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PubMed1989

Cleavage by RNase III in the transcripts of the met Y-nus-A-infB operon of Escherichia coli releases the tRNA and initiates the decay of the downstream mRNA.

The metY gene coding for a minor form of the initiator tRNA is the first gene of a complex polycistronic operon also encoding the transcription termination factor NusA and the translation initiation factor IF2. The mixed tRNA-mRNA polycistronic transcript is cleaved by RNase III in a hairpin structure downstream from the tRNA. This cleavage separates the tRNA from the mRNA and initiates the rapid degradation of the 5' extremity of the downstream mRNA. Dissociation of the structural (tRNA) and informational (mRNA) RNAs from this operon is also achieved by independent transcription in vivo. The presence of two transcription terminators located downstream from metY produces a small tRNAMetf2 precursor transcript, whereas an internal promoter situated between metY and the first open reading frame directs the transcription of only the protein-coding part of the operon.

Bacterial Proteins↗
PubMed1985

Poly(A) site cleavage in a HeLa nuclear extract is dependent on downstream sequences.

Efficient utilization of the early SV40 poly(A) site in vivo requires sequences between 5 bp and 18 bp downstream of the cleavage site. We have used a HeLa nuclear extract to examine the sequence requirements for in vitro cleavage. DNA segments containing the SV40 poly(A) site were cloned into an SP6 vector. SP6 RNAs, accurately cleaved and polyadenylated, were detected by primer extension. Cleavage was enhanced by the presence of a cap on the primary transcript, and was inhibited by the addition of 10 microM 7meGpppG. In close agreement with the in vivo results, efficient processing at the poly(A) site in vitro required the specific downstream sequences in the SP6 RNA transcript. These experiments indicate that the sequence in the RNA precursor downstream of the cleavage site, shown to be important for efficient processing in vivo, is recognized in vitro.

Base Sequence↗
PubMed1993

Operons in C. elegans: polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions.

The mRNAs of six C. elegans genes are known to be trans-spliced to SL2. We report here that a similarly oriented gene is located 100-300 bp upstream of each. We present evidence that the genes in these clusters are cotranscribed and downstream mRNAs are formed by cleavage at the polyadenylation site and trans-splicing. From one three-gene cluster we isolated cDNA clones representing both polycistronic RNAs and mRNAs polyadenylated at the free 3' end created by trans-splicing, suggesting that polycistronic RNAs can be processed by trans-splicing. Several experiments indicate that SL2 trans-splicing is a consequence of a gene's downstream location in an operon. In particular, when an SL1-accepting gene was moved to a downstream location, its mRNA was trans-spliced largely to SL2. The possible regulatory significance of cotranscription of C. elegans genes is discussed.

Animals↗
PubMed1981

Downstream regulation of int gene expression by the b2 region in phage lambda.

Expression of the int gene after phage lambda infection normally requires the products of genes cII and cIII. However, when the phage carries a deletion in the nonessential b2 region adjacent to int, efficient synthesis of active Int protein does not require cII and cIII function. This inhibition of Int synthesis by nucleotide sequences downstream from the int structural gene behaves in a cis-dominant fashion in mixed infections. It is specific for PL- and not pI-initiated transcripts. Based on these observations, and those of others, a model is proposed in which Int translation from the pL transcript is inhibited by the interaction of downstream b2 nucleotide sequences and nucleotide sequences in the int region. The data imply a novel temporal mechanism regulating prophage lambda induction: circularization of the prophage genome results in the transposition of inhibitory b2 region sequences next to int and blocks further Int protein synthesis beyond the low level required for excision. As a consequence of this process, the control of int expression is transferred from the pL promoter to pI and the cII/cIII system. Such a genetic regulatory mechanism involving the rearrangement of genetic elements downstream from a structural gene may be of general use during development in other systems.

Bacteriophage lambda↗
PubMed1987

Conserved 12-bp element downstream from mRNA polyadenylation sites.

Sequences downstream from the AATAAA motif in a number of cellular and viral transcription units have been compared. A 12-bp conserved element was identified in approximately half of the cases studied, and a consensus sequence TTGANNNTTTTTT was derived from a comparison of 74 such sequences. This element is located immediately (5-20 bp) downstream from the poly(A)-addition site in every case where this is known, and it is suggested that this element may be involved in the cleavage/polyadenylation reaction. This proposal is consistent with published studies on deletion mutants of downstream regions.

Animals↗
PubMed1989

Human ribosomal DNA: conserved sequence elements in a 4.3-kb region downstream from the transcription unit.

The sequence of 4366 bp of nontranscribed spacer (NTS) human ribosomal DNA (rDNA) located downstream from the 3' end of the transcription unit has been determined. The NTS rDNA is rich in pyrimidine nucleotides (31% T and 30% C) that tend to occur on the coding strand in runs of simple sequence repeats. Other highly repetitive sequence elements are also represented, including tracts of (dA-dC)26 and (dG-dT)29 on the coding strand downstream from the putative termination of transcription. Still farther downstream, two Alu repeat sequences are found. Such sequences are also found in rat DNA at comparable locations, consistent with the possibility of a comparable functional role.

