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PubMed1995

Cellular proteins bind to the downstream component of the lytic origin of DNA replication of Epstein-Barr virus.

The lytic origin of DNA replication of Epstein-Barr virus, oriLyt, is a complex eukaryotic origin which is activated during the lytic phase of the viral life cycle. It consists of at least two independent cis-acting components, one of which plays a dual role in transcription and DNA replication. The binding of the viral factor BZLF1, a member of the AP1 family of transcription factors, to this upstream component is crucial for oriLyt function (A. Schepers, D. Pich, and W. Hammerschmidt, EMBO J. 12:3921-3929, 1993). The second cis-acting element, the downstream component of oriLyt, is equally indispensable; however, its function is unknown. In this study, the downstream component was found to be the binding target of several cellular proteins. One could be identified as Sp1 or as a related protein which binds twice to the downstream component of oriLyt. Mutational analysis indicated that Sp1 alone is not directly involved in mediating DNA replication; however, other factors which share the same binding sequence or bind closely to one of the Sp1 binding sites are likely candidates to contribute to a replication protein complex at the downstream component of oriLyt. The sequence requirements for the downstream component are remarkably stringent, indicating that at least one of the putative factors is a sequence-specific DNA-binding protein which is required for the activation of oriLyt.

Base Sequence↗
PubMed1994

The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location.

The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked to the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.

Adenoviridae↗
PubMed1987

Expression of mouse histone genes: transcription into 3' intergenic DNA and cryptic processing sites downstream from the 3' end of the H3 gene.

Introduction of the mouse histone H3.1 gene into tk- mouse L cells by cotransfection with the herpesvirus thymidine kinase gene resulted in the production of two mRNAs from the transfected gene, one with a normal 3' end and the other one with a longer 3'-untranslated region, ending at site X, which was poly(A)+. In contrast, the endogenous histone H3.1 gene only produced a single mRNA. The cryptic poly(A)+ site was only used when the histone H3.1 gene was transfected. To localize possible downstream cryptic processing sites, the hairpin loop at the end of the histone gene was deleted and the resulting deletions were introduced into L cells. Two major mRNAs were produced from this gene, one ending at site X and the major one ending at site Y, which was located 150 nucleotides before site X. Transcription extended downstream of site X efficiently in the endogenous gene, as judged by the extent of transcription of downstream sequences in isolated nuclei. Transcription extended downstream of site X in the transfected gene because the placement of a normal histone 3' end downstream of site X resulted in transcripts that ended at site X and longer transcripts that ended with the new histone 3' end. These results indicate that transcription may normally proceed a substantial distance past the hairpin loop (greater than 500 bases). The formation of the different 3' ends in these transfected genes was due to competition between different processing mechanisms.

Animals↗
PubMed1992

A downstream sequence of the rpL32 promoter competes with the glucocorticoid responsive element for a protein factor.

The murine ribosomal protein (rp) L32 gene contains essential promoter sequences located both upstream and downstream of the cap site. A combination of gel mobility shift, UV cross-linking, and cell-free transcription assays were used to analyze the interaction of factors binding to a downstream element (located at position +25 to +37). The rpL32 downstream element identified polypeptides (transcription factors) ranging in size from 45 to 25 kilodaltons (kDa). Four base pair changes in the wild-type sequence of the downstream element eliminated binding. An oligonucleotide containing the glucocorticoid responsive element sequence competed specifically for the 45-kDa protein in both the gel mobility shift assay and in the UV cross-linking studies. Our data also indicate that the downstream binding factors contribute to cell-free transcription of the rpL32 gene.

Animals↗
PubMed2001

Heart motion-adapted MR velocity mapping of blood velocity distribution downstream of aortic valve prostheses: initial experience.

PURPOSE: To investigate blood flow velocities and shear rates at two distances downstream of an artificial aortic valve in patients. MATERIALS AND METHODS: Blood velocity was quantified downstream of the valve prosthesis (for replacement after aortic valve stenosis or combined stenosis and regurgitation) in 10 patients by using a magnetic resonance (MR) cine velocity mapping method in which the imaging section position is adapted according to the excursion of the valvular plane of the heart. Two acquisitions were performed to display the blood velocity distributions one-fourth valve diameter and one valve diameter downstream of the valve and to quantify blood volumes and shear rates. RESULTS: The velocity profiles measured during flow acceleration one-fourth valve diameter downstream were characterized by a distinct pattern of two lateral jets and one central jet of antegrade flow. High shear rates were found along the leaflet tips. The profiles obtained one valve diameter downstream were skewed, with varying velocity patterns among patients. Peak shear rates were found close to the vessel wall. With correction for through-plane motion of the valve, the mean apparent regurgitant fraction (+/- SD) was 14% +/- 6; the mean regurgitant fraction without correction was 9% +/- 5. CONCLUSION: The described noninvasive procedure for velocity mapping enables measurements close to the valve and thus evaluation of blood flow patterns with respect to valve design in humans.

