Homologous Promoter Research Articles

Polyethylene glycol (PEG)-mediated transformation of the fused egfp-hph gene into Pleurotus ostreatus

A transformation system for the basidiomycete Pleurotus ostreatus was established using PEG-mediated transformation. The homologous glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter and terminator regions of P. ostreatus were amplified and cloned onto the restriction site of the pUC19 vector. Enhanced green fluorescent protein-encoding gene (egfp) and a selection marker, the hygromycin phosphotransferase gene (hph), were then cloned and an expression vector, pUEGFP-hph, was constructed. Protoplasts of P. ostreatus were prepared and used as the recipients in transformation procedures. An improved transformation method was established in the present study and about 100 to 200 resistant colonies per microgram DNA per 107 viable protoplasts were obtained. After subculturing, putative transformants were screened by PCR and verified by Southern blot and RT-PCR analyses showing the correct insertion into the genomic DNA and transcription of the transformed DNA. Moreover, the quantitative PCR and green fluorescence observation were carried out, thus demonstrating expression of the egfp gene driven by the homologous gpd promoter. This was the first report of transformation of P. ostreatus using homologous gpd promoter by PEG-mediated methods. These data will be helpful in future investigations using PEG-mediated transformation for functional characterisation of genes in the edible fungi basidiomycete P. ostreatus. Key words : Pleurotus ostreatus, PEG-mediated transformation, EGFP.

Sequence analysis and potentials of the native RbcS promoter in the development of an alternative eukaryotic expression system using green Microalga Ankistrodesmus convolutus.

The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS) has been used to drive the expression of β-glucuronidase (gusA) gene in this microalga. Besides the determination of the transcription start site by 5′-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2), which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif), which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.

Genetic analysis of RNA-mediated gene silencing in Arabidopsis thaliana

In plants, a particular class of small non-coding RNAs, short interfering RNAs, can serve as a signal to induce cytosine methylation at homologous genomic DNA regions in the nucleus. If the targeted DNA regions have promoter function, this RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing of transgenes provides a versatile system for the study of epigenetic gene regulation in plants. In our experimental setup in Arabidopsis thaliana, transcription of a promoter-inverted repeat provides a RNA signal that triggers de novo cytosine methylation and TGS of a homologous nopaline synthase promoter (proNOS) in trans. Utilising this two component transgene system in a forward-genetic screen for “suppressor of silencing” mutations, we were able to identify new candidates for factors involved in RdDM of transgenic as well as endogenous target regions.

Use of ChIP-Seq data for the design of a multiple promoter-alignment method

We address the challenge of regulatory sequence alignment with a new method, Pro-Coffee, a multiple aligner specifically designed for homologous promoter regions. Pro-Coffee uses a dinucleotide substitution matrix estimated on alignments of functional binding sites from TRANSFAC. We designed a validation framework using several thousand families of orthologous promoters. This dataset was used to evaluate the accuracy for predicting true human orthologs among their paralogs. We found that whereas other methods achieve on average 73.5% accuracy, and 77.6% when trained on that same dataset, the figure goes up to 80.4% for Pro-Coffee. We then applied a novel validation procedure based on multi-species ChIP-seq data. Trained and untrained methods were tested for their capacity to correctly align experimentally detected binding sites. Whereas the average number of correctly aligned sites for two transcription factors is 284 for default methods and 316 for trained methods, Pro-Coffee achieves 331, 16.5% above the default average. We find a high correlation between a method's performance when classifying orthologs and its ability to correctly align proven binding sites. Not only has this interesting biological consequences, it also allows us to conclude that any method that is trained on the ortholog data set will result in functionally more informative alignments.

GPMiner: an integrated system for mining combinatorial cis-regulatory elements in mammalian gene group