Base Composition↗
PubMed1993

Sequence analysis of the region downstream from a peptidoglycan hydrolase-encoding gene from Staphylococcus aureus NCTC8325.

The nucleotide (nt) sequence of a 4.7-kb DNA fragment downstream from a peptidoglycan hydrolase-encoding gene (lytA) from Staphylococcus aureus NCTC8325 was determined. Sequencing revealed three open reading frames (ORFs) of 513, 447 and 879 bp with consensus ribosome-binding sites located upstream from the ATG start codons. Results from in vitro transcription-translation analysis and maxicell experiments suggested that the 447-bp ORF was the one being actively expressed. Comparison of the amino acid (aa) sequences of the ORFs with the aa sequences in the NCBI Entrez database (Release 4.0, April 1993) did not show any significant homology to any sequenced polypeptides. However, nt sequences downstream from lytA showed perfect homology to the bacteriophage phi 11 attachment site (attP) and integration site (attB), and significant homology to downstream regions of the staphylokinase (sak) and exfoliative toxin A (eta) genes of S. aureus.

Amino Acid Sequence↗
PubMed1995

TBP binding to the TATA box induces a specific downstream unwinding site that is targeted by pluramycin.

BACKGROUND: The TATA-binding protein (TBP) is one of the major components of the human TFIID multiprotein complex. It is important in directing the initiation of RNA transcription at a site immediately downstream of the TATA sequence (TATA box) found in many eukaryotic promoters. The crystal structure of TBP complexed with an oligonucleotide containing the TATA box revealed a protein with an approximate two-fold symmetry which apparently has symmetrical interactions with DNA. It is not known how an asymmetric effect involving downstream activation can be produced by an apparent symmetric complex. We set out to examine the state of DNA in the TBP-DNA complex using pluramycin, a small molecular weight probe of DNA accessibility. RESULTS: Binding of TBP to the TATA box facilitates intercalation of pluramycin at a defined site immediately downstream of the TATA sequence through an apparent transient unwinding of the DNA. Pluramycin adducts are detected by the production of DNA strand breakage products upon heating. Incubation of pluramycin with the TBP-DNA complex facilitates the trapping of the specific complex by intercalation. Gel mobility shift and circularization assays reveal that the binding of pluramycin on the 3'-side of the TATA box region considerably stabilizes the TBP-DNA complex. CONCLUSIONS: We propose that the TBP-DNA-pluramycin ternary complex is a 'specific' binding mode in which TBP and pluramycin make compensatory alterations in DNA, accounting for the improved stability of the ternary complex. We also propose a model of the ternary complex that explains the observed asymmetric effect of TBP binding to the TATA box.

Aminoglycosides↗
PubMed2006

The presence of a common downstream box enables the simultaneous expression of multiple proteins in an E. coli extract.

In our experiments to produce different combinations of recombinant proteins in a cell-free protein synthesis system derived from Escherichia coli, we found that certain pairs of ORFs were not expressed equally. Instead, only a single DNA species was expressed dominantly, while the expression of the others was almost completely repressed. This bias during the co-expression of the DNA pairs was eliminated when an identical downstream box sequence was added to the 5'-ends of the template DNA pairs. By introducing identical nucleotide sequences of the his-tag or the downstream box of chloramphenicol acetyltransferase (CAT-DB) in front of the target genes that were otherwise not expressed compatibly, both of the encoded proteins were produced at similar productivities. Moreover, in the presence of a common downstream box, multiple genes were simultaneously expressed in the same reaction mixture. We expect that the proposed approach will offer a powerful tool for the preparation of unbiased protein libraries, as well as for studying the structure and functions of interacting proteins.

Cell Extracts↗
PubMed2004

Integration of multiple downstream signals determines the net effect of insulin on MAP kinase vs. PI 3'-kinase activation: potential role of insulin-stimulated H(2)O(2).

