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PubMed1999

Identification and characterization of a cAMP-responsive element in the region upstream from promoter 1.3 of the human aromatase gene.

Aromatase converts androgens to estrogens. The expression of this enzyme is driven by multiple tissue-specific promoters which are differentially regulated. Aromatase expression in breast cancer and the surrounding adipose cells is directed mainly by promoters I.3 and II, while its expression in the normal breast adipose tissue is driven by promoter I.4. Like promoter II, promoters I.3 is thought to be a cAMP-driven promoter, demonstrated previously by cell culture experiments. In the present study, we have identified and characterized a cAMP-responsive element (CREaro) upstream from promoter 1.3. This positive element, TGAAGTCA, between -66 and -59 bp relative to the transcriptional start site of promoter 1.3 was identified by DNA deletion and mutation analyses. The sequence of CREaro is one base different from the consensus CRE sequence (CREpal; TGACGTCA), and the mutational analysis revealed that CREaro had a higher enhancer activity to promoter I.3 than CREpal. Nuclear proteins from both WS3TF breast tumor fibroblasts and SK-BR-3 breast cancer cells bound to this CREaro, as demonstrated by DNA mobility shift assay. The molecular weight of the major binding protein in fibroblasts was determined to be approximately 60 kDa, as shown by UV crosslinking, which is different from those of known CRE-binding proteins. It is thought that CREB1 is not expressed in tumor fibroblasts because the Western blot analysis using anti-CREB1 antibody was not able to detect CREB1 in the nuclear protein extract from these cells. DNA mobility shift analysis using a nuclear protein extract from SK-BR-3 cells revealed that at least two proteins bound to the CREaro and that one of these proteins was identified to be CREB1. These studies provide direct evidence that promoter I.3 is a cAMP-responsive promoter.

Aromatase↗
PubMed1993

Effect of SV40 and MMTV promoters on intermolecular homologous recombination in rat cells.

The effect of SV40 and MMTV promoters on intermolecular homologous recombination between neomycin (neo) genes carrying deletions proximal and distal to the promoters has been examined in rat XC cells. One deleted neo gene linked to either promoter and the other deleted one without any promoter were co-transfected to cells so that the number of resulting G418-resistant colonies reflected the frequency at which the promoter-linked neo allele had been corrected by homologous recombination. We found that when the the distal deletion was placed under the SV40 promoter, it was corrected over 20-fold more frequently than the proximal. In contrast, when the MMTV promoter replaced the SV40 promoter, the distal deletion was corrected only 2-fold frequently compared with the proximal, and this result did not alter by activating the promoter with a glucocorticoid hormone dexamethasone. These results suggest that the preferential gene correction event observed with the SV40 promoter is attributed not to the transcriptional activity but to some specific sequence of the promoter.

Animals↗
PubMed1997

Dual promoters of mouse mu-opioid receptor gene.

Two transcriptional initiation sites, distal and proximal, located at approximately 500 bp apart in mouse mu-opioid receptor gene were identified recently. Using deletional and transfection analyses, the distal or proximal promoter alone displayed similar activity in neuronal cells constitutively expressing mu-opioid receptors (SH-SY-5Y). The presence of both promoters did not result in an increase in activity. However, when distal promoter linked with 3'-downstream sequences without the proximal promoter region, the distal promoter activity was abolished. Transfection analysis using various cell lines further suggested that only the proximal promoter preferentially directed the neuronal subtype expression. To verify the physiological importance of each promoter, the ratio of mu-receptor transcripts initiated by either promoter was examined by quantitative RT-PCR using mouse adult brain mRNA. We found that receptor mRNA was predominantly initiated by the proximal promoter. These studies suggested that the proximal promoter was the major functional promoter.

Animals↗
PubMed1996

Effects of Fis on ribosome synthesis and activity and on rRNA promoter activities in Escherichia coli.

