Consensus Promoter Sequence Research Articles

Identification of Promoters Recognized by RNA Polymerase-σ E Holoenzyme from Thermus thermophilus HB8

Thermus thermophilus sigma(E), an extracytoplasmic function sigma factor from the extremely thermophilic bacterium Thermus thermophilus HB8, bound to the RNA polymerase core enzyme and showed transcriptional activity. With the combination of in vitro transcription assay and GeneChip technology, we identified three promoters recognized by sigma(E). The predicted consensus promoter sequence for sigma(E) is 5'-CA(A/T)(A/C)C(A/C)-N(15)-CCGTA-3'.

Expression of Corynebacterium glutamicum glycolytic genes varies with carbon source and growth phase

A basic pattern of gene expression and of relative expression levels during different growth phases was obtained for Corynebacterium glutamicum R grown on different carbon sources. The gapA-pgk-tpi-ppc gene cluster was transcribed as a mono- or polycistronic mRNA, depending on the growth phase. The 1.4 kb (gapA) and 2.3 kb (pgk-tip) mRNAs were expressed in the early through late exponential phases, whereas the 3.7 kb (gapA-pgk-tpi) and 5.4 kb (pgk-tpi-ppc) mRNAs were only detected in the mid-exponential phase. All other glycolytic genes except pps, glk and pgi were transcribed as monocistronic mRNAs under all tested conditions. Identification and alignment of the promoter regions of the transcriptional start sites of glycolytic genes revealed strong similarities to the sigma(A) consensus promoter sequences of Gram-positive bacteria. All genes involved in glycolysis were coordinately expressed in medium containing glucose. Growth in the presence of glucose gave rise to abundant expression of most glycolytic genes, with the level of gapA transcript being the highest. Glucose depletion led to a rapid repression of most glycolytic genes and a corresponding two- to fivefold increased expression of the gluconeogenic genes pps, pck and malE, which are induced by pyruvate, lactate, acetate and/or other organic acids.

Promoter and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus gordonii

An optimally cross-linked peptidoglycan requires both transglycosylation and transpeptidation, provided by class A and class B penicillin-binding proteins (PBPs). Streptococcus gordonii possesses three class A PBPs (PBPs 1A, 1B, and 2A) and two class B PBPs (PBPs 2B and 2X) that are important for penicillin resistance. High-level resistance (MIC, > or =2 microg/ml) requires mutations in class B PBPs. However, although unmutated, class A PBPs are critical to facilitate resistance development (M. Haenni and P. Moreillon, Antimicrob. Agents Chemother. 50:4053-4061, 2006). Thus, their overexpression might be important to sustain the drug. Here, we determined the promoter regions of the S. gordonii PBPs and compared them to those of other streptococci. The extended -10 box was highly conserved and complied with a sigma(A)-type promoter consensus sequence. In contrast, the -35 box was poorly conserved, leaving the possibility of differential PBP regulation. Gene expression in a penicillin-susceptible parent (MIC, 0.008 microg/ml) and a high-level-resistant mutant (MIC, 2 microg/ml) was monitored using luciferase fusions. In the absence of penicillin, all PBPs were constitutively expressed, but their expression was globally increased (1.5 to 2 times) in the resistant mutant. In the presence of penicillin, class A PBPs were specifically overexpressed both in the parent (PBP 2A) and in the resistant mutant (PBPs 1A and 2A). By increasing transglycosylation, class A PBPs could promote peptidoglycan stability when transpeptidase is inhibited by penicillin. Since penicillin-related induction of class A PBPs occurred in both susceptible and resistant cells, such a mutation-independent facilitating mechanism could be operative at each step of resistance development.