BackgroundSequence features in promoter regions are involved in regulating gene transcription initiation. Although numerous computational methods have been developed for predicting transcriptional start sites (TSSs) or transcription factor (TF) binding sites (TFBSs), they lack annotations for do not consider some important regulatory features such as CpG islands, tandem repeats, the TATA box, CCAAT box, GC box, over-represented oligonucleotides, DNA stability, and GC content. Additionally, the combinatorial interaction of TFs regulates the gene group that is associated with same expression pattern. To investigate gene transcriptional regulation, an integrated system that annotates regulatory features in a promoter sequence and detects co-regulation of TFs in a group of genes is needed.ResultsThis work identifies TSSs and regulatory features in a promoter sequence, and recognizes co-occurrence of cis-regulatory elements in co-expressed genes using a novel system. Three well-known TSS prediction tools are incorporated with orthologous conserved features, such as CpG islands, nucleotide composition, over-represented hexamer nucleotides, and DNA stability, to construct the novel Gene Promoter Miner (GPMiner) using a support vector machine (SVM). According to five-fold cross-validation results, the predictive sensitivity and specificity are both roughly 80%. The proposed system allows users to input a group of gene names/symbols, enabling the co-occurrence of TFBSs to be determined. Additionally, an input sequence can also be analyzed for homogeneity of experimental mammalian promoter sequences, and conserved regulatory features between homologous promoters can be observed through cross-species analysis. After identifying promoter regions, regulatory features are visualized graphically to facilitate gene promoter observations.ConclusionsThe GPMiner, which has a user-friendly input/output interface, has numerous benefits in analyzing human and mouse promoters. The proposed system is freely available at http://GPMiner.mbc.nctu.edu.tw/.

A Machine Learning Approach To Identify Hydrogenosomal Proteins in Trichomonas vaginalis

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most widespread nonviral sexually transmitted disease in humans. It possesses hydrogenosomes-anaerobic mitochondria that generate H(2), CO(2), and acetate from pyruvate while converting ADP to ATP via substrate-level phosphorylation. T. vaginalis hydrogenosomes lack a genome and translation machinery; hence, they import all their proteins from the cytosol. To date, however, only 30 imported proteins have been shown to localize to the organelle. A total of 226 nuclear-encoded proteins inferred from the genome sequence harbor a characteristic short N-terminal presequence, reminiscent of mitochondrial targeting peptides, which is thought to mediate hydrogenosomal targeting. Recent studies suggest, however, that the presequences might be less important than previously thought. We sought to identify new hydrogenosomal proteins within the 59,672 annotated open reading frames (ORFs) of T. vaginalis, independent of the N-terminal targeting signal, using a machine learning approach. Our training set included 57 gene and protein features determined for all 30 known hydrogenosomal proteins and 576 nonhydrogenosomal proteins. Several classifiers were trained on this set to yield an import score for all proteins encoded by T. vaginalis ORFs, predicting the likelihood of hydrogenosomal localization. The machine learning results were tested through immunofluorescence assay and immunodetection in isolated cell fractions of 14 protein predictions using hemagglutinin constructs expressed under the homologous SCSα promoter in transiently transformed T. vaginalis cells. Localization of 6 of the 10 top predicted hydrogenosome-localized proteins was confirmed, and two of these were found to lack an obvious N-terminal targeting signal.

Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci

The strength of recombinant gene expression is a key property of cell lines for biopharmaceutical protein production. In most stable cell lines the expression vector is stably introduced into the host chromosomal DNA. Apart from the copy number and the used expression control elements the performance of recombinant expression vectors is modulated by genetic and epigenetic features provided by flanking host elements. Since targeted integration is very difficult cell clones with high expression of a recombinant vector are created by random integration and large scale screening for gene expression. This allows the isolation of those rare recombinant cells in which gene expression is optimal. This is usually due to locus-specific influences of the chromosomal surroundings. We have developed an efficient methodology for targeting expression cassettes to specific chromosomal sites [1,2]]. The method (Flp recombinase mediated cassette exchange - RMCE) allows the repeated use of defined loci by targeting constructs for expression of proteins and viruses, thereby allowing to exploit the positive features of a given integration site [3,4]]. Thereby, a systematic evaluation of the performance of a set of expression vectors in various chromosomal sites becomes feasible. In this study we screened for high performance integration sites in HEK293 and CHO-K1 cells supporting expression cassettes driven by a potent promoter. As a read out, production of antibodies and recombinant retroviral vectors were used. Thereby, we could show that high level expression of a given promoter is restricted to defined integration sites, while other sites show only moderate expression (data not shown). An important new finding was that a given chromosomal site is not flexible with respect to the integrated cassette but requires the integration of specific promoters. As illustrated in figure ​figure1,1, an integration site, identified for supporting high level expression of an SV40 promoter driven cassette fails to adequately support expression of MPSV and adenoviral major late promoter (AdmlP). Vice versa, another site, initially screened for supporting MSCV promoter expression, could restore expression of the highly homologous MPSV promoter while the SV40 promoter and the AdmlP promoter give only moderate expression. Figure 1 Mode of tagging defines the optimal targeting cassette. Two highly potent chromosomal integration sites were screened upon random integration of an expression cassette driven by the SV40 promoter and the MSCV promoter, respectively. By means of Flp recombinase ... Finally, we tested the impact of the orientation of the cassette in a specific chromosomal site. We found that some integration sites are flexible with respect to the orientation of the expression cassettes while others support expression only in one direction (data not shown). While classical enhancer elements are known to activate promoters largely independent from the relative position this finding suggests that other cis acting elements affect the incoming cassettes in an orientation dependent manner. Together, this shows that not the nature of integration site and the design of the vector as such define the performance of a producer cell clone. Rather, the interplay between these components defines the level and stability of expression. Since these interactions cannot be predicted, the performance of a vector in a given site has to be evaluated empirically. In order to exploit favourable sets of chromosomal sites and vectors we made use of bacterial artificial chromosome (BAC) vectors. By recombineering, expression cassettes were integrated into pre-selected chromosomal sites as encoded by BAC vectors. These vectors were randomly integrated into cells by standard transfection protocols. Clones were isolated and evaluated for expression. As expected, highly reproducible expression characteristics were found in individual clones (data not shown). In conclusion, the definition of favorable combinations of specific integration sites and vector design allow the rational exploitation of given chromosomal sites. For this purpose, technologies for site specific genetic manipulation of mammalian cells are essential. This concerns both targeted integration of expression cassettes into defined loci (such as RMCE or site specific nuclease induced homologous recombination) or by transduction of large chromosomal domains (as provided by BAC vectors). These technologies pave the way for predictable and high expression of biotechnologically relevant products such as antibodies and recombinant viral vectors.

AGO6 Functions in RNA-Mediated Transcriptional Gene Silencing in Shoot and Root Meristems in Arabidopsis thaliana

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

Co-expression of multiple target proteins in plants from a tobacco mosaic virus vector using a combination of homologous and heterologous subgenomic promoters

To co-express multiple target proteins, we engineered a single-component chimeric tobacco mosaic virus (TMV)-based vector containing homologous and heterologous capsid protein subgenomic RNA promoters. Delivery of this vector into Nicotiana benthamiana plants via agroinfiltration resulted in co-expression of two reporter genes within a single cell. Furthermore, co-expression of a host-specific antisense RNA or a silencing suppressor protein from this vector augmented the accumulation of green fluorescent protein or a vaccine antigen, hemagglutinin from avian influenza virus A/Vietnam/1194/04. These findings suggest that this chimeric vector utilizing the homologous and heterologous subgenomic TMV promoters has a potential for high-level production of multiple therapeutic proteins including monoclonal antibodies.

An Evolutionarily Conserved TNF-α–Responsive Enhancer in the Far Upstream Region of Human CCL2 Locus Influences Its Gene Expression

Comparative cross-species genomic analysis has served as a powerful tool to discover novel noncoding regulatory regions that influence gene expression in several cytokine loci. In this study, we have identified several evolutionarily conserved regions (ECRs) that are shared between human, rhesus monkey, dog, and horse and that are upstream of the promoter regions that have been previously shown to play a role in regulating CCL2 gene expression. Of these, an ECR that was ~16.5 kb (-16.5 ECR) upstream of its coding sequence contained a highly conserved NF-κB site. The region encompassing the -16.5 ECR conferred TNF-α responsiveness to homologous and heterologous promoters. In vivo footprinting demonstrated that specific nucleotide residues in the -16.5 ECR were protected or became hypersensitive after TNF-α treatment. The footprinted regions were found to bind NF-κB subunits in vitro and in vivo. Mutation/deletion of the conserved NF-κB binding site in the -16.5 ECR led to loss of TNF-α responsiveness. After TNF-α stimulation, the -16.5 ECR showed increased sensitivity to nuclease digestion and loss of histone signatures that are characteristic of a repressive chromatin. Chromosome conformation capture assays indicated that -16.5 ECR physically interacts with the CCL2 proximal promoter after TNF-α stimulation. Taken together, these results suggest that the -16.5 ECR may play a critical role in the regulation of CCL2.