Cellular insulin stimulation generates a burst of H(2)O(2) that modulates protein-tyrosine phosphorylation in the insulin action pathway, in part by the inhibition of redox-sensitive protein-tyrosine phosphatases [J. Biol. Chem. 276 (2001) 21938]. Blocking the insulin-induced rise in H(2)O(2) with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) strongly attenuated the activation of phosphatidylinositol 3' (PI 3')-kinase, Akt and GLUT4 translocation by insulin in 3T3-L1 adipocytes; however, under identical conditions, we observed a paradoxical increase in the activation of p42/p44 mitogen-activated protein (MAP) kinase. DPI inhibited the insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1/2, and also reduced the association of Grb2 with IRS-1, suggesting that the effect of DPI on MAP kinase activation occurred downstream of the IR and IRS proteins. DPI increased the insulin-stimulated phosphorylation of p42/p44 MAP kinase with no change in basal, and increased insulin-stimulated MAP kinase kinase (MEK) activity by a similar degree. DPI enhanced basal Grb2-Sos binding and reduced the effect of insulin to potentiate the dissociation of the Grb2-Sos complex, suggesting that the effect of DPI was mediated upstream of Raf-1. Cell treatment with dibutyryl cAMP significantly reduced the enhancement of MAP kinase activation in the presence of DPI. However, forskolin, acting in a PKA-independent manner, increased the insulin stimulation of MAP kinase and MEK, but fully abrogated the effect of DPI to enhance these insulin responses. PLCgamma inhibition with U73122 blocked the insulin stimulation of MAP kinase and MEK as well as the enhancing effect of DPI on these responses. PKC activation strongly stimulated MAP kinase and MEK activation, even in the presence of U73122, consistent with PKC acting downstream of PLCgamma. These data show that the insulin-stimulated oxidant signal differentially affects the two major downstream components of the insulin signaling pathway, PI 3'-kinase and MAP kinase, and cross-talk between insulin action, PLCgamma and, to a lesser extent, PKA modulates the net cellular effects of insulin-stimulated cellular H(2)O(2).

3T3-L1 Cells↗
PubMed2005

Sorption-desorption behavior of polycyclic aromatic hydrocarbons in upstream and downstream river sediments.

Sorption and desorption behaviors of phenanthrene and naphthalene were studied with the whole sediment, humic acid (HA) and humin samples from downstream and upstream sites along the Kishon River, Israel. The 13C nuclear magnetic resonance spectra and the sorption coefficients suggest that sorption occurs to both aromatic and aliphatic moieties of the sedimentary organic matter and that rigid paraffinic domains probably contribute to the sorption non-linearity. The carbon-normalized Freundlich affinity values for the two sorbates were significantly higher for the whole sediment and humin samples from the downstream region of the river than for the upstream sediment samples. On the basis of the measured affinity values, the sorbents can be arranged in the following order: humin>HA>whole sediment. Phenanthrene exhibited the lowest desorption from the whole sediment samples compared with the other sorbents. For naphthalene, the desorption hysteresis obtained with the whole sediment and humin samples were similar: both exhibited a decrease in desorption with decreasing solute concentration. The higher sorption affinities observed for all the organic fractions from the downstream sediment are suggested to be related to the low levels of polar domains and humin content. It is concluded that in bulk sediment samples, the overall contribution of the HA fraction to short-term sorption is of high importance, but the sorption non-linearity is controlled mainly by the humin complexes. The low desorption potential recorded for the whole sediment samples could affect the natural attenuation of the sorbed hydrophobic organic compounds.

Adsorption↗
PubMed2006

The p53 network: p53 and its downstream genes.

The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network. p53 is a key molecular node in the network, which is activated in response to several cellular signals resulting in the maintenance of genetic stability. Several cellular signals may activate the p53 network. When the expression of P53 is elevated, P53-MDM2 module and the ubiquitin system can accurately regulate the expression level of P53. P53 can bind to specific DNA sequence, activate its downstream genes expression, and control cell-cycle arrest, DNA repair, and apoptosis. Elucidating the function of p53 gene network will help understand the interaction mechanisms of p53 and its downstream genes.

Animals↗
PubMed2006

Turbulent stress measurements downstream of three bileaflet heart valve designs in pigs.

OBJECTIVE: Mechanical heart valves can cause thromboembolic complications, possibly due to abnormal flow patterns that produce turbulence downstream of the valve. The objective of this study was to investigate whether three different bileaflet valve designs would exhibit clinically relevant differences in downstream turbulent stresses. METHODS: Three bileaflet mechanical heart valves (Medtronic Advantage), CarboMedics Orbis Universal and St. Jude Medical Standard) were implanted into 19 female 90 kg pigs. Blood velocity was measured during open chest conditions in the cross sectional area downstream of the valves with 10 MHz ultrasonic probes connected to a modified Alfred Pulsed Doppler equipment. As a measure of turbulence, Reynolds normal stress (RNS) was calculated at three different cardiac output ranges (3-4, 4.5-5.5, 6-7 L/min). RESULTS: Data from 12 animals were obtained. RNS correlated with increasing cardiac outputs. The highest instantaneous RNS observed in these experiments was 47 N/m2, and the mean RNS taken spatially over the cross sectional area of the aorta during systole was between 3 N/m2 and 15 N/m2. In none of the cardiac output ranges RNS values exceeded the lower critical limit for erythrocyte or thrombocyte damage for any of the valve designs. CONCLUSIONS: Reynolds normal stress values were below 100 N/m2 for all three valve designs and the difference in design was not reflected in generation of turbulence. Hence, it is unlikely that any of the valve designs causes flow induced damage to platelets or erythrocytes.

Animals↗
PubMed2006

Randomized comparison of