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PubMed2001

Truncated and dispersed rpl2 and rps19 pseudogenes are co-transcribed with neighbouring downstream genes in wheat mitochondria.

The wheat mitochondrial genome contains only partial coding sequences for the L2 and S19 ribosomal proteins, unlike in rice or liverwort mitochondria, where these genes are functional and have a bacterial-type linkage. A single-copy stretch corresponding to the extreme 3' terminus of the wheat rpl2 gene is co-transcribed with the trans-splicing nad1 exon 4; and, at another unique location, the rps19 segment lacking the 5' coding region is co-transcribed with the downstream nad4L gene. In both cases, the 5' termini of these transcripts map to promoter consensus motifs acquired through genomic reorganization, enabling continued expression of essential downstream genes. In both wheat and rice, the rpl2 and rps19 genomic regions differ in their RNA profiles between germinating embryos and seedlings. The absence of intact rpl2 and rps19 genes in wheat mitochondria is consistent with their inactivation through DNA rearrangement/deletion after the successful transfer of functional copies to the nucleus.

Amino Acid Sequence↗
PubMed1998

Downstream elements from the pea albumin 1 gene confer sulfur responsiveness on a reporter gene.

The levels of mRNAs for some of the sulfur-rich proteins in seeds are regulated by the level of sulfur supplied to the plants. In peas, there is a mechanism that lowers the level of mRNA for legumin and pea albumin 1 (PA1) when plants are grown under sulfur-deficient conditions. This mechanism acts after transcription initiation. In this study, a gene encoding PA1 was expressed in leaves of transgenic tobacco. Expression of the gene was controlled by the level of sulfur supplied to the plants, mimicking the behaviour of the intact gene in peas. A gene encoding a different high-sulfur protein, ovalbumin, was unresponsive to sulfur status and was used as a reporter gene to test defined regions of the PA1 gene for sulfur responsiveness. These constructs, together with a set of PA1 gene deletions, were tested in transgenic tobacco and yielded the following observations: the PA1 gene was sensitive to sulfur status in the leaf as well as the seed; intron processing of the PA1 transcript was not required for sensitivity to sulfur stress; both the coding region and the 3' flanking regions of the PA1 gene contained sequences which conferred sensitivity to sulfur stress; the sulfur-responsive sequence in the 3' region was contained within a 134-nucleotide segment downstream of the end of the coding sequence. We conclude that there are at least two downstream elements which confer sensitivity to sulfur supply.

Albumins↗
PubMed2000

Characterization of TH1 and CTSZ, two non-imprinted genes downstream of GNAS1 in chromosome 20q13.

The clustering and coordinate regulation of many imprinted genes justifies positional searches for imprinted genes adjacent to known ones. We recently characterized a locus on 20q13, containing GNAS1, which has a highly complex imprinted expression pattern. In a search for neighbouring genes, we have now characterized a new gene, TH1, downstream of GNAS1. TH1 and GNAS1 are separated by more than 70 kb consisting largely of interspersed repetitive DNA. TH1 is the homologue of a gene that, in Drosophila, lies adjacent to the DNA repair gene mei-41. We have determined the full-length structures of human, mouse and Drosophila TH1. Though of unknown function, TH1 is highly conserved and widely expressed. Nonetheless, there is no similar Caenorhabditis elegans protein. We have also determined the complete genomic structures of human and Drosophila TH1. The Drosophila gene has five exons spanning 2.6 kb. The last three introns have precise equivalents in the human gene, which has 15 exons spanning 14 kb and is transcribed away from GNAS1. Using a single-nucleotide polymorphism in the 3' untranslated region, we have demonstrated biallelic TH1 expression in human fetal tissues, suggesting that, unlike GNAS1, TH1 is probably not imprinted. Immediately downstream of TH1 lies CTSZ, encoding the recently described cysteine protease, cathepsin Z. We have also elucidated the genomic structure of this gene; it has six exons spanning 12 kb and is oriented tail-to-tail with TH1, only 70 bp separating their polyadenylation sites. A polymorphism was again identified within the CTSZ 3' untranslated region and used to demonstrate biallelic expression in fetal tissues.

Amino Acid Sequence↗
PubMed2006

Gefitinib and the modulation of the signaling pathways downstream of epidermal growth factor receptor in human liver cancer cells.