Adult↗
PubMed1997

Do plaques grow upstream or downstream?: an angiographic study in the femoral artery.

Although the distribution of atherosclerosis has been described, little is known about the direction of growth of plaques. In this study, 237 patients with slight or moderate atherosclerosis underwent femoral angiography twice at a 3-year interval, and the films were studied with computerized image analysis. First, atherosclerosis was measured as edge roughness, and the change in roughness of each 1-cm segment over the 3-year period was related to the edge roughness of the segments immediately upstream and downstream. On the medial side of the artery, the change in edge roughness was found to be more strongly related to the roughness values upstream than to those downstream of the segment studied. This suggests that growth in the downstream direction is more common than growth in the upstream direction. On the lateral side, more equivocal results were obtained. Atherosclerosis was also assessed by study of the cross-sectional area of the artery as a function of distance along the vessel. A mathematical model of plaque growth was formulated as a nonlinear filtering of this curve. Growth in the downstream direction was significantly (P<.001) more frequent than growth in the upstream direction. The findings are compatible with an atherogenic effect of fluid mechanical disturbances, such as flow separation, that may occur downstream of a stenosis.

Adult↗
PubMed2006

Comparative analysis of Hox downstream genes in Drosophila.

Functional diversification of body parts is dependent on the formation of specialized structures along the various body axes. In animals, region-specific morphogenesis along the anteroposterior axis is controlled by a group of conserved transcription factors encoded by the Hox genes. Although it has long been assumed that Hox proteins carry out their function by regulating distinct sets of downstream genes, only a small number of such genes have been found, with very few having direct roles in controlling cellular behavior. We have quantitatively identified hundreds of Hox downstream genes in Drosophila by microarray analysis, and validated many of them by in situ hybridizations on loss- and gain-of-function mutants. One important finding is that Hox proteins, despite their similar DNA-binding properties in vitro, have highly specific effects on the transcriptome in vivo, because expression of many downstream genes respond primarily to a single Hox protein. In addition, a large fraction of downstream genes encodes realizator functions, which directly affect morphogenetic processes, such as orientation and rate of cell divisions, cell-cell adhesion and communication, cell shape and migration, or cell death. Focusing on these realizators, we provide a framework for the morphogenesis of the maxillary segment. As the genomic organization of Hox genes and the interaction of Hox proteins with specific co-factors are conserved in vertebrates and invertebrates, and similar classes of downstream genes are regulated by Hox proteins across the metazoan phylogeny, our findings represent a first step toward a mechanistic understanding of morphological diversification within a species as well as between species.

Animals↗
PubMed1997

VP16-activation of the C. elegans neural specification transcription factor UNC-86 suppresses mutations in downstream genes and causes defects in neural migration and axon outgrowth.

The POU homeobox gene unc-86 specifies many neuroblast and neural fates in the developing C. elegans nervous system. Genes regulated by unc-86 are mostly unknown. Here we describe a genetic strategy for the identification of downstream pathways regulated by unc-86. We activate UNC-86 transcription activity by inserting the VP16 activation domain into an unc-86 genomic clone that bears all regulatory sequences necessary for normal expression in C. elegans. unc-86/VP16 complements unc-86 mutations in the specification of neuroblast and neural cell fates, but displays novel genetic activities: it can suppress non-null mutations in the downstream genes mec-3 and mec-7 that are necessary for mechanosensory neuron differentiation and function. These data suggest that UNC-86/VP16 increases the expression of mec-3 and mec-7 to compensate for the decreased activities of mutant MEC-3 or MEC-7 proteins. The suppression of mutations in downstream genes by an activated upstream transcription factor should be a general strategy for the identification of genes in transcriptional cascades. unc-86/VP16 also causes neural migration and pathfinding defects and novel behavioral defects. Thus, increased or unregulated expression of genes downstream of unc-86 can confer novel neural phenotypes suggestive of roles for unc-86-regulated genes in neural pathfinding and function. Genetic suppression of these unc-86/VP16 phenotypes may identify the unc-86 downstream genes that mediate these events in neurogenesis.

Animals↗
PubMed2006

COX-2 expression in hepatocellular carcinoma is an initiation event; while EGF receptor expression with downstream pathway activation is a prognostic predictor of survival.