Transcription Initiation by Mix and Match Elements: Flexibility for Polymerase Binding to Bacterial Promoters

Bacterial RNA polymerase is composed of a core of subunits (β β′, α1, α2, ω), which have RNA synthesizing activity, and a specificity factor (σ), which identifies the start of transcription by recognizing and binding to sequence elements within promoter DNA. Four core promoter consensus sequences, the –10 element, the extended –10 (TGn) element, the –35 element, and the UP elements, have been known for many years; the importance of a nontemplate G at position -5 has been recognized more recently. However, the functions of these elements are not the same. The AT-rich UP elements, the –35 elements (–35TTGACA–30), and the extended –10 (15TGn–13) are recognized as double-stranded binding elements, whereas the –5 nontemplate G is recognized in the context of single-stranded DNA at the transcription bubble. Furthermore, the –10 element (–12TATAAT–7) is recognized as both double-stranded DNA for the T:A bp at position –12 and as nontemplate, single-stranded DNA from positions –11 to –7. The single-stranded sequences at positions –11 to –7 as well as the –5 contribute to later steps in transcription initiation that involve isomerization of polymerase and separation of the promoter DNA around the transcription start site. Recent work has demonstrated that the double-stranded elements may be used in various combinations to yield an effective promoter. Thus, while some minimal number of contacts is required for promoter function, polymerase allows the elements to be mixed and matched. Interestingly, which particular elements are used does not appear to fundamentally alter the transcription bubble generated in the stable complex. In this review, we discuss the multiple steps involved in forming a transcriptionally competent polymerase/promoter complex, and we examine what is known about polymerase recognition of core promoter elements. We suggest that considering promoter elements according to their involvement in early (polymerase binding) or later (polymerase isomerization) steps in transcription initiation rather than simply from their match to conventional promoter consensus sequences is a more instructive form of promoter classification.

Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System

We developed techniques for the genetic manipulation of Flavobacterium species and used it to characterize several promoters found in these bacteria. Our studies utilized Flavobacterium hibernum strain W22, an environmental strain we isolated from tree hole habitats of mosquito larvae. Plasmids from F. hibernum strain W22 were more efficiently (approximately 1,250-fold) transferred by electroporation into F. hibernum strain W22 than those isolated from Escherichia coli, thus indicating that an efficient restriction barrier exists between these species. The strong promoter, tac, functional in proteobacteria, did not function in Flavobacterium strains. Therefore, a promoter-trap plasmid, pSCH03, containing a promoterless gfpmut3 gene was constructed. A library of 9,000 clones containing chromosomal fragments of F. hibernum strain W22 in pSCH03 was screened for their ability to drive expression of the promoterless gfpmut3 gene. Twenty strong promoters were used for further study. The transcription start points were determined from seven promoter clones by the 5' rapid amplification of cDNA ends technique. Promoter consensus sequences from Flavobacterium were identified as TAnnTTTG and TTG, where n is any nucleotide, centered approximately 7 and 33 bp upstream of the transcription start site, respectively. A putative novel ribosome binding site consensus sequence is proposed as TAAAA by aligning the 20-bp regions upstream of the translational start site in 25 genes. Our primary results demonstrate that at least some promoter and ribosome binding site motifs of Flavobacterium strains are unusual within the bacterial domain and suggest an early evolutionary divergence of this bacterial group. The techniques presented here allow for more detailed genetics-based studies and analyses of Flavobacterium species in the environment.

Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial sigma factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for sigma(F), sigma(C), and sigma(K), and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of sigma factor genes. Our results revealed that many sigma factors are expressed from autoregulated promoters.

Identification of a Novel Core Promoter Element That Enhances Transcription: Application to Ankyrin Promoters for Globin Gene Therapy.

Gene Therapy for the hemoglobin β-chain disorders, Sickle Cell Disease (SCD) or beta thalassemia (β-thal), requires both efficient globin gene transfer into hematopoietic stem cells (HSC) as well as expression of globin mRNA and protein at levels that are >20% of the level of α-globin. Expression of the β-like globin genes in either transgenic mice or from RNA virus vectors integrated into the genome of erythroid cells requires sequences from the Locus Control Region (LCR). The LCR sequences contain cryptic splicing and polyadenylation sequences that contribute to inefficient production and low titers of recombinant virus particles. In addition, the powerful LCR enhancer elements pose a significant risk of insertional activation of leukemia genes. We hypothesized that enhancer-independent promoters from other genes expressed in erythrocytes could be used to express sufficient amounts of β-like globin to treat SCD or β-thal. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the 4 different tissue-specific promoters used to express the ANK-1 gene. We have previously demonstrated that a “double copy” Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. In mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein was expressed at uniform level of 7.5 % of α-globin per vector copy (Sabatino et al. PNAS 97:13294–9, 2000). To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG (−73/−72) dinucleotide in the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID (Gallagher et al. Hum Mol Gen 14:2501–9, 2005). We hypothesized that altering the sequence in the TFIID binding region of the ANK-1E promoter would increase TFIID binding, resulting in the increased level of ANK-1E/γ-globin transcription needed for an effective therapy for SCD and β-thal. We generated a library of ANK-1E promoters with degenerate sequence in the TFIID binding region, preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained from the sequencing results: the wild type sequence (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones: (T/G)(G/C)(G/C)GG(T/A)GAG was found in similar locations relative to the transcriptional start site in the three other ANK-1 promoter regions, as well as in 22% of >4000 human loci with GC-rich promoters that lack TATA boxes and other consensus promoter sequences. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase mRNA and protein than the wild type promoter (p=0.001; 0.005 respectively). We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.