The DNA-bending protein Fis of Escherichia coli is required for efficient initiation of chromosome replication and for activation of the P1 promoters of stable RNA (rRNA and tRNA) genes. Using fis+ and fis- bacterial strains, we have determined ribosome synthesis and activity, rRNA gene dosage, and transcript initiation rates at the rrnB P1 and P2 promoters. Different growth media were used to achieve growth rates between 0.7 and 2.7 doublings/hour for the fis+ strain, and between 0.7 and 2.2 doublings/hour for the fis- strain. In minimal media, the Fis deficiency only reduced the DNA and rrn gene concentration (DNA, oriC copies and rrn genes per protein); in amino acid-supplemented media, it also reduced rRNA synthesis rates per protein. Under all conditions, the ribosome activity (protein synthesis rate/ribosome) remained unchanged by the absence of Fis. In the presence of Fis, the absolute activities of the isolated rrnB P1 and P2 promoters increased from 2 to 88 and 10 to 50 initiations/minute, respectively, with increasing growth-rate. In the absence of Fis, these activities increased from 3 to 70 and 10 to 80 initiations/minute, respectively. Relative to the isolated rrnB P2 promoter, the strength of the rrnB P1 promoter was found to increase with increasing growth rate tenfold (from 0.17 to 1.7) in the presence, but only fivefold (from 0.17 to 0.85) in the absence of Fis. An evaluation of the data leads us to propose estimates of kcat and relative KM values for the two rRNA promoters, and relative values for free RNA polymerase concentrations during growth in different media. The analysis suggests that the reduced strength of P1 promoters of stable RNA genes in the absence of Fis, together with the reduced rrn gene concentration, increases the concentration of free RNA polymerase. In addition, the lower rrn P1 promoter activity in the absence of Fis reduces the probability that the downstream P2 promoter is blocked ("occluded") by a transcription elongation complex originating at the P1 promoter. The increased polymerase concentration and reduced P2 promoter occlusion both help to compensate for the Fis-deficiency, but during growth in rich media when the demand for ribosomes is high, this compensation is insufficient for fis- bacteria to achieve the wild-type level of ribosome synthesis and growth.

Carrier Proteins↗
PubMed1997

Regulation of Escherichia coli topA gene transcription: involvement of a sigmaS-dependent promoter.

To investigate the regulation of Escherichia coli topA gene transcription, primer extension was employed to determine the transcription initiation sites from the chromosomal topA gene. When cells were grown in LB medium to log phase, four transcription initiation sites could be identified. Three of these sites corresponded to promoters P1, P2 and P4 previously characterized using topA-galK fusion plasmids. The P3 promoter that is active on the plasmid was not utilized at the chromosomal topA gene under the conditions employed. There was a new transcription initiation site corresponding to a new promoter Px1. When cells started to enter stationary phase, promoter Px1 gradually became the major transcription initiation site for topA, while transcription from promoters P2 and P4 decreased. In an E. coli mutant lacking sigmaS (the rpoS gene product), the stationary phase specific sigma factor, the induction of transcription from promoter Px1 was abolished. In another mutant lacking H-NS activity, resulting in increased sigmaS level in log-phase, the transcription from promoter Px1 during log phase was increased. Thus Px1 appeared to be regulated by sigmaS. The activity of promoter P1 on the chromosome increased during heat shock, consistent with the previous result obtained using the topA-galK fusion plasmid showing that P1 is a sigma32-dependent heat shock promoter. Promoters P2 and P4 were most likely to be recognized by sigma70. The total level of topoisomerase I protein in the rpoS mutant was not reduced significantly in stationary phase due to increased transcription initiation from the other topA promoters. The utilization of multiple sigma factors for transcription initiation of topA could be important for adaptation of E. coli to change in growth conditions.

Bacterial Proteins↗
PubMed1999

Activities of constitutive promoters in Escherichia coli.

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+ spoT+ strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient DeltarelADeltaspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the beta-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage lambda; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of a lac deletion strain. The amount of 5'-terminal lacZ mRNA and of beta-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combining lacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+ spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity of rrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+ spoT+) relative to ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains.

Cell Division↗
PubMed2002

Identification of two middle promoters upstream DNA ligase gene 30 of bacteriophage T4.

Bacteriophage T4 DNA ligase gene 30 lies in the cluster of prereplicative genes located counterclockwise from map units 149 to 121. Based on the early transcription studies this gene has been considered as a typical early gene of bacteriophage T4. In agreement with this assignment, two strong T4 early promoters, P(E )30.8 (128.6) and P(E )30.7 (128.2), located about 3.1 and 2.7 kb upstream from gene 30 have been revealed by promoter mapping and sequence analysis. In addition, the existence of a putative early promoter just upstream of gene 30 was proposed from the sequence data. However, here we show that the putative early promoter just upstream of gene 30 is, in fact, a T4 middle promoter. Furthermore, we detected one more middle promoter located in the genomic region between early promoter P(E )30.7 (128.2) and DNA ligase gene 30 in the coding region of gene 30.3. Both new middle promoters have differences from the consensus MotA box, while their -10 regions match the sigma(70) consensus sequence very well. The 5' ends of MotA-dependent transcripts directed from these promoters, as well as the kinetics of 5' end accumulation in the cells, have been determined by primer extension analysis. The results of these analyses indicate that both MotA-dependent and MotA-independent promoters control the transcription of T4 DNA ligase gene 30 in vivo. Moreover, we show that the first transcripts for gene 30 are directed from its own middle promoter, P(M)30.