BACKGROUND: The transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGFR) signaling pathway has been demonstrated to have a pivotal role in hepatocarcinogenesis. We examined whether abrogation of the TGF-alpha/EGFR signaling pathway with a selective EGFR tyrosine kinase inhibitor, gefitinib, could inhibit the proliferation of human hepatocellular carcinoma (HCC) cells. METHODS: Cellular growth was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell-cycle distribution was analyzed by flow cytometric analysis. Activities of signaling molecules were evaluated by Western blot analysis. RESULTS: HCC cells expressed EGFR at variable levels; however, extracellular signal-regulated kinase (ERK)1/2 and AKT, key signaling molecules downstream of EGFR, were not constitutively active in the cells. When HCC cells were treated with TGF-alpha, cellular growth was accelerated in a manner dependent on activation of ERK1/2 and AKT. When the cells were co-treated with gefitinib and TGF-alpha, enhanced proliferation and activation of ERK1/2 and AKT were canceled, and the cell-cycle promotion by TGF-alpha was inhibited by co-treatment with gefitinib and TGF-alpha, independently of expression levels of EGFR. In contrast, gefitinib did not show an antiproliferative effect on HCC cells cultivated under the 10% serum condition. CONCLUSIONS: The present data demonstrated that gefitinib exerted an antiproliferative action on HCC cells under a limited condition when signaling pathways downstream of EGFR were activated by TGF-alpha.

ErbB Receptors↗
PubMed2006

Protein kinase C and downstream signaling pathways in a three-dimensional model of phorbol ester-induced angiogenesis.

Angiogenesis, a critical process in both health and disease, is mediated by a number of signaling pathways. Although proangiogenic stimuli, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and the phorbol ester phorbol-12 myristate-13 acetate (PMA) are known to promote blood vessel formation, their downstream targets are ill defined. We sought to investigate the signaling pathways required for vessel assembly by utilizing a three-dimensional collagen matrix in which human umbilical vein endothelial cells (HUVECs) form tubular structures. Our data show that PMA is sufficient for the induction of angiogenesis, and that protein kinase C (PKC) is necessary for this process. Evaluation of PKC isoforms alpha and sigma revealed that these proteins are uniquely regulated. Characterization of an additional PMA target, protein kinase D (PKD) demonstrated that this enzyme becomes phosphorylated in HUVECs, and may therefore be involved in proangiogenic signaling. Further examination of downstream effectors of PKC showed that extracellular signal-regulated kinase (ERK) is critical for angiogenesis, and is accordingly phosphorylated in response to PMA. Surprisingly however, phosphorylation of ERK is independent of PKC activity. In addition, we show that the PKC target sphingosine kinase (SPK) is required for vessel formation. These findings illustrate the complexities of blood vessel formation, and suggest that activators utilize multiple independent pathways to invoke a complete angiogenic response.

Cell Culture Techniques↗
PubMed2005

Hox genes and their candidate downstream targets in the developing central nervous system.

1. Homeobox (Hox) genes were originally discovered in the fruit fly Drosophila, where they function through a conserved homeodomain as transcriptional regulators to control embryonic morphogenesis. Since then over 1000 homeodomain proteins have been identified in several species. In vertebrates, 39 Hox genes have been identified as homologs of the original Drosophila complex, and like their Drosophila counterparts they are organized within chromosomal clusters. Vertebrate Hox genes have also been shown to play a critical role in embryonic development as transcriptional regulators. 2. Both the Drosophila and vertebrate Hox genes have been shown to interact with various cofactors, such as the TALE homeodomain proteins, in recognition of consensus sequences within regulatory elements of their target genes. These protein-protein interactions are believed to contribute to enhancing the specificity of target gene recognition in a cell-type or tissue- dependent manner. The regulatory activity of a particular Hox protein on a specific regulatory element is highly variable and dependent on its interacting partners within the transcriptional complex. 3. In vertebrates, Hox genes display spatially restricted patterns of expression within the developing CNS, both along the anterioposterior and dorsoventral axis of the embryo. Their restricted gene expression is suggestive of a regulatory role in patterning of the CNS, as well as in cell specification. Determining the precise function of individual Hox genes in CNS morphogenesis through classical mutational analyses is complicated due to functional redundancy between Hox genes. 4. Understanding the precise mechanisms through which Hox genes mediate embryonic morphogenesis requires the identification of their downstream target genes. Although Hox genes have been implicated in the regulation of several pathways, few target genes have been shown to be under their direct regulatory control. Development of methodologies used for the isolation of target genes and for the analysis of putative targets will be beneficial in establishing the genetic pathways controlled by Hox factors. 5. Within the developing CNS various cell adhesion molecules and signaling molecules have been identified as candidate downstream target genes of Hox proteins. These targets play a role in processes such as cell migration and differentiation, and are implicated in contributing to neuronal processes such as plasticity and/or specification. Hence, Hox genes not only play a role in patterning of the CNS during early development, but may also contribute to cell specification and identity.