A 200 kb Survey of Chromatin in the ANK-1 Locus Demonstrates an Erythroid-Specific Chromatin Hub That Activates the Erythrocyte Ankyrin (ANK-1E) Promoter.

Mammals express a variety of erythroid and nonerythroid ankyrin-1 isoforms generated by alternate mRNA splicing and by expression from promoters upstream of 3 known alternate first exons. ANK-1 Exon 1B is located 138 kb 5′ of exon 2 and is expressed only in neuronal and muscle cells. Exon 1E is located 39 kb 5′ of exon 2 and is expressed only in erythroid cells. Exon 1A is located 27 kb 5′ of exon 2 and is expressed in many cell types. We have previously shown that the ANK-1E promoter is flanked by DNase I Hypersensitive Sites (HS), one immediately upstream of the RNA initiation sites (5′HS) and a pair of closely spaced HS 5kb downstream (3′HS1 and 3′HS2). To determine the location of additional HS in the ANK-1 locus, we designed PCR primers spaced ~250 bp apart that span a 200 kb region from exon 2 to 60 kb upstream of Exon 1B for use in a high throughput DNase I HS assay. We identified the HS surrounding Exon 1E, as well as HS that flank Exons 1A and 1B. In both the Exon 1A and Exon 1B promoters, the 5′HS is located immediately upstream of the mRNA initiation sites and the 3′HS is located 4–7 kb downstream. An additional pair of HS were identified 70 kb upstream of exon 2 between the ANK-1B and ANK-1E promoters. This region contains the 5′ ends of at least 5 human ESTs. We used 5′ RACE to show that the homologous region in the mouse is transcribed and splices to exon 2. This putative promoter is designated ANK-1C. All 4 ANK-1 promoters lack consensus promoter sequences involved in the binding of the transcription initiation complex, TFIID, including TATA, InR, DPE or DCE elements. We have recently identified a novel consensus sequence that binds TFIID: (T/G)(G/C)(G/C)GGTGAG. This sequence is present multiple times in all 4 ANK-1 promoters as well as in 22% of >4000 mammalian promoters lacking TFIID-binding consensus sequences, strong evidence of functional significance. To understand the relationship of the flanking HS to the ANK-1 promoters we used the activation of ANK-1E promoter in erythroid cells as a model and have undertaken a molecular dissection of the elements in the ANK-1E region. Using transgenic mice and K562-based assays we have shown that both ANK-1E 5′HS and 3′HS2 are barrier elements that prevent gene silencing. In K562 cells, ANK-1E 3′HS1 increases expression only when located adjacent to the ANK-1E or thymidine kinase promoters (p=0.0009), but not in SY5Y neuronal cells (p=0.35). DNase I footprinting, gel shift, and reporter gene assays demonstrated that 3′HS1 binds the erythroid-specific transcription factor NF-E2. Mutation of the NF-E2 binding site abolished the ability of 3′HS1 to increase gene expression (p=0.08) in K562 cells. Chromatin Conformation Capture (3C) analysis demonstrated the formation of a 5 kb erythroid-specific chromatin loop that brings 5′HS into close proximity with 3′HS1/2. In agreement with the 3C results, Chromatin Immune Precipitation analysis demonstrated a hub in which Brg-1 and CTCF (associated with barrier elements), NF-E2, GATA-1 and RNA Pol II occupy both 5′HS and 3′HS1/2, despite the lack of consensus sites for NF-E2 in 5′HS, or GATA-1 or RNA initiation sites in 3′HS1/2. Our current model is that the formation of the erythroid-specific ANK-1E chromatin loop is mediated by the binding of the erythroid-specific transcription factors GATA-1 to 5′HS and NF-E2 to 3′HS1.