Bacteriophage T4↗
PubMed1993

Viral and cellular factors influence the activity of the Epstein-Barr virus BCR2 and BWR1 promoters in cells of different phenotype.

Transformation of B-lymphocytes by Epstein-Barr virus (EBV) is characterized by the expression of six viral nuclear antigens (EBNA1 to EBNA6) which are encoded by messages derived from long primary transcripts initiated at one of two promoters located in the BamHI C (BCR2) and BamHI W (BWR1) regions of the viral genome. The BWR1 promoter is preferentially utilized during the initial phases of EBV infection, whereas the BCR2 promoter is almost invariably used in transformed lymphoblastoid cell lines (LCLs). In order to gain some insight into the molecular mechanisms underlying promoter usage we have analyzed the activity of reporter plasmids carrying different parts of the BWR1 and BCR2 regulatory sequences in EBV-negative and EBV-carrying B cell lines that, on the basis of their surface marker expression, are representative of different stages of B cell activation/differentiation. We show that: (i) there is an inverse correlation between the activity of BWR1 and oriP-containing BCR2 reporter plasmids in cell lines expressing a BL group I versus a group III phenotype, the BWR1 promoter being virtually inactive in group III cells; (ii) BCR2 reporter plasmids devoid of the oriP region are active in EBV-negative cell lines and EBV-positive cells expressing a group I or group II phenotype and virtually inactive in BL group III cells and LCLs, suggesting that cellular factors are required for activation of BCR2 promoter elements. These factors are lost upon progression to a group III phenotype); (iii) expression of EBNA2 is sufficient to activate reporter plasmids containing the proximal part of the BCR2 promoter in EBV negative cells, whereas coexpression of EBNA2 and EBNA1 is required to activate the promoter in oriP-containing plasmids; (iv) the 30-bp repeat region of oriP acts as a negative cis-element on downstream promoters but is transformed into a transcriptional enhancer by the concerted action of EBNA1 and cellular factors. There was a poor correlation between the activity of exogenous reporter plasmids and endogenous BWR1 and BCR2 promoters in phenotypically different EBV-positive cell lines. The presence of the appropriate trans-acting factors was not sufficient to activate the endogenous BWR1 and BCR2 promoters in BL cells expressing a group I phenotype.

Antigens, Viral↗
PubMed1993

Mutational analysis of the herpes simplex virus type 1 glycoprotein E promoter.

A set of linker-scanning mutations was constructed in the herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promoter to define cis-acting regulatory elements common to late viral promoters. The gE promoter is a late viral promoter that has some activity in the absence of viral DNA replication and is representative of the gamma 1 class of HSV-1 promoters. Each promoter mutation was inserted upstream of the Escherichia coli lacZ gene in a recombinant virus, and the relative activities of beta-galactosidase expressed from individual recombinant viruses were compared. This analysis identified two sequence elements, a TATA element and an element at the start of transcription, that corresponded to similarly positioned elements previously identified in the gC and gH late (gamma 2) HSV-1 promoters. Mutation of the initiation element reduced expression from this promoter during normal lytic infection, but had no appreciable effect on expression in the absence of viral DNA replication, suggesting a specific role in late gene expression. Analysis of expression from hybrid gE/gC promoters revealed that the TATA and the initiation elements of these two promoters were interchangeable and that expression from the gE promoter in the absence of viral DNA replication was due to regulatory elements upstream from TATA element.

Animals↗
PubMed1995

Deletion analysis of the promoter for the cucumber necrosis virus 0.9-kb subgenomic RNA.

