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PubMed1983

A protected region upstream and limited nucleosomal positioning downstream of the transcription initiation region of the yeast 35S ribosomal gene.

To locate nucleosome positions around the transcription initiation site of 35S rDNA in yeast, single and double cleavage site data for staphylococcal nuclease and single cleavage site data for DNase I were obtained by methods involving some modifications of the usual indirect end-labeling procedure. Results show that immediately upstream of the initiation site there is an approximately 225 base pair (bp) stretch of chromatin which is quite resistant to nuclease digestion. The nucleosomal domain begins on the coding sequences, at the downstream end of the resistant region, with the major locus 30 bp and a minor locus 5 bp downstream of the initiation site in growing cells. The location of the major locus is corroborated by analysis within the coding sequences, although results within the gene are less convincing because of a strong similarity of chromatin and naked DNA digestion profiles. The staphylococcal nuclease cleavage maps were tested by their ability to reproduce chromatin digest profiles and to explain the presence or absence, depending on which parts of the region were used as probes, of specific bands within the pattern. The site of the major upstream locus of the nucleosomal domain differs in growing and stationary cells. This shift changes the accessibility of approximately 25 bp of DNA and may be associated with the expression of these genes.

Base Composition↗
PubMed2003

Elements located upstream and downstream of the major splice donor site influence the ability of HIV-2 leader RNA to dimerize in vitro.

An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1, a 5' leader region site termed stem-loop 1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5'-end of the primer-binding site (PBS) and a stem-loop region homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization and electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5'- and/or 3'-ends and by making compensatory base substitutions, we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1-dependent tight dimers, probably by sequestering SL1 in a stable intramolecular arrangement. Moreover, we found that nucleotides downstream of SL1 decreased the rate of tight dimerization. Interestingly, dimerization at 37 degrees C in the presence of nucleocapsid protein increased the yield of SL1-mediated tight dimerization in vitro, even in the presence of the two interfering elements, suggesting a relationship between the nucleocapsid protein and activation of the SL1 dimerization signal in vivo.

3' Untranslated Regions↗
PubMed1997

Evidence supporting a role for microfilaments in regulating the coupling between poorly dissociable IgE-Fc epsilonRI aggregates downstream signaling pathways.

Aggregation of Fc epsilonRI, the high-affinity receptor for IgE, on RBL-2H3 mast cells caused by reversible ligands such as multivalent antigen causes cellular responses that can be halted by subsequent addition of excess monovalent ligand. In contrast, Ca2+ and degranulation responses elicited by effectively irreversible streptavidin cross-linking of biotinylated IgE-Fc epsilonRI are not stopped by addition of excess biotin after stimulation is initiated. These results support previous conclusions based on studies with covalent oligomers of IgE that stable cross-links can continue to deliver stimulatory signals for extended periods of time. Dissociation measured in the presence of monovalent hapten reveals two populations of IgE-Fc epsilonRI cross-linked by multivalent antigen that differ in functional effectiveness. Aggregates with readily dissociable cross-links are normally responsible for triggering essentially all of the degranulation response, whereas aggregates with poorly dissociable cross-links apparently do not trigger this response. Treatment of RBL-2H3 cells with cytochalasin D, an inhibitor of actin polymerization, enhances downstream signaling and enables the less readily dissociable aggregates to stimulate Ca2+ and degranulation responses. Under these conditions, cytochalasin D does not affect hapten-mediated dissociation of multivalent antigen, nor does it prevent hapten from reversing tyrosine phosphorylation of Syk. Cytochalasin D alone causes tyrosine phosphorylation of a protein at approximately 75 kDa, and it reduces hapten-induced reversal of antigen-stimulated tyrosine phosphorylation of several other proteins. Taken together, these results indicate that stimulated actin polymerization normally regulates the coupling of aggregated Fc epsilonRI to downstream signaling pathways, and they provide an explanation for seeming discrepancies between responses to stable and reversible cross-links.