Transformation of, and Heterologous Protein Expression in, Lactobacillus agilis and Lactobacillus vaginalis Isolates from the Chicken Gastrointestinal Tract

Lactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derived Lactobacillus strains, Lactobacillus agilis La3, Lactobacillus vaginalis Lv5, and Lactobacillus crispatus Lc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 10(8) and 10(6) transformants per microgram of plasmid DNA, respectively. The third strain tested, L. crispatus Lc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter gene gfp was analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3 ldhL promoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression in L. agilis and L. vaginalis. The ability of these strains to express heterologous proteins in vitro indicates their potential utility as in vivo delivery vectors for therapeutic peptides to the chicken gastrointestinal tract.

Abstract 5734: Prolonged activation of NFκB (RelA and RelB), BCL10, and IL-8 following exposure to common food additive carrageenan in human colonic epithelial cells may be attributable to NFκB binding site in BCL10 promoter

Abstract The common food additive carrageenan (CGN) is a sulfated polysaccharide that resembles the naturally occurring sulfated glycosaminoglycans (sGAG), particularly chondroitin sulfate and keratan sulfate. Unlike the naturally occurring sGAG, CGN contains alpha-1,3-galactosidic linkages that are immunogenic in humans. Previously, we reported that CGN activation of a TLR4-MyD88 mediated inflammatory cascade proceeds through BCL10 and NFκB (p65), leading to increased secretion of IL-8 in human colonic epithelial cells. Translocations involving BCL10 and MALT1 are found in mucosa-associated lymphoid tissue (MALT) lymphomas, associated with constitutive activation of NFκB. Since CGN has been widely used to induce colitis in animal models of inflammatory bowel disease, we addressed whether CGN exposure can lead to sustained activation of BCL10 and NFκB, consistent with a model of constitutive activation of inflammation and possible pre-malignant changes. Experiments were performed with NCM460 cells, a human colonic epithelial cell line derived from normal colon, in M3:10A medium (Incell) with λCGN (1μg/ml). Triplicate cell preparations and spent media from untreated control cell preparations, cells treated with CGN for only the initial 12 hour time interval, and cells treated for six 12-hour periods were harvested every 12 hours over 72 hours, and media, with or without CGN was replenished. IL-8 in the spent media, and BCL10 in the cytosolic preparations and NFκB [RelA (p65) and RelB] in the nuclear preparations were measured at successive 12-hour periods. IL-8 remained significantly increased for an additional 36 hours following withdrawal of CGN. BCL10 and RelA remained significantly increased for an additional 24 hours, and RelB was significantly increased for an additional 12 hours. These findings are consistent with previous data that demonstrate that CGN activates both canonical and non-canonical pathways of NFκB. The presence of a sequence with high homology to the established NFκB consensus oligonucleotide sequence in the BCL10 promoter may be responsible for an ongoing process of activation following withdrawal of the CGN exposure. In vitro testing of binding of RelA and RelB to the NFκB consensus oligonucleotide, the homologous BCL10 promoter sequence, a mutated BCL10 promoter sequence, and a scrambled NFκB consensus sequence indicates over 50% binding of either RelA or RelB to the BCL10 promoter sequence, compared to 100% to the established NFκB consensus sequence. Hence, the study findings demonstrate that an environmental exposure to a common food additive (CGN) can trigger prolonged activation of an inflammatory loop involving BCL10, RelA, RelB, and IL-8 in human colonic epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5734.

Two Highly Homologous Promoters of a Squash Aspartic Protease Inhibitor (SQAPI) Multigene Family Exhibit Differential Expression in Transgenic Tobacco Phloem and Trichome Cells

Two variants of the promoter of the squash aspartic acid protease inhibitor multigene family were isolated from Cucurbita maxima cv. ‘Supermarket’ Hybrid genomic DNA. The isolated promoters, possibly not full length, comprised a 5′-untranslated region (UTR) of 202–208 bp, contained a 63-bp upstream open reading frame (uORF) and the immediate upstream sequences of 441–445 bp. The two promoters contained several small deletions relative to each other and 22 single base differences but exhibit overall 92.5% homology over 654 bp. When the promoters were fused to a β-glucuronidase reporter gene and expressed in tobacco, one variant was highly expressed in the companion cells of the inner and outer phloem of leaves and at lower levels in other organs. The other variant was expressed at high levels in the long glandular trichomes of the leaf. Deletion analysis identified a region of ~280 bp immediately upstream of the 5′-UTR containing the TATA box that was responsible for phloem specific expression and a further region of ~180 bp that enhanced expression in one promoter and conferred trichome expression in the other. Removal of the 5′-UTR, including the uORF, inactivated the phloem promoter.