INTRODUCTION: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and is a leading cause of cancer mortality with over 90% of HCC patients succumbing to the disease. Current systemic therapies have had no measurable impact on survival in this disease; however there are small subsets of patients who benefit from systemic therapy who have been difficult to identify. Improvements in patient stratification and the development of biological therapies have resulted from the elucidation of the molecular mechanisms integral to tumor development and progression. Recent studies have found that COX-2 and EGFR are frequently inappropriately expressed in HCC compared to normal liver expression; however the presence of surface receptors does not always mean that the downstream pathway is active. In this study, we investigate the incidence and impact of activated EGFR downstream messengers phosphorylated akt (pakt) and/or phosphorylated MAPK (pMAPK) on survival in patients with HCC. METHOD: Thirty consecutive HCC patients treated at a single institution were retrospectively reviewed. Patient data including age, sex, Child's score, histological type, grade, stage, and survival were analyzed. Immunohistochemical staining was performed on formalin fixed, paraffin embedded tissues using monoclonal antibodies to COX-2, EGF receptor, pMAPK, and pakt. Histoscores were determined for each marker and evaluated for impact in survival, stage, and tumor grade. RESULTS: The median age was 67 years (39-83) and 67% of patients were male. Median survival was 9.8 months (1-47 months) for the whole group. COX-2 and EGFR expression was present in 90 and 67% of the tumors, respectively. Expression of activated downstream EGFR messengers was present in 53% of tumors (pMAPK 41%, pakt 31%). Median survival was significantly better in patients with downstream messenger expression, 24.4 months, compared to no expression, 4.7 months (P = 0.03). These groups were matched in age, stage, and Child's score. CONCLUSION: COX-2 and EGFR expression are commonly seen in HCC. Activated downstream EGFR expression is also common in HCC and is a predictor of improved survival. There may be a therapeutic role for EGFR tyrosine kinase inhibitors in this subset of patients and further investigation is warranted.

Adult↗
PubMed2006

Use of differentiating adult stem cells (marrow stromal cells) to identify new downstream target genes for transcription factors.

We developed a strategy for use of microarray data to rapidly identify new downstream targets of transcription factors known to drive differentiation by following the time courses of gene expression as a relatively homogeneous population of stem/progenitor cells are differentiated to multiple phenotypes. Microarray assays were used to follow the differentiation of human marrow stromal cells (MSCs) into chondrocytes or adipocytes in three different experimental conditions. The steps of the analysis were the following: (a) hierarchical clustering was used to define groups of similarly behaving genes in each experiment, (b) candidates for new downstream targets of transcription factors that drive differentiation were then identified as genes that were consistently co-expressed with known downstream target genes of the transcription factors, and (c) the list of candidate new target genes was refined by identifying genes whose signal intensities showed a highly significant linear regression with the signal intensities of the known targets in all the data sets. Analysis of the data identified multiple new candidates for downstream targets for SOX9, SOX5, CCAAT/enhancer binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma. To validate the analysis, we demonstrated that PPAR-gamma protein specifically bound to the promoters of four new targets identified in the analyses. The same multistep analysis can be used to identify new downstream targets of transcription factors in other systems. Also, the same analysis should make it possible to use MSCs from bone marrow to define new mutations that alter chondogenesis or adipogenesis in patients with a variety of syndromes.

Adipogenesis↗
PubMed2003

Examination of the responses of slimy sculpin (Cottus cognatus) and white sucker (Catostomus commersoni) collected on the Saint John River (Canada) downstream of pulp mill, paper mill, and sewage discharges.

As part of a larger survey on cumulative effects within the Saint John River basin (Canada), a fish survey was conducted near Edmundston (NB, Canada) in the fall of 1999 using slimy sculpin (Cottus cognatus) and white sucker (Catostomus commersoni). The discharge environment receives effluent from the pulp mill, a paper mill, three sewage discharges, and tributaries receiving agricultural runoff. Sculpin collected downstream of the sewage discharges and pulp mill effluent had greater growth, condition, and liver size but no significant differences in gonad size. Stable isotope data indicated slimy sculpin did not move between sites. Female sculpin collected downstream of the paper mill showed no significant differences in length, body weight, age, condition factor, liver size, and gonad size compared to fish from reference sites. Female white sucker collected downstream of the pulp mill did not differ significantly in any measured parameter compared to reference fish. Liver sizes of white sucker from the Saint John River were outside the range considered to be indicative of uncontaminated riverine sites. In 2000, sculpin collected downstream from a poultry-processing facility had larger livers and lower condition factors, suggesting that the site is contaminated. We found no significant differences in sculpin length, weight, condition (except for males), and liver size in sculpin collected downstream from the pulp mill in October 2001. The responses of slimy sculpin and white sucker differed, perhaps in relation to differences in life history characteristics. Results from this study indicate the slimy sculpin is a suitable fish species for monitoring rivers that receive multiple industrial and municipal effluents.