Tetracycline-inducible gene expression in mycobacteria within an animal host using modified Streptomyces tcp830 regulatory elements

Inducible expression systems are powerful tools for studying gene function. Though several inducible expression systems are now available for mycobacteria, none have been used to modulate bacterial gene expression during an animal infection. A tetracycline-inducible expression system from Streptomyces coelicolor was successfully adapted for use in mycobacteria. To prevent baseline expression without induction, S. coelicolor tetR gene was overexpressed using the acetamidase promoter and regulatory gene block. Target gene expression was controlled by the S. coelicolor tcp830 promoter and operator allele. The -10 promoter consensus sequence of the tcp830 promoter was modified to better resemble known strong mycobacterial promoters. Using this system, induction of tetR fully repressed tcp830-dependent expression of green fluorescent protein (GFP) to baseline levels. Addition of anhydrotetracycline led to a 62-fold induction of GFP expression in vitro and 15-fold induction in a mouse mycobacterial peritonitis model in the presence of maximal tetR expression. Chemically regulatable gene expression during animal infection may be a useful tool in studying mycobacterial pathogenesis.

Identification of the σ E regulon of Salmonella enterica serovar Typhimurium

The extracytoplasmic function sigma factor, sigma(E), has been shown to play a critical role in virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium). The previously optimized two-plasmid system has been used to identify S. Typhimurium promoters recognized by RNA polymerase containing sigma(E). This method allowed identification of 34 sigma(E)-dependent promoters that direct expression of 62 genes in S. Typhimurium, 23 of which (including several specific for S. Typhimurium) have not been identified previously to be dependent upon sigma(E) in Escherichia coli. The promoters were confirmed in S. Typhimurium and transcriptional start points of the promoters were determined by S1-nuclease mapping. All the promoters contained sequences highly similar to the consensus sequence of sigma(E)-dependent promoters. The identified genes belonging to the S. Typhimurium sigma(E)-regulon encode proteins involved in primary metabolism, DNA repair systems and outer-membrane biogenesis, and regulatory proteins, periplasmic proteases and folding factors, proposed lipoproteins, and inner- and outer-membrane proteins with unknown functions. Several of these sigma(E)-dependent genes have been shown to play a role in virulence of S. Typhimurium.

Handbook of genome research: genomics, proteomics, metabolomics, bioinformatics, ethical and legal issues

Handbook of genome research: genomics, proteomics, metabolomics, bioinformatics, ethical and legal issues

The rubrerythrin-like protein Hsp21 of Clostridium acetobutylicum is a general stress protein

The small heat shock protein Hsp21 of Clostridium acetobutylicum was recently identified as a rubrerythrin-like protein with a rubredoxin-like FeS(4) domain at the N-terminus and a ferritin-like diiron domain at the C-terminus. Here, we report that the two identical tandem genes rbr3A and rbr3B, which encode the heat shock protein Hsp21, show the transcription pattern of general stress genes. Northern blot analysis indicated that the transcription of the rbr3AB operon is induced by various environmental stress conditions: in addition to heat and oxidative stress, an increase of the pH of the growth medium from 4.5 to 6.2, addition of the salt NaCl (400 mM) or of the solvent butanol (3.5% v/v), and lowering the incubation temperature from 37 to 25 degrees C resulted in transiently increased transcript levels. The promoter region deduced from the 5' end of the mRNA has only limited similarity to the consensus promoter sequence of Gram-positive bacteria. A conserved inverted repeat between this promoter and the initiation codon is proposed to have a regulatory role. Although C. acetobutylicum is regarded as a strictly anaerobic bacterium, live/dead staining demonstrated that it can survive exposure to air or H(2)O(2) and other stressors to various extents.

