signaling properties are largely determined by the quantity and combination of ion channels expressed. The Drosophila slowpoke gene encodes a Ca(2+)-activated K(+) channel used throughout the nervous system. The slowpoke transcriptional control region is large and complex. To simplify the search for sequences responsible for tissue-specific expression, we relied on evolutionary conservation of functionally important sequences. A number of conserved segments were found between two Drosophila species. One led us to a new 5' exon and a new transcriptional promoter: Promoter C0. In larvae and adults, Promoter C0 was demonstrated to be neural-specific using flies transformed with reporter genes that either contain or lack the promoter. The transcription start site of Promoter C0 was mapped, and the exon it appends to the 5' end of the mRNA was sequenced. This is the second neural-specific slowpoke promoter to be identified, the first being Promoter C1. Promoter choice does not alter the encoded polypeptide sequence. RNAase protection assays indicate that Promoter C0 transcripts are approximately 12 times more abundant that Promoter C1 transcripts. Taken together, these facts suggest that promoter choice may be a means for cells to control channel density.

Animals↗

PubMed1999

Characterization of promoters integrated in the genome of bovine herpesvirus-1 (BHV-1).

Bovine herpesvirus-1 (BHV-1) has been used as a vector of live recombinant vaccines for cattle which express the genes of other pathogens. Because of the importance of the choice of the promoter which allows the efficient expression of the foreign genes in the BHV-1 vector, we compared the relative efficacy of various promoters integrated in the BHV-1 genome. The promoter sequences of the BHV-1 thymidine kinase (tk), gB, gC, SV40 early, and pseudorabies virus (PRV) immediate early (IE) genes were placed at the upstream of the open reading frame of the chloramphenycol acetyl transferase (CAT) gene and the promoter-CAT sequences were integrated into the tk gene of BHV-1 by homologous recombination. The promoter activity was assayed by measuring the CAT activity in the extracts of Madin Darby bovine kidney (MDBK) cells infected with the recombinant BHV-1. The PRV IE promoter was activated earlier and maintained at a higher level activity than the BHV-1 gB or gC promoters throughout the most of the growth phase of BHV-1. At the late phase, however, the activities of the BHV-1 gB and gC promoters reached the higher level. The BHV-1 tk promoter activity was low and the SV40 early promoter was hardly activated when integrated into the BHV-1 genome. promoter, recombinant BHV-1.

Animals↗

PubMed2004

Controlled expression of homologous genes by genomic promoter replacement in the yeast Saccharomyces cerevisiae.

Exchange of the promoter of a gene in the genome for another promoter whose expression can be controlled easily can overcome problems associated with the expression of the same gene from a promoter on a plasmid. Some genes are difficult or impossible to clone in plasmid-based vectors and often a stable expression and maintenance of the gene during cell proliferation is desirable. We present a method by which any genomic promoter can be replaced by a promoter of choice to achieve controlled (or constitutive and strong) expression of the gene concerned. The new promoter and a marker gene of choice are amplified by PCR using primers with a tail homologous to the regions adjacent to the site of integration in the genome and primers with a restriction site allowing ligation of the promoter and marker PCR products. After ligation of these PCR products, the ligated construct is transformed into yeast cells and allowed to exchange for the original promoter by homologous recombination. The transformants are selected based on the presence of the marker gene and proper exchange of the original promoter for the new promoter is checked by means of PCR amplification using primers in the new promoter and in the gene under its control.

Cloning, Molecular↗

PubMedDate not supplied

Grapevine protoplasts as a transient expression system for comparison of stilbene synthase genes containing cGMP-responsive promoter elements.

A method for preparing elicitor-responsive protoplasts from grapevine cells kept in suspension culture was established. The protoplasts were employed in order to perform transient gene expression experiments produced by externally added plasmids. Using the gene coding for bacterial beta-glucuronidase as the reporter gene, the transient expression under the control of various promoters of stilbene synthase genes were analyzed. The elicitor-responsiveness of promoters from grapevine genes and heterologous promoters were assayed: the grapevine stilbene synthase gene VST-1 and pine stilbene synthase genes PST-1, PST-2 and PST-3. Compared to the expression effected by the cauliflower mosaic virus 35S RNA-promoter, the stilbene synthase promoters caused a 2-5-fold increase in GUS-activity. Incubation of transformed protoplasts with fungal cell wall further stimulated the stilbene synthase promoters but not the 35S RNA-promoter. An even more pronounced differentiation between the promoters was observed when cGMP was included in the transient expression assays. Instead of treating transformed protoplasts with fungal cell wall we administered simultaneously cGMP and the plasmid to be tested. The cGMP-responsive increase was (a) specific concerning the nucleotide applied, (b) characteristic of grapevine protoplasts, and (c) not seen with shortened promoter-GUS constructs or GUS under the control of the 35S RNA-promoter. The highest cGMP-dependent response to stress was shown by the promoter of the grapevine stilbene synthase gene VST-1.