Actin Cytoskeleton↗
PubMedDate not supplied

Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation.

Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg.L(-)(1) in the whole broth and a biomass concentration of 150 g.L(-)(1) cell dry weight, thus comprising a space-time yield of 5.2 mg.L(-)(1).h(-)(1). Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased space-time yield of 25 mg.L(-)(1).h(-)(1) while reducing the cell dry weight concentration from 150 g.L(-)(1) in fed-batch to 65 g.L(-)(1) in continuous cultivation.

Chymotrypsinogen↗
PubMedDate not supplied

MINLP models for the synthesis of optimal peptide tags and downstream protein processing.

The development of systematic methods for the synthesis of downstream protein processing operations has seen growing interest in recent years, as purification is often the most complex and costly stage in biochemical production plants. The objective of the work presented here is to develop mathematical models based on mixed integer optimization techniques, which integrate the selection of optimal peptide purification tags into an established framework for the synthesis of protein purification processes. Peptide tags are comparatively short sequences of amino acids fused onto the protein product, capable of reducing the required purification steps. The methodology is illustrated through its application on two example protein mixtures involving up to 13 contaminants and a set of 11 candidate chromatographic steps. The results are indicative of the benefits resulting by the appropriate use of peptide tags in purification processes and provide a guideline for both optimal tag design and downstream process synthesis.

Amino Acid Sequence↗
PubMedDate not supplied

Downstream process synthesis for biochemical production of butanol, ethanol, and acetone from grains: generation of optimal and near-optimal flowsheets with conventional operating units.

Manufacturing butanol, ethanol, and acetone through grain fermentation has been attracting increasing research interest. In the production of these chemicals from fermentation, the cost of product recovery constitutes the major portion of the total production cost. Developing cost-effective flowsheets for the downstream processing is, therefore, crucial to enhancing the economic viability of this manufacturing method. The present work is concerned with the synthesis of such a process that minimizes the cost of the downstream processing. At the outset, a wide variety of processing equipment and unit operations, i.e., operating units, is selected for possible inclusion in the process. Subsequently, the exactly defined superstructure with minimal complexity, termed maximal structure, is constructed from these operating units with the rigorous and highly efficient graph-theoretic method for process synthesis based on process graphs (P-graphs). Finally, the optimal and near-optimal flowsheets in terms of cost are identified.

Acetone↗
PubMed1999

Upstream and downstream sequence elements determine the specificity of the rice tungro bacilliform virus promoter and influence RNA production after transcription initiation.

The contribution of sequences upstream and downstream of the transcription start site to the strength and specificity of the promoter of rice tungro bacilliform virus was analysed in transgenic rice plants. The promoter is strongly stimulated by downstream sequences which include an intron and is active in all vascular and epidermal cells. Expression in the vascular tissue requires a promoter element located between -100 and -164 to which protein(s) from rice nuclear extracts bind. Elimination of this region leads to specificity for the epidermis. Due to the presence of a polyadenylation signal in the intron, short-stop RNA is produced from the promoter in addition to full-length primary transcript and its spliced derivatives. The ratio between short-stop RNA and full-length or spliced RNA is determined by upstream promoter sequences, suggesting the assembly of RNA polymerase complexes with different processivity on this promoter.

Badnavirus↗
PubMed2003

Matrix attachment regions enhance transcription of a downstream transgene and the accessibility of its promoter region to micrococcal nuclease.