Animals↗
PubMed2001

Effect of Downstream Sequence on the Cleavage of Envelop Protein 1 Signal Sequence in Hepatitis C Virus.

The RNA genome of hepatitis C virus encodes a polyprotein of 3 000 amino acids, which is processed into 10 viral proteins by proteases provided by host cells and virus itself. Multiple precursors are produced due to inefficient processing. Here, the study of E1 signal sequence (C/E1 site) processing in eukaryotic vaccinia virus/T7 system is reported. Differently truncated HCV structural proteins were expressed in this system. It was found that the efficient cleavage of E1 signal sequence was affected by downstream envelope protein sequences. When the lacZ gene encoding a product with similar size was engineered downstream to the E1 signal sequence, the inefficient cleavage of signal sequence was also observed, suggesting that the effect of downstream sequence on the cleavage was due to the presence of the envelop protein sequences. Computer-aided analysis clearly showed that E1 signal sequences was a typical signal sequence. The influence of downstream sequences to signal sequence cleavage demonstrated here was uncommon. To date, similar observations were only reported for the processing of IL-12 signal sequence and the C/prM site of flavivirus. As both flavivirus and HCV are classified into the same Flaviviridae family, this downstream-sequence-related cleavage of signal sequence worths further studying.

Journal Article↗
PubMed1988

A functionally redundant downstream sequence in SV40 late pre-mRNA is required for mRNA 3'-end formation and for assembly of a precleavage complex in vitro.

In eukaryotes, mRNA 3' termini are formed by endonucleolytic cleavage of a long primary transcript and polyadenylation of the new end. Here we show that sequences downstream of the poly(A) site are required for cleavage of simian virus 40 (SV40) late pre-mRNAs in vitro in a crude nuclear extract from HeLa cells. The critical sequences are functionally redundant: extensive deletions or substitutions of downstream sequences prevent cleavage, but small substitutions do not. This functional redundancy is not due to a repetition of the same sequence. Either two or more different sequences can promote cleavage, or a single element exists which is long and diffuse. Although pre-mRNAs transcribed from certain genes require a U- or UG-rich sequence downstream of the poly(A) site for efficient cleavage, SV40 does not. Removal of these sequences from SV40 late pre-mRNAs does not significantly reduce cleavage efficiency. Downstream sequences also are required for formation of a specific precleavage complex between SV40 pre-mRNA and components present in the extract. Mutant RNAs that are cleaved efficiently form such complexes, while those that are cleaved inefficiently do not. Based on these and previous results (Zarkower, D., and Wickens, M. (1987b) EMBO J. 6, 4185-4192), we propose that a critical role of the region downstream of the poly(A) site is to facilitate formation of a specific precleavage complex in which cleavage subsequently occurs.

Animals↗
PubMed1987

Two regions downstream of AATAAA in the human antithrombin III gene are important for cleavage-polyadenylation.

We have investigated the sequence requirements for the cleavage-polyadenylation reaction in the human antithrombin III (ATIII) gene. A series of 5'-3' and 3'-5' deletions were produced around the AATAAA site using Bal31 nuclease. Ligation of appropriate pairs of such mutations resulted in the generation of varying sized deletions or duplications of sequences either upstream of, downstream of, or within the region encompassing the poly(A) site. Whereas a large deletion 3' to the AATAAA signal abolished cleavage and polyadenylation of ATIII transcripts, smaller deletions, all of which were subsets of the large one, did not. This indicated that the ATIII gene contains at least two independently acting poly(A)-cleavage signals 3' of AATAAA. When one of these signals was eliminated and the other was partially deleted at its 3'-end, we were able to disrupt the normal spacing between AATAAA and the cleavage site without substantially affecting the efficiency of the cleavage reaction. This suggested that the distance between AATAAA and the cleavage site is determined by the same sequence which, along with AATAAA, specifies cleavage and polyadenylation. The duplication of regions either upstream or downstream of AATAAA affected neither the efficiency nor the site of cleavage of the ATIII transcript. When a duplication included a large region containing AATAAA as well as downstream sequences, both sites were chosen for cleavage reactions. With a more delimited duplication, which included AATAAA but not the downstream cleavage signals, both sites were again used. However, two new cleavage sites were now detected. These results suggested that the distance between AATAAA sites and critical downstream cleavage-spacing sequences may also be important in determining the site of cleavage. This may in part stem from spatial constraints imposed by RNA-protein complexes which have been postulated to be critical in catalyzing the cleavage-polyadenylation reaction.