Animals↗
PubMed2005

Populations of a Connecticut River midge structured by geological history and downstream gene flow.

Connecticut River midges in the genus Axarus are constrained to semi-isolated populations associated with submerged clay exposures, and gene flow between populations is restricted. I sampled larval midges from fifteen spatially separated sites in the Connecticut River and determined karyotypes for 3111 larvae in two closely related species. In one of these species, an undescribed species referred to as Axarus species varvestris, 5 chromosomal rearrangements were present as polymorphisms. Four of these are paracentric inversions, and the fifth is a terminal insertion or deletion. I used chromosomal rearrangement frequencies to calculate Nei's genetic distance measures for all pairwise population comparisons and I constructed a neighbor-joining phenetic tree for the populations using these distances. Genetic distance between populations of A. sp. varvestris increases with geographic separation, and tree structure indicates that restricted gene flow only occurs in a downstream direction. The effect of unidirectional (downstream) gene flow on population genetic structure in riverine midges is described here for the first time. A genetic break is apparent between populations associated with 12 000-year-old sediments originating in different ancient lakes. The geological break between lakes also coincides with a break in the species composition of the Axarus populations.

Animals↗
PubMed1991

Does the brachial artery Doppler flow velocity waveform reflect changes in downstream impedance?

Brachial artery Doppler flow velocity waveforms were recorded from 10 nonpregnant women and 19 women with uncomplicated pregnancies. The pregnant group showed higher peak systolic frequencies (3.24 compared with 2.26, p = 0.01), higher maximum end-diastolic frequencies (0.62 compared with 0.25, p = 0.01), and lower pulsatility indices (2.57 compared with 3.95, p = 0.06) when compared with the nonpregnant group. These Doppler changes could reflect the increase in maternal cardiac output and decline in systemic vascular resistance that occur in normal pregnancy. The effect on brachial artery Doppler of acutely increasing downstream impedance by hand immersion in ice-cold water was studied in nine women with uncomplicated pregnancies at 32 to 38 weeks' gestation. Immersion for 15 seconds was associated with a significant reduction in peak systolic frequencies (from 3.06 to 1.97, p less than or equal to 0.005) and maximum end-diastolic frequencies (from 0.82 to 0.03, p less than or equal to 0.005) and a significant increase in the pulsatility index (from 1.87 to 5.62, p less than or equal to 0.005). These changes persisted for the duration of immersion (60 seconds). After immersion, the brachial artery flow velocity waveform returned to its preimmersion pattern by 60 seconds. We conclude that in normal pregnancy, the maternal brachial artery Doppler flow velocity waveform reflects acute and chronic changes in downstream impedance.

Blood Flow Velocity↗
PubMed1992

Downstream region of the human tyrosinase-related protein gene enhances its promoter activity.

We have cloned and sequenced the human genomic DNA segments encoding the 5'-flanking region and the first two exons of the tyrosinase-related protein (TRP) gene, a pigment cell-specific gene. Functional analysis of its promoter suggests that the downstream region of the TRP gene, including the first intron, enhances the transient expression of the luciferase gene under control of the TRP gene promoter about 16- to 20-fold. This enhancer-like activity is detected not only in melanoma cells but also in HeLa cells whose TRP gene expression is assumed to be repressed. We suggest a possibility that the downstream region is not sufficient to confer pigment cell-specific expression, but is required for efficient transcription of the TRP gene in pigment cells.

Animals↗
PubMed1990

Estimation of turbulent shear stresses in pulsatile flow immediately downstream of two artificial aortic valves in vitro.