Nuclear matrix attachment regions (MARs) are thought to influence the expression of flanking genes. In this study, we investigated the activation of genes by tobacco MARs that had previously been identified in the 5' region of the basic class I chitinase gene, CHNS0. In transgenic tobacco cells, a construct consisted of the 35S promoter of cauliflower mosaic virus (CaMV) fused to a beta-glucuronidase gene (uidA) with 5' MAR elements was expressed at a 10-fold higher level than a similar construct without MAR sequences. However, expression of a similar construct with 3' MARs and of a construct with a truncated (-46) 35S minimal promoter and uidA with 5' MARs was not similarly enhanced, suggesting that MARs might act by increasing the activity of downstream enhancers. Deletion analysis of the MAR sequences revealed that the function of the MARs that increased the expression of the transgene was redundant. Moreover, assays of the transient expression of transgenes suggested that MAR elements might be involved in the structure and organization of chromatin. To examine the influence of MARs on chromatin structure, we investigated the effects of micrococcal nuclease (MNase) on the DNA in the reporter gene around the MARs. Analysis of the time-course of digestion of nuclei with MNase revealed that the 35S promoter region with 5' MARs was much more sensitive to MNase than the same region without MARs, suggesting that MARs might mediate the opening of chromatin in the region of a downstream promoter, with consequent enhancement of transcription.

Chitinases↗
PubMed2003

A vector with the downstream box of the initiation codon can highly enhance protein expression in Escherichia coli.

An expression vector, pET-DB, with a perfectly matching downstream box of the initiation codon has been constructed on the basis of the pET system. Any gene of interest can then be inserted into the vector. Four genes were used to test the expression efficiency of the vector. The results show that the vector pET-DB can further increase protein expression level at least up to 35-70% as compared with the initial T7 expression system, indicating that the downstream box can enhance protein expression in Escherichia coli.

Adenylate Kinase↗
PubMed2004

Nucleotides downstream of start codons show marked non-randomness in Escherichia coli but not in Bacillus subtilis.

This study aimed at measuring the nucleotide non-randomness in the region downstream of start codons in bacterial genes and to see if the non-randomness differs between biased and unbiased genes, in terms of the effective number of codons (Nc) and the codon adaptation index (CAI). In Escherichia coli, there was a marked elevation in nucleotide conservation for the genes having low Nc-values compared to the genes having high Nc-values, i.e the more biased genes showed a higher level of non-randomness. Likewise, the genes displaying high CAI-values showed stronger nucleotide conservation than the genes of low CAI-values. This elevated conservation is visible up to approximately 15-17 nucleotides downstream of the start codon, after which there is little difference. This indicates that there may be distinct selectional mechanisms acting upon the first 5-6 codons within genes in E. coli. In B. subtilis, these effects are less pronounced, if present at all. Furthermore, analyses of codons used in this region were not in support of the hypothesis that the elevation in nucleotide non-randomness is a question of selection for certain optimal codons.

Bacillus subtilis↗
PubMed2001

Telomere looping permits gene activation by a downstream UAS in yeast.

In yeast (Saccharomyces cerevisiae), transcriptional activators, such as Gal4 and Gal4-VP16, work ordinarily from sites located in the upstream activating sequence (UAS) positioned about 250 base pairs upstream of the transcription start site. In contrast to their behaviour in mammalian cells, however, such activators fail to work when positioned at distances greater than approximately 600-700 base pairs upstream, or anywhere downstream of the gene. Here we show that, in yeast, a gene bearing an enhancer positioned 1-2 kilobases downstream of the gene is activated if the reporter is linked to a telomere, but not if it is positioned at an internal chromosomal locus. These observations are explained by the finding that yeast telomeres form back-folding, or looped, structures. Because yeast telomeric regions resemble the heterochromatin found in higher eukaryotes, these findings might also explain why transcription of some higher eukaryotic genes depends on their location in heterochromatin.

Chromatin↗
PubMed1997

pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila.