Antithrombin III↗
PubMed2025

Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF.

Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the "main" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus-like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.

Humans↗
PubMed1998

Multiple sequences from downstream of the J kappa cluster can combine to recruit somatic hypermutation to a heterologous, upstream mutation domain.

Recruitment of somatic hypermutation to the Ig kappa locus has previously been shown to depend on the enhancer elements, Ei/MAR and E3'. Here we show that these elements are not sufficient to confer mutability. However, hypermutation is effectively targeted to a chimeric beta-globin/Ig kappa transgene whose 5' end is composed of the human beta-globin gene (promoter and first two exons) and whose 3' end consists of selected sequences derived from downstream of the J kappa cluster (Ei/MAR, C kappa + flank and E3'). Thus, multiple downstream Ig kappa sequences (all derived from 3' of the J kappa cluster) can combine to recruit mutation to a heterologous mutation domain. The location of this hypermutation domain is defined by the position of the transcription start site and this applies even if the Ig kappa Ei/MAR is positioned upstream of the promoter. Hotspots within the mutation domain are, however, defined by local DNA sequence as evidenced by a new hotspot being created within the beta-globin domain by a mutation within the transgene. We propose that multiple, moveable Ig kappa sequences (that are normally located downstream of the transcription start site) cooperate to bring a hypermutation priming factor to the transcription initiation complex; a mutation domain is thereby created downstream of the promoter but the local sequence defines the detailed pattern of mutation within that domain.

Animals↗
PubMed1997

Identification of a homolog of the C alpha 3'/hs3 enhancer and of an allelic variant of the 3'IgH/hs1,2 enhancer downstream of the human immunoglobulin alpha 1 gene.

Although four regulatory elements are known downstream of the mouse IgH alpha gene, a single enhancer homologous to hs1,2 has been thus far described downstream of each human alpha gene (Chen, C. and Birshtein, B. K., J. Immunol. 1997. 159: 1310). We characterized a 10-kb region downstream of the human alpha 1 gene. Two B cell-specific regulatory elements homologous to the murine C alpha 3'/hs3 and hs1,2,3' enhancers were found, which are duplicated downstream of alpha 2. The hs1,2 element is in inverted orientation by comparison with a recently reported alpha 1 hs1,2 element: it appears as a common allelic variant carrying an internal tandem repeat insertion and its prevalence in the human population is 60%. As in the mouse, the human hs1,2 enhancer is flanked with long inverted repeats which may have promoted inversion events through homologous recombination. Although the palindromic organization of the region is maintained in human, sequence identity with rodents focuses on core enhancer elements rather than on flanking repeats. Concerted divergence of both sides of the dyad symmetry suggests that inverted repeats are not just evolutionary remnants but rather play an architectural role in the LCR function.

Alleles↗
PubMed1991

Poly(A) site efficiency reflects the stability of complex formation involving the downstream element.

A critical step in mRNA biogenesis is the generation of the mRNA 3' end through an endonucleolytic cleavage of the primary transcript followed by the addition of a approximately 200 nucleotide (nt) poly(A) tail. The efficiency of poly(A) site function can vary widely and for those genes with multiple poly(A) sites, the choice can be a regulated event. A functional poly(A) site is characterized by cis-acting RNA sequences including the well-conserved AAUAAA hexamer, located 10-30 nt upstream of the cleavage site, and a highly variable downstream GU- or U-rich element. The gene specific nature of the downstream sequence suggests that it may be a primary determinant of poly(A) site efficiency. Several recent studies have detailed the purification of factors that mediate the cleavage and polyadenylation reaction and that recognize the cis-acting signals. Two of these factors are responsible for the formation of a stable, committed ternary complex with the pre-RNA. In order to define the role of this stable complex in poly(A) site function, we have compared the processing efficiency of several pre-mRNAs with the stability of the complex that forms on these RNAs. We show that ternary complex stability reflects both the in vivo and the in vitro efficiency of the poly(A) site and that the stability of this complex is dependent on the nature of the downstream sequence element. We conclude that the stability of these protein--RNA interactions, dictated by the downstream element, plays a major role in determining the processing efficiency of a particular poly(A) site.

Base Sequence↗