Measuring turbulent shear stresses is of major importance in artificial heart valve evaluation. Bi- and unidirectional fluid velocity measurements enable calculation of Reynolds shear stress [formula: see text] and Reynolds normal stress [formula: see text]. tau is important due to the relation to hemolysis and thrombus formation, but sigma is the only obtainable parameter in vivo. Therefore, determination of a correlation factor between tau and sigma is pertinent. In a pulsatile flow model, laser Doppler (LDA) and hot-film (HFA) anemometry were used for simultaneous bi- and unidirectional fluid velocity measurements downstream of a Hall Kaster and a Hancock Porcine aortic valve. Velocities were registered in two flow field locations and at four cardiac outputs. The velocity signals were subjected to analog signal processing prior to digital turbulence analysis, as a basis for calculation of tau and sigma. A correlation factor of 0.5 with a correlation coefficient of 0.97 was found between the maximum Reynolds shear stress and Reynolds normal stress, implying [formula: see text]. In vitro estimation of turbulent shear stresses downstream of artificial aortic valves, based on the axial velocity component alone, seems possible.

Aortic Valve↗
PubMed1991

Requirement of gene VII in cis for the expression of downstream genes on the major transcript of figwort mosaic virus.

The six major conserved genes of figwort mosaic virus (FMV), a caulimovirus, appear in tandem array on an RNA transcript that spans the entire viral genome. Gene VI, the only cistron that appears as a separate subgenomic RNA, has been reported to transactivate the expression of downstream genes of the full-length transcript. This transcript has a long 5'-leader of about 600 nucleotides followed by a small nonconserved region (gene VII), a smaller intergenic region (57 nucleotides), and the major conserved genes in a closely spaced array. In our present experiments we have constructed expression units containing the promoter for the full-length transcript followed by the 5' leader region, gene VII, and a reporter gene. These have been tested for expression with and without gene VI as a separate plasmid by electroporation into plant protoplasts. A series of these expression units containing truncated versions of the 5' leader region placed upstream of a reporter gene (CAT) showed that gene VI transactivation occurred only when gene VII sequences were present in cis between the leader region and the reporter gene. In addition, a more complete version of the FMV genome containing the reporter gene further downstream (in viral gene IV) showed CAT expression only when gene VII sequences were present in an upstream position. A similar construct failed to express CAT activity when gene VII was absent.

Chloramphenicol O-Acetyltransferase↗
PubMed1985

Transcription of mouse rDNA terminates downstream of the 3' end of 28S RNA and involves interaction of factors with repeated sequences in the 3' spacer.

RNA polymerase I terminates transcription of mouse rDNA 565 bp downstream of the 3' end of mature 28S rRNA. This specific termination event can be duplicated in a nuclear extract system. RNA molecules with authentic 3' ends are transcribed from ribosomal minigene constructs provided the templates retain a minimal length of downstream spacer sequences. The nucleotide sequence of the region of transcription termination contains a set of repetitive structural elements consisting of 18 bp conserved nucleotides surrounded by stretches of pyrimidines. Termination in vivo occurs within the first element. This site is preferentially used in vitro at low template concentrations. At increasing DNA concentrations a termination site within the second repetitive element is used. Competition experiments with defined 3'-terminal fragments suggest that transcription termination by RNA polymerase I requires interaction of some factor (or factors) with the repetitive structural elements in the 3' nontranscribed spacer.

Animals↗
PubMed1986

Transcription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence.

We describe an in vitro transcription system in which the polyadenylated 3' ends of vaccinia virus mRNAs are formed by termination downstream of a regulatory signal. When linear DNA templates containing vaccinia early genes were incubated with soluble enzymes extracted from vaccinia virions, mature-size mRNAs were synthesized within 1 min, whereas longer run-off transcripts were not detected until later. Deleting a series of thymidylate residues located downstream of the coding region abolished termination. The 3' ends of the transcripts formed heterogeneously, between 50 and 70 nucleotides past the T-rich termination signal, in either viral- or vector-derived sequences. Polyadenylation of the transcripts occurred without regard to the terminal sequence and was inhibited by adding exogenous RNA. Although exogenous RNAs were also polyadenylated, processing of 3' ends was not observed.

Base Sequence↗
PubMed1986

Mutations downstream of the polyadenylation site of a Xenopus beta-globin mRNA affect the position but not the efficiency of 3' processing.

In order to identify nucleotide sequences required for efficient and accurate polyadenylation of mRNA precursors, we have constructed a series of mutations in the X. laevis beta 1-globin gene and analyzed transcripts produced upon microinjection into Xenopus oocytes. Small deletion and linker replacement mutations, which lie in the region from 8 to 39 bp downstream of the AATAAA sequence and which effectively remove previously identified second components of the polyadenylation signal, do not greatly reduce the efficiency of processing, but in some cases alter the precise site of cleavage. We conclude that sequences downstream of the polyadenylation site affect the position of 3' RNA processing, but have minimal effects on its efficiency.