Members of the Wnt/Wingless (Wg) family of signalling proteins organize many aspects of animal development by regulating the expression of particular target genes in responding cells. Recent biochemical studies indicate that the vertebrate HMG-domain proteins Lef-1 and XTcf-3 can physically interact with beta-catenin, a homologue of Drosophila Armadillo (Arm), the most downstream component known in the Wnt signal transduction pathway. However, these studies do not address whether the endogenous Lef/Tcf family members are required in vivo to transduce Wnt signals. Using genetic methods in Drosophila, we define a new segment polarity gene, pangolin (pan), and show that its product is required in vivo for Wg signal transduction in embryos and in developing adult tissues. In addition, we show that pan encodes a Lef/Tcf homologue and provide evidence that its protein product binds to the beta-catenin homologue Armadillo in vivo. Finally, we demonstrate that Pan functions downstream of Arm to transduce the Wg signal. Thus, our results indicate that Pan is an essential component of the Wg transduction pathway and suggest that it acts directly to regulate gene transcription in response to Wg signalling.

Amino Acid Sequence↗
PubMed2001

Activation of caspase-8 in 3-deazaadenosine-induced apoptosis of U-937 cells occurs downstream of caspase-3 and caspase-9 without Fas receptor-ligand interaction.

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.

Amino Acid Chloromethyl Ketones↗
PubMed2006

UBD, a downstream element of FOXP3, allows the identification of LGALS3, a new marker of human regulatory T cells.

Here, we report the identification of the ubiquitin-like gene UBD as a downstream element of FOXP3 in human activated regulatory CD4(+)CD25(hi) T cells (T(reg)). Retroviral transduction of UBD in human allo-reactive effector CD4(+) T helper (T(h)) cells upregulates CD25 and mediates downregulation of IL4 and IL5 expression similar to overexpression of FOXP3. Moreover, UBD impairs T(h) cell proliferation without upregulation of FOXP3 and impairs calcium mobilization. In the presence of ionomycin, overexpression of UBD in T(h) cells leads to the induction of IL1R2 that resemble FOXP3-transduced T(h) cells and naturally derived T(reg) cells. A comparison of the transcriptome of FOXP3- and UBD-transduced T(h) cells with T(reg) cells allowed the identification of the gene LGALS3. However, high levels of LGALS3 protein expression were observed only in human CD4(+)CD25(hi) derived T(reg) cells and FOXP3-transduced T(h) cells, whereas little was induced in UBD-transduced T(h) cells. Thus, UBD contributes to the anergic phenotype of human regulatory T cells and acts downstream in FOXP3 induced regulatory signaling pathways, including regulation of LGALS3 expression. High levels of LGALS3 expression represent a FOXP3-signature of human antigen-stimulated CD4(+)CD25(hi) derived regulatory T cells.

Biomarkers↗
PubMed2004

Aberration of epidermal growth factor receptor expression in bone and soft-tissue tumors: protein overexpression, gene amplification and activation of downstream molecules.

In order to evaluate the involvement of epidermal growth factor receptor, and to analyze the correlation between gene aberration and protein expression in mesenchymal tumors, we examined protein expression by immunohistochemistry in 125 cases of bone and soft-tissue tumors. Furthermore, amplification of epidermal growth factor receptor gene was determined by fluorescence in situ hybridization. Positive immunostaining was found in 23 cases (18.4%). Among these 23 cases, one of malignant fibrous histiocytoma showed the highest degree (3+) of protein overexpression and gene amplification as clusters of hybridization signals, indicating homogeneously staining regions. The second case of malignant fibrous histiocytoma also showed a higher degree (2+) of overexpression and coamplification of the epidermal growth factor receptor gene with the centromeric regions, indicating polysomy of chromosome 7. The levels of expression observed in immunohistochemistry were confirmed by immunoblotting and found to be comparable. Moreover, although expression of phosphorylated epidermal growth factor receptor was detected in those two cases of malignant fibrous histiocytoma, constitutive activation of extracellular signal-related protein kinase 1/2 was not observed, suggesting that activation of epidermal growth factor receptor does not necessarily and constantly lead to signal transduction to the downstream molecules. In the remaining 123 cases, including 21 cases exhibiting weak (1+) immunoreactivity, no gene amplification nor polysomy was found. Collectively, expression of epidermal growth factor receptor was observed not infrequently in mesenchymal tumors, but 'overexpression' is rare and can be attributed to an increase in gene copy number, resulting from amplification or polysomy. Although cases that scored positive for protein expression and/or gene amplification could be qualified candidates for antiepidermal growth factor receptor therapies, further examination of the status of downstream molecules in the signal cascade, such as phosphorylated epidermal growth factor receptor and extracellular signal-related protein kinase 1/2, may be required as the process of therapeutic strategy.