Sequences comprising the core promoter for the cucumber necrosis virus (CNV) 0.9-kb subgenomic RNA have been determined using deletion analysis and site-directed mutagenesis. The deletion studies indicate that the promoter lies within a region located 20 nucleotides upstream and 6 nucleotides downstream and including the subgenomic start site. Sequences further upstream or downstream of the core promoter do not appear to strongly affect promoter activity or viral RNA accumulation. Results of site-directed mutagenesis studies indicate that nucleotides immediately surrounding the subgenomic start site regulate promoter activity. Comparison of sequences within the CNV promoter region with the corresponding region of other tombusviruses shows that the tombusvirus promoter shares a region of near complete identity in 14 of the 26 core promoter nucleotides. Little similarity exists between the CNV 0.9-kb subgenomic RNA promoter and the region surrounding the transcription initiation site for the CNV 2.1-kb subgenomic RNA. Likewise, limited similarity occurs with the 5' region of CNV genomic RNA. Sequences similar to the ICR2-like motifs found in the promoters of several alphavirus-like (supergroup III) plant and animal viruses are not apparent. This study represents the first analysis of a subgenomic promoter from a member of supergroup II of positive-strand RNA viruses.

Base Sequence↗
PubMed1995

Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana.

We compared the organ specificity and the strength of different constitutive (CaMV-35S, CaMV-35Somega, Arabidopsis ubiquitin UBQ1, and barley leaf thionin BTH6 promoter) and one inducible promoter (soybean heat-shock promoter Gmhsp17.3) in stably transformed Arabidopsis thaliana plants. For this purpose we constructed a set of plant expression vectors equipped with the different promoters. Using the uidA reporter gene we could show that the CaMV-35S promoter has the highest expression level which was enhanced two- to threefold by the addition of a translational enhancer (TMV omega element) without altering the organ specificity of the promoter. The barley leaf thionin promoter was almost inactive in the majority of lines whereas the ubiquitin promoter exhibited an intermediate strength. The heat-shock promoter was inducible up to 18-fold but absolute levels were lower than in the case of the ubiquitin promoter. Conclusive quantitative results for different organs and developmental stages were obtained by the analysis of 24 stably transformed lines per promoter construct.

Antimicrobial Cationic Peptides↗
PubMed1991

Structural and functional analysis of promoter from gliadin, an endosperm-specific storage protein gene of Triticum aestivum L.

To identify cis-regulatory elements of the gliadin gene, a study of the gliadin gene promoter was conducted by transient expression analysis of plasmid DNAs which were introduced into plant protoplasts by electroporation. The promoter region (-592 bp to +18 bp from the translational start) of this developmentally regulated gene, when fused upstream to the chloramphenicol acetyl transferase (CAT) reporter cassette was unable to direct significant CAT expression in wheat or tobacco suspension cells. Because this monocot gene promoter appeared to be under stringent tissue-specific control, a hybrid promoter approach using a nopaline synthase (nos) promoter was employed. A series of 3' deletions of the gliadin promoter were placed upstream of either a nonfunctional -101 nos or a nearly wild-type -155 nos promoter fused in turn to a CAT reporter gene cassette. Transient expression analysis of these plasmid DNAs in tobacco cells showed that the gliadin fragment could either restore the activity of the non-functional nos promoter (series I) or enhance the activity of the functional nos promoter (series II). The degree of restoration of the promoter function conferred by gliadin fragments of the first series was proportional to the enhancing effect of the same fragments in the second series of constructs. The transcriptional activity of the gliadin (-592 bp to -77 bp) -nos hybrid promoter was reduced by 26% upon 3' deletion of sequences in the region -141 bp to -77 bp, which contains both the TATA and CCAAT boxes.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Mitochondrial transcription initiation: promoter structures and RNA polymerases.