Combinatorial Analysis of Transcription Factor Partners Reveals Recruitment of c-MYC to Estrogen Receptor-α Responsive Promoters

In breast cancer and normal estrogen target tissues, estrogen receptor-alpha (ERalpha) signaling results in the establishment of spatiotemporal patterns of gene expression. Whereas primary target gene regulation by ERalpha involves recruitment of coregulatory proteins, coactivators, or corepressors, activation of these downstream promoters by receptor signaling may also involve partnership of ERalpha with other transcription factors. By using an integrated, genome-wide approach that involves ChIP-chip and computational modeling, we uncovered 13 ERalpha-responsive promoters containing both ERalpha and c-MYC binding elements located within close proximity (13-214 bp) to each other. Estrogen stimulation enhanced the c-MYC-ERalpha interaction and facilitated the association of ERalpha, c-MYC, and the coactivator TRRAP with these estrogen-responsive promoters, resulting in chromatin remodeling and increased transcription. These results suggest that ERalpha and c-MYC physically interact to stabilize the ERalpha-coactivator complex, thereby permitting other signal transduction pathways to fine-tune estrogen-mediated signaling networks.

Cloning and transcriptional analysis of the lipA (lipoic acid synthetase) gene from Rhizobium etli.

We report here the isolation of a Rhizobium etli gene involved in lipoic acid metabolism, the lipA gene, which complements a lipA mutant strain of Escherichia coli. A promoter region (lipAp) was mapped immediately upstream of lipA and two in vivo transcription initiation sites were identified, preceded by sequences showing some homology to the -10/-35 promoter consensus sequences. The activity of the lipAp was found not to be regulated either by the carbon source or by the addition of lipoic acid. Moreover, quantitative analysis of the lipA transcript by RNase protection assays indicated its down-regulation during entry into stationary phase.

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7.0-9.0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9.0. Growth still occurred at pH 9.5 but at a reduced rate. The expression of the pH-regulated F0 F1 ATPase operon (containing the eight genes atpBEFHAGDC) was induced at alkaline pH. A 7.5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9.0. The same occurred with a 1.2 kb transcript corresponding to the atpB gene. RT-PCR studies confirmed the alkaline pH induction of the F0 F1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F0 F1 operon, was expressed at a lower level than the polycistronic 7.5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F0 F1 operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional in C. glutamicum and Escherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetative sigma factor of C. glutamicum, whereas the -35 and -10 boxes of P-atp2 fitted the consensus sequence for sigma(H)-recognized Mycobacterium tuberculosis promoters C(C)/(G)GG(A)/(G)AC 17-22 nt (C)/(G)GTT(C)/(G), known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F0 F1 operon is highly expressed at alkaline pH, probably using a sigma (H) RNA polymerase.

Role of the spacer between the −35 and −10 regions in σs promoter selectivity in Escherichia coli

In vitro, the sigma(s) subunit of RNA polymerase (RNAP), RpoS, recognizes nearly identical -35 and -10 promoter consensus sequences as the vegetative sigma70. In vivo, promoter selectivity of RNAP holoenzyme containing either sigma(s) (Esigma(s)) or sigma70 (Esigma70) seems to be achieved by the differential ability of the two holoenzymes to tolerate deviations from the promoter consensus sequence. In this study, we suggest that many natural sigma(s)-dependent promoters possess a -35 element, a feature that has been considered as not conserved among sigma(s)-dependent promoters. These -35 hexamers are mostly non-optimally spaced from the -10 region, but nevertheless functional. A +/- 2 bp deviation from the optimal spacer length of 17 bp or the complete absence of a -35 consensus sequence decreases overall promoter activity, but at the same time favours Esigma(s) in its competition with Esigma70 for promoter recognition. On the other hand, the reduction of promoter activity due to shifting of the -35 element can be counterbalanced by an activity-stimulating feature such as A/T-richness of the spacer region without compromising Esigma(s) selectivity. Based on mutational analysis of sigma(s), we suggest a role of regions 2.5 and 4 of sigma(s) in sensing sub-optimally located -35 elements.

Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi) gene and characterization of its protein

Chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. We used touchdown PCR to clone the chitinase (Schi) gene from Bacillus thuringiensis serovar sotto (Bt sotto) chromosomal DNA. Our DNA sequencing analysis revealed that the Bt sotto Schi gene consists of an open reading frame (ORF) of 2067 nucleotides with codes for the chitinase precursor. We also found that the putative promoter consensus sequences (the -35 and -10 regions) of the Bt soto Schi gene are identical to those of the chiA71 gene from Bt Pakistani, the chiA74 gene from Bt kenyae and the ichi gene from Bt israelensis. The Schi chitinase precursor is 688 amino acids long with an estimated molecular mass of 75.75 kDa and a theoretical isoelectric point of 5.74, and contains four domains, which are, in sequence, a signal peptide, an N-terminal catalytic domain, a fibronectin type III like domain and a C-terminal chitin-binding domain. Sequence comparison and the evolutionary relationship of the Bt sotto Schi chitinase to other chitinase and chitinase-like proteins are also discussed.

Chemokine regulation in response to beryllium exposure in human peripheral blood mononuclear and dendritic cells

Exposure to beryllium (Be) induces a delayed-type hypersensitivity immune reaction in the lungs of susceptible individuals, which leads to the onset of Be sensitivity and Chronic Beryllium Disease (CBD). Although some mechanistic aspects of CBD have begun to be characterized, very little is known about the molecular mechanisms by which Be activates the host immune response. To gain insight into the cellular response to Be exposure, we have performed global microarray analysis using a mixture of peripheral blood mononuclear and dendritic cells (PBMC/DCs) from a non-CBD source to identify genes that are specifically upregulated in response to BeSO 4 stimulation, compared to a control metal salt, Al 2(SO 4) 3. We identified a number of upregulated immunomodulatory genes, including several chemokines in the MIP-1 and GRO families. Using PBMC/DCs from three different donors, we demonstrate that BeSO 4 stimulation generally exhibits an increased rate of both chemokine mRNA transcription and release compared to Al 2(SO 4) 3 exposure, although variations among the individual donors do exist. We show that MIP-1α and MIP-1β neutralizing antibodies can partially inhibit the ability of BeSO 4 to stimulate cell migration of PBMC/DCs in vitro. Finally, incubation of PBMC/DCs with BeSO 4 altered the binding of the transcription factor RUNX to the MIP-1α promoter consensus sequence, indicating that Be can regulate chemokine gene activation. Taken together, these results suggest a model in which Be stimulation of PBMC/DCs can modulate the expression and release of different chemokines, leading to the migration of lymphocytes to the lung and the formation of a localized environment for development of Be disease in susceptible individuals.

Structural and Transcriptional Variation of Mitochondrial DNA in Pollen-Sterile Agamospermous Sugar Beet (Beta vulgaris L.) Progeny

The cytoplasm status according to mitochondrial sequence tags was determined in agamospermous sugar beet progenies characterized by unstable manifestation of cytoplasmic male sterility. The detected variations in the ratios of homologous sequences related to the N and S mtDNA types did not correlate with pollen phenotypes of the plants. Polymerase chain reaction allowed semiquantitative evaluation of these variations and their detection. A cDNA corresponding to a hitherto unknown minor RNA and containing a consensus sequence of dicotyledonous plant promoters was detected by mtRNA display. Probable sources of CMS variability in agamospermous sugar beet progenies are discussed.

Athena: a resource for rapid visualization and systematic analysis of Arabidopsis promoter sequences

To better understand the regulatory networks that control plant gene expression, tools are needed to systematically analyze and visualize promoter regulatory sequences in Arabidopsis thaliana. We have developed the Athena database, which contains 30,067 predicted Arabidopsis promoter sequences and consensus sequences for 105 previously characterized transcription factor (TF) binding sites. Athena provides four novel tools to facilitate the analysis of promoter sequences: a promoter visualization tool to enable the rapid inspection of key regulatory sequences in multiple promoters; a TF binding site enrichment tool to identify statistically over-represented TF sites occurring in a user-selected subset of promoters; a data-mining tool to rapidly select promoter sequences containing the specified combination of TF binding sites; and a tool to display the distribution of TF binding site positions in a selected set of promoter sequences.