Animals↗
PubMed1989

The ultrastructure of upstream and downstream regions of an active Balbiani ring gene.

When active, the 37 kb Balbiani ring genes are known to form transcription loops with an almost fully extended chromatin axis. Here we examine the upstream and downstream regions of such transcription loops by electron microscopy. We demonstrate that a loop starts and ends in tightly packed chromatin; the two anchoring sites are clearly separated from each other in space. The upstream, nontranscribed region consists of a thin, extended, apparently flexible and nucleosome-free fiber corresponding to about 0.5 kb DNA. The downstream, nontranscribed region appears as a 200 nm long nucleofilament loosely coiled into a short, thick chromatin fiber and estimated to contain about 3 kb DNA.

Animals↗
PubMed1990

Regulatory elements downstream of the promoter of an rRNA gene of E. coli.

Previously we have shown that plasmid constructs carrying a reporter gene fused to the P2 promoter of the E. coli rrnB gene exhibited a strange two-phase kinetics of expression depending on the physiological conditions of the cell if a short DNA region downstream of the promoter was present between the promoter and the reporter gene (Lukacsovich et al. (1987) J. Bacteriol. 169, 272-277). Insertion of a synthetic oligonucleotide corresponding to the first half of this region into constructs where the reporter directly follows the promoter, leads to a complete block of expression in vivo, while in vitro--in a purified system--transcription is not inhibited. Band-shift experiments indicate that the putative regulatory region downstream of the promoter specifically binds protein(s) present in total bacterial extracts.

Base Sequence↗
PubMed1993

Cyclic AMP inhibits the respiratory burst of electropermeabilized human neutrophils at a downstream site of protein kinase C.

We studied a signaling pathway for the activation of the superoxide (O2-)-generating NADPH oxidase and effects of cAMP on the pathway using electropermeabilized human neutrophils. The permeabilized cells produced O2- by the addition of protein kinase C (PKC) activator, phorbol myristate acetate (PMA), and a non-hydrolyzable GTP analogue, GTP gamma S in the presence of ATP and Mg2+. The O2- production by PMA not by GTP gamma S was inhibited by inhibitors of PKC. The production by PMA and GTP gamma S was inhibited by a GDP analogue, GDP beta S, in the same dose-dependent manner and the production by PMA was not enhanced by the addition of GTP gamma S and vice versa. These findings suggest the presence of a GTP-binding protein which follows PKC in the activation pathway. The O2- production by PMA and GTP gamma S was dose-dependently inhibited by cAMP and the inhibition was completely restored by an inhibitor of cAMP-dependent protein kinase, H-89, indicating that cAMP blocks the activating pathway at the site between the GTP-binding protein located downstream of PKC and the NADPH oxidase by activating cAMP-dependent protein kinase. The activation of the oxidase by sodium dodecyl sulfate (SDS) seemed to be different from the above pathway. It needed higher concentrations of GDP beta S for inhibition, did not absolutely need ATP and was inhibited by neither cAMP nor protein kinase C inhibitors. Moreover, the O2- production by the combination of GTP gamma S and SDS or of PMA and SDS was essentially the same as the sum of the production by each stimulant alone. We may conclude from the observations that the signaling pathway involving PKC for the activation of the oxidase is distinct from the pathway induced by SDS: the former is blocked by cAMP at the site between the GTP-binding protein located downstream of PKC and the oxidase and the latter is cAMP-insensitive.

Binding Sites↗
PubMedDate not supplied

The maximum rate of gene expression is dependent on the downstream context of unfavourable codons.

Presented here is an experimental demonstration of our theoretical predictions on the role of the downstream context of unfavourable codons in a gene on its expression level. Six non clustered AGG codons were inserted in the chloramphenicol acetyltransferase (cat) gene of E. coli and the expression of this modified gene (cat4) was compared with that of a cat gene in which four clustered AGG codons were inserted (cat2 gene). As predicted, the rate of production of the corresponding CAT4 and CAT2 proteins is equal as long as the rate of transcription of the gene does not exceed a given limit. When this limit is exceeded, production of CAT4 continues to increase, whereas CAT2 production decreases dramatically. Various consequences and possible applications of this downstream context effect are discussed.

Base Sequence↗