A diversity of promoter structures. It is evident that tremendous diversity exists between the modes of mitochondrial transcription initiation in the different eukaryotic kingdoms, at least in terms of promoter structures. Within vertebrates, a single promoter for each strand exists, which may be unidirectional or bidirectional. In fungi and plants, multiple promoters are found, and in each case, both the extent and the primary sequences of promoters are distinct. Promoter multiplicity in fungi, plants and trypanosomes reflects the larger genome size and scattering of genes relative to animals. However, the dual roles of certain promoters in transcription and replication, at least in yeast, raises the interesting question of how the relative amounts of RNA versus DNA synthesis are regulated, possibly via cis-elements downstream from the promoters. Mitochondrial RNA polymerases. With respect to mitochondrial RNA polymerases, characterization of human, mouse, Xenopus and yeast enzymes suggests a marked degree of conservation in their behavior and protein composition. In general, these systems consist of a relatively non-selective core enzyme, which itself is unable to recognize promoters, and at least one dissociable specificity factor, which confers selectivity to the core subunit. In most of these systems, components of the RNA polymerase have been shown to induce a conformational change in their respective promoters and have also been assigned the role of a primase in the replication of mtDNA. While studies of the yeast RNA polymerase have suggested it has both eubacterial (mtTFB) and bacteriophage (RPO41) origins, it is not yet clear whether these characteristics will be conserved in the mitochondrial RNA polymerases of all eukaryotes. mtTFA-mtTFB; conserved but dissimilar functions. With respect to transcription factors, mtTFA has been found in both vertebrates and yeast, and may be a ubiquitous protein in mitochondria. However, the divergence in non-HMG portions of the proteins, combined with differences in promoter structure, has apparently relegated mtTFA to alternative, or at least non-identical, physiological roles in vertebrates and fungi. The relative ease with which mtTFA can be purified (Fisher et al. 1991) suggests that, where present, it should be facile to detect. mtTFB may represent a eubacterial sigma factor adapted for interaction with the mitochondrial RNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Functional in vivo verification in E. coli of promoter activities from the rDNA/tDNA(Val)(GAC) leader region of Zea mays chloroplasts.

Restriction fragments containing upstream sequences of the rRNA operon from Zea mays chloroplasts were tested for promoter activity in vivo by insertion into an E. coli promoter-probe vector. The expression of this vector's reporter gene, which codes for alkaline phosphatase, was stimulated more than 1,500-fold upon linkage with the chloroplast rRNA promoter. Site specific mutagenesis of the invariant T of the -10 sequence of this promoter reduced the expression of the reporter gene to 2% of the wild type. This indicates that the chloroplast rRNA promoter, which directs transcriptional initiation 117 bp upstream of the 16S rRNA gene, is also active in the bacterial system. A restriction fragment further upstream containing the gene for tRNA(Val) (GAC) also showed strong promoter activity (29% as compared with the rRNA promoter). This promoter activity probably reflects the chloroplast promoter directing the synthesis of the tRNA(Val) (GAC) primary transcript. Surprisingly, this restriction fragment also displayed promoter activity (13% compared with the rRNA promoter) in reverse orientation.

Base Sequence↗
PubMed2003

Variation in the FABP2 promoter affects gene expression: implications for prior association studies.

AIMS/HYPOTHESIS: An Ala54Thr polymorphism in the FABP2 gene has previously been associated with insulin resistance and lipid oxidation rates in Pima Indians. Ala54Thr functionally alters the protein's ability to bind and transport dietary fatty acids. In the current report, we sought additional functional variation in FABP2 by sequencing putative regulatory regions. METHODS: More than 1.2 Kb of the putative promoter of FABP2 was sequenced in 20 Pima subjects. Variations were genotyped in 84 additional Pima Indian subjects to assess haplotype combinations. Functional activities of variant and nonvariant promoters were compared in Caco-2 cells transfected with luciferase reporter constructs. RESULTS: Seven variations were identified in the FABP2 promoter in Pima Indians. Genotypes of these variants were in complete concordance with each other, and were in complete concordance with Ala54Thr. Therefore, only two promoter alleles were observed in Pima Indians, an Ala54-associated promoter and a Thr54-associated promoter. In contrast, genotyping of these variants in Caucasian DNA showed multiple genotypic combinations. In vitro reporter assays indicated that the Thr54-associated promoter in Pima Indians resulted in a threefold reduction in promoter activity as compared to Ala54-associated promoter. CONCLUSION/INTERPRETATION: Two functional variations exist in FABP2--the coding Ala54Thr and the variant promoter. In the Pima Indian population, but not the Caucasian population, these two functional variants are always carried on the same allele. Therefore, some of the in vivo phenotypic associations previously attributed to the Ala54Thr substitution, which alters binding characteristics of the protein, could instead be due to promoter variation, which alters expression levels.

Alanine↗
PubMed2005

TP53 promoter methylation in human gliomas.