Blotting, Western↗
PubMed2006

Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence.

p53 limits the proliferation of primary diploid fibroblasts by inducing a state of growth arrest named replicative senescence - a process which protects against oncogenic transformation and requires integrity of the p53 tumour suppressor pathway. However, little is known about the downstream target genes of p53 in this growth-limiting response. Here, we report that suppression of the p53 target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference (RNAi) leads to escape from replicative senescence both in primary mouse embryo fibroblasts and primary human BJ fibroblasts. PAI-1 knockdown results in sustained activation of the PI(3)K-PKB-GSK3beta pathway and nuclear retention of cyclin D1, consistent with a role for PAI-1 in regulating growth factor signalling. In agreement with this, we find that the PI(3)K-PKB-GSK3beta-cyclin D1 pathway is also causally involved in cellular senescence. Conversely, ectopic expression of PAI-1 in proliferating p53-deficient murine or human fibroblasts induces a phenotype displaying all the hallmarks of replicative senescence. Our data indicate that PAI-1 is not merely a marker of senescence, but is both necessary and sufficient for the induction of replicative senescence downstream of p53.

Animals↗
PubMed1997

Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis.

Apoptotic cell death is driven by ICE family proteases (caspases) and negatively regulated by Bcl-2 family proteins. Although it has been shown that Bcl-2 exerts anti-apoptotic activity by blocking a step(s) leading to the activation of caspases, a role for Bcl-2 and Bcl-xL downstream of the caspase cascade has remained unclear. Here, we show that purified active caspase-3 (CPP32/Yama/apopain) and caspase-1 (ICE) induces apoptosis when microinjected into the cytoplasm of cells, confirming our recent observations, and that the apoptosis is not at all prevented by Bcl-2 and Bcl-xL, which are overexpressed more than sufficiently to prevent Fas-mediated and overexpressed procaspase-1-mediated apoptosis. Thus, Bcl-2 and Bcl-xL do not act downstream of the caspase cascade.

Apoptosis↗
PubMed2001

Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription.

Human annexin A5 is a ubiquitous protein implicated in diverse signal transduction processes associated with cell growth and differentiation, and its gene regulation is an important component of this function. Promoter transcriptional activity was determined for a wide 5' portion of the human annexin A5 gene, from bp -1275 to +79 relative to the most 5' of several discrete transcription start points. Transfection experiments carried out in HeLa cells identified the segment from bp -202 to +79 as the minimal promoter conferring optimal transcriptional activity. Two canonical Sp1 sites in the immediate 5' flanking region of a CpG island were required for significant transcription. Strong repressive activity in the distal promoter region between bp -717 to -1153 was attributed to the presence of an endogenous retroviral long terminal repeat, homologous with long terminal repeat 47B. The downstream sequence from bp position +31 to +79 in untranslated exon 1 was also essential for transcription, as its deletion from any of the plasmid constructs abolished activity in transfection assays. Electrophoretic mobility-shift assays, Southwestern-blot analysis and affinity chromatography were used to identify a protein doublet of relative molecular mass 35 kDa that bound an octanucleotide palindromic sequence in exon 1. The DNA cis-element resembled an E-box, but did not bind higher molecular mass transcription factors, such as upstream stimulatory factor or activator protein 4. The discovery of a downstream element crucial for annexin A5 gene transcription, and its interaction with a potentially novel transcription factor or complex, may provide a clue to understanding the initiation of transcription by TATA-less, multiple start site promoters.

Amino Acid Motifs↗