Acyltransferases↗

PubMedDate not supplied

The ecdysteroid UDP-glucosyltransferase gene promoter from Autographa californica multicapsid nucleopolyhedrovirus.

The ecdysteroid UDP-glucosyltransferase (egt) gene promoter fragments of different lengths were generated from the genomic DNA of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) by PCR. After being purified and enzymatic digestion, they were cloned into the pGEM-3Z vector for construction of reporter plasmids pAcegt542-luc, pAcegt309-luc and pAcegt159-luc with the luciferase gene driven by the AcMNPV egt promoter. The results of transient expression in the Spodoptera frugiperda cell line-21 (Sf21) showed that the transcriptional activity of the AcMNPV egt promoter required the transactivation of viral factor(s). The expression of luciferase gene driven by the AcMNPV egt promoter was first detected at 24 h post infection. The egt promoter from the Bombyx mori nucleopolyhedrovirus (BmNPV), closely related to AcMNPV, revealed similar properties to that of the AcMNPV egt promoter. When BmNPV homologous region 3 was subcloned downstream the luciferase gene, the luciferase activity of the reporter plasmid was enhanced by over 1000-fold, but the property of the promoter was not changed. As a substrate of ecdysteroid UDP-glucosyltransferase (EGT), foreign insect ecdysone showed negative effects on egt promoter transcriptional activity. Ecdysone of 1.0-2.0 microg/ml significantly down-regulated the promoter activity. Promoter activities of different lengths showed that an AcMNPV egt promoter fragment of 159 bp has the basal transcriptional activity but it was almost abolished only about 0.2% of that of 309 bp and 542 bp, respectively, and the nucleotide sequence from - 159 to - 309 nt upstream the translation initiation site includes the main cis-acting elements interacting with viral factors.

Animals↗

PubMed2004

Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters.

Despite important advances in human therapeutics, no specific treatment for both non-functioning gonadotroph and resistant somatotroph adenomas is available. Gene transfer by viral vectors can be considered as a promising way to achieve a specific and efficient treatment. Here we show the possibility of efficient gene transfer in human pituitary adenoma cells in vitro using a human immunodeficiency virus (HIV)-type 1-derived vector. Using enhanced green fluorescent protein (eGFP) gene as a marker placed under the phosphoglycerate kinase (PGK) promoter, gonadotroph and somatotroph adenomas were transduced even with moderate viral loads. The expression started at day 2, reached a peak at day 5, and it was still present at day 90. For targeting somatotroph and gonadotroph adenomas, human growth hormone (GH) promoter (GH -481, +54 bp) and two fragments of the human glycoprotein hormone alpha-subunit promoter (alpha-subunit 1 -520, +33 bp, and alpha-subunit 2 -907, +33 bp) were tested. In gonadotroph adenomas, the percentage of identified fluorescent cells and the fluorescence intensity analyzed by fluorescence-activated cell sorting indicated that the strength of the alpha-subunit 1 and alpha-subunit 2 promoters were comparable to that of the PGK promoter. Primary cultures of rat pituitary cells showed that alpha-subunit 1 is more selective to thyreotroph and gonadotroph phenotypes than alpha-subunit 2. GH promoter activity appeared weak in somatotroph adenomas. The human GH enhancer did not increase the GH promoter activity at all but the human prolactin promoter (-250 bp) allowed 4-fold more fluorescent cells to be obtained than the GH promoter. Several cell lines appeared too permissive to test cell-specificity of pituitary promoters. However, on human non-pituitary cell cultures, the tested pituitary promoters seemed clearly selective to target endocrine pituitary phenotypes. This study gives a starting point for a gene-therapy program using lentiviral vectors to transfer therapeutic genes in human pituitary adenomas.

Adenoma↗