Methylation of the promoter region of tumor suppressor genes may be associated with transcriptional silencing and tumor progression. The 5' region of the TP53 gene does not contain a CpG island, but a basal promoter region of 85 bp is essential for its full promoter activity. In the present study, we assessed whether TP53 promoter methylation is present in malignant glioma cells and whether this is associated with reduced TP53 expression. Methylation-specific PCR revealed TP53 promoter methylation in three (U87MG, LNT-229, T98G) out of six malignant glioma cell lines studied. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) led to up-regulated expression of TP53 mRNA and protein in U87MG and T98G cells, suggesting that promoter methylation is associated with reduced expression in some malignant glioma cells. We then assessed TP53 promoter methylation in primary tissue of low-grade gliomas, and observed TP53 promoter methylation in 29/48 (60%) low-grade astrocytomas, 11/18 (61%) oligoastrocytomas, and 31/42 (74%) oligodendrogliomas. Promoter methylation of the p14ARF gene, another gene involved in the TP53 pathway, was detected by methylation-specific PCR in 5/49 (10%) low-grade astrocytomas, 7/18 (39%) oligoastrocytomas, and 15/41 (37%) oligodendrogliomas. Our previous and present data show alterations of at least one of TP53 promoter methylation, p14ARF promoter methylation, and TP53 mutations in 43/49 (88%) of low-grade astrocytomas, 15/18 (83%) of oligoastrocytomas, and 35/42 (83%) oligodendrogliomas, suggesting that disruption of the TP53/p14ARF pathway is frequent in all histological types of low-grade glioma.

Base Sequence↗
PubMed2002

Activation of three pathogen-inducible promoters of tobacco in transgenic pear (Pyrus communis L.) after abiotic and biotic elicitation.

In order to improve pear resistance against fire blight caused by Erwinia amylovora, a search for promoters driving high-level expression of transgenes specifically in response to this bacterial pathogen has been undertaken. We have examined the ability of hsr203J, str246C and sgd24 tobacco (Nicotiana tabacum L.) promoters to drive expression of the uidA reporter gene in pear. Transgenic pear clones were obtained by Agrobacterium tumefaciens-mediated transformation. Beta-glucuronidase activity was determined quantitatively and qualitatively in these plants grown in vitro using fluorometric and histochemical assays and compared to cauliflower mosaic virus (CaMV) 35S promoter-driven activity. The hsr203J promoter appeared to be very weakly activated following inoculation in pear, which is the converse of the situation in tobacco. The str246C promoter was rapidly activated in pear during compatible and incompatible interactions, by wounding and following the application of several elicitors (capsicein, cryptogein, harpin, salicylic acid and jasmonic acid). The sgd24 promoter, a deletion derivative of str246C, exhibited a low level of expression after bacterial inoculation, was weakly activated by wounding and elicitors, and was not activated by phytohormones (salicylic acid and jasmonic acid). Interestingly, the sgd24 promoter was locally activated in pear, whereas the str246C promoter was activated systemically from the infection site. Taken together, these data show that, although the s tr246C and sgd24 promoters are less active than the CaMV35S promoter in pear, their pathogen-responsiveness would permit them to be used to drive the expression of transgenes to promote bacterial disease resistance.

Bacteria↗
PubMed2004

The MsPRP2 promoter enables strong heterologous gene expression in a root-specific manner and is enhanced by overexpression of Alfin 1.

Promoter specificity and efficiency of utilization are essential for endogenous and transgene expression. Selective root expression remains to be defined in terms of both promoter elements and transcription factors that provide high levels of ubiquitous expression. We characterized expression from the MsPRP2 promoter with the green fluorescent protein (GFP) reporter transgene in alfalfa (Medicago sativa) and found that a promoter fragment (+1 to -652 bp) retained the root and callus specificity of the endogenous MsPRP2 gene and hence this promoter fragment contains elements necessary for root-specific expression. The strong ubiquitous expression obtained from this promoter was comparable to that of the CaMV 35S promoter in roots and was enhanced by transgenic overexpression of Alfin 1, a root- and callus-specific transcription factor in alfalfa. No transgenic expression was obtained in leaves with this promoter in the presence or absence of Alfin 1. The increased expression of GFP in alfalfa containing the Alfin 1 transgene confirms the function of Alfin 1 binding sites in the MsPRP2 promoter fragment and also indicates that Alfin 1 concentrations are limiting for maximal expression in calli and roots. These findings characterize the MsPRP2 promoter as a novel root- and callus-specific promoter of plant origin that can be used as an effective tool for strong root-directed gene expression. In addition, we have demonstrated that the signal sequence of MsPRP2 can be used for efficient secretion of transgene products from callus and roots.

DNA-Binding Proteins↗