Transfection of Eimeria and Toxoplasma using heterologous regulatory sequences

Eimeria tenella and Toxoplasma gondii are Apicomplexan protozoa and share many similarities in biology and genomics. While the latter parasites are easily cultured in vitro and genetically manipulated, many Eimeria species are difficult to grow in vitro. We hypothesised that molecular tools for the genetic manipulation of T. gondii could be applied to the study of Eimeria parasites . Here we show that three different promoter sequences originating from E. tenella could function effectively not only in other species of the Eimeria genus (histone H4) but also in T. gondii (histone H4, actin and tubulin). Similarly, promoters of the “housekeeping” gene (tubulin) and differentially regulated gene (surface antigen gene, sag1) of T. gondii were effective in driving the expression of the yellow fluorescent protein (YFP) maker gene in E. tenella. The transfection efficiency with heterologous regulatory sequences was similar to that with homologous promoters; while the promoter strength of heterologous vectors is slightly weaker than the homologous vectors in both E. tenella and T. gondii. The results suggest that 5′ regulatory sequences are functionally conserved not only among the Eimeria species, but also between T. gondii and E. tenella, and that T. gondii could be used as a novel transfection check system for Eimeria-rooted vectors, accelerating the development of reverse genetics in Eimeria spp.

Microarray transfection analysis of conserved genomic sequences from three immediate early genes

In an effort to define novel transcriptional regulatory elements, microarray cotransfection was used to functionally characterize conserved non-coding sequences (CNSs) of three immediate early genes: c- fos, JunB and EGR-1. Cotransfection of fluorescent CNS reporter constructs and expression vectors for constitutively active signaling proteins demonstrated that many of the CNSs alter both the basal and regulated expressions of reporter constructs, but the effects of these CNSs were usually specific for their homologous promoter. One CNS located in the first intron of the c- fos gene conferred regulation by cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG) and Raf. Mutagenesis and cotransfection experiments showed that PKA regulation of this c- fos intronic element was mediated by two adjacent CRE-like sequences and the transcription factor CREB. In the context of a reporter containing previously characterized regulatory elements, the novel intronic sequence contributed 50% of the transcriptional response to PKA. These studies suggest that microarray transfection studies may be useful in functional characterization of conserved genomic sequences on a larger scale.

Modulation of Mrps12/ Sarsm promoter activity in response to mitochondrial stress

Transcription from the bidirectional promoter of two mouse genes encoding components of the mitochondrial translational apparatus, mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm), was shown previously to be dependent upon an array of four CCAAT boxes, interacting with the transcription factor NF-Y. Here we report that the homologous human promoter is governed by a CCAAT box array acting in an essentially similar manner. Analysis of the transcriptional response of both the human and mouse promoters to various mitochondrially acting toxins, including inhibitors of mitochondrial protein synthesis, and agents that bring about uncoupling or respiratory chain inhibition, produced either of two distinct outcomes, depending on the cell type and the conditions used. In mouse C2C12 myoblasts, human HEK293 cells or U2OS osteosarcoma cells, plus HeLa cells at high drug doses or mouse 3T3 fibroblasts subjected to prolonged drug exposure, a dose-dependent, bidirectional suppression of transcription was observed. In 3T3 cells subjected only to pre-treatment with the drugs, bidirectional Mrps12/ Sarsm promoter activity was strongly stimulated. A similar, though weaker stimulation was observed at lower drug doses in HeLa cells. Reporter studies using mutated variants of the mouse promoter construct indicated that the stimulation of promoter activity in response to mitochondrial OXPHOS stress in 3T3 cells was independent of the CCAAT box array and of putative binding sites for NRF-2, AP-1 and other transcription factors, whereas transcriptional downregulation under prolonged mitochondrial stress was CCAAT box-dependent. Promoter stimulation was correlated with mitochondrial ROS production, which may be a crucial component in its signalling.

Barley grain maturation and germination: metabolic pathway and regulatory network commonalities and differences highlighted by new MapMan/PageMan profiling tools.

Plant seeds prepare for germination already during seed maturation. We performed a detailed transcriptome analysis of barley (Hordeum vulgare) grain maturation, desiccation, and germination in two tissue fractions (starchy endosperm/aleurone and embryo/scutellum) using the Affymetrix Barley1 GeneChip. To aid data evaluation, Arabidopsis thaliana MapMan and PageMan tools were adapted to barley. The analyses allow a number of conclusions: (1) Cluster analysis revealed a smooth transition in transcription programs between late seed maturation and germination within embryo tissues, but not in the endosperm/aleurone. (2) More than 12,000 transcripts are stored in the embryo of dry barley grains, many of which are presumably activated during germination. (3) Transcriptional activation of storage reserve mobilization events occurs at an early stage of germination, well before radicle protrusion. (4) Key genes of gibberellin (GA) biosynthesis are already active during grain maturation at a time when abscisic acid peaks suggesting the formation of an endogenous store of GA in the aleurone. This GA probably acts later during germination in addition to newly synthesized GA. (5) Beside the well-known role of GA in gene activation during germination spatiotemporal expression data and cis-element searches in homologous rice promoters confirm an equally important gene-activating role of abscisic acid during this developmental period. The respective regulatory webs are linked to auxin and ethylene controlled networks. In summary, new bioinformatics PageMan and MapMan tools developed in barley have been successfully used to investigate in detail the transcriptome relationships between seed maturation and germination in an important crop plant.

Transcriptional co-expression and co-regulation of genes coding for components of the oxidative phosphorylation system

BackgroundThe mitochondrial oxidative phosphorylation (OXPHOS) is critical for energy (ATP) production in eukaryotic cells. It was previously shown that genes coding for mitochondrial proteins involved in energy production co-express at the RNA level. Because the OXPHOS enzymes are multimeric complexes, we tested the hypothesis that genes coding for components of specific complexes are also co-regulated at the transcriptional level, and share common regulatory elements in their promoters.ResultsWe observed for the first time that, not only OXPHOS genes as a group co-express, but there is a co-expression of genes within each of the five OXPHOS enzyme complexes, showing a higher degree of complexity in gene co-regulation. In silico analysis of homologous promoter sequences in mammals identified the likely core promoter elements for most genes encoding OXPHOS subunits/assembly factors. The results included a significant abundance of previously identified sites (e.g. NRF1, NRF2, ERRA and YY1), as well as several sites that had not been previously detected. Although we identified patterns that correlated to OXPHOS gene expression, we did not detect an OXPHOS complex-specific arrangement of transcription factor binding sites within the core promoter that could explain the tight co-expression of these functionally related genes.ConclusionThis study mapped the core promoters of most OXPHOS related genes and provided an example of gene expression regulation based on the final protein arrangement within a linear metabolic pathway.

Efficient and stable transgene suppression via RNAi in field-grown poplars

The efficiency and stability of RNA interference (RNAi) in perennial species, particularly in natural environments, is poorly understood. We studied 56 independent poplar RNAi transgenic events in the field over 2 years. A resident BAR transgene was targeted with two different types of RNAi constructs: a 475-bp IR of the promoter sequence and a 275-bp IR of the coding sequence, each with and without the presence of flanking matrix attachment regions (MARs). RNAi directed at the coding sequence was a strong inducer of gene silencing; 80% of the transgenic events showed more than 90% suppression. In contrast, RNAi targeting the promoter resulted in only 6% of transgenic events showing more than 90% suppression. The degree of suppression varied widely but was highly stable in each event over 2 years in the field, and had no association with insert copy number or the presence of MARs. RNAi remained stable during a winter to summer seasonal cycle, a time when expression of the targeted transgene driven by an rbcS promoter varied widely. When strong gene suppression was induced by an IR directed at the promoter sequence, it was accompanied by methylation of the homologous promoter region. DNA methylation was also observed in the coding region of highly suppressed events containing an IR directed at the coding sequence; however, the methylation degree and pattern varied widely among those suppressed events. Our results suggest that RNAi can be highly effective for functional genomics and biotechnology of perennial plants.

Production of l-alanine by metabolically engineered Escherichia coli

Escherichia coli W was genetically engineered to produce L: -alanine as the primary fermentation product from sugars by replacing the native D: -lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native D: -lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of L: -alanine to D: -alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l(-1) glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g(-1) glucose) with a chiral purity greater than 99.5% L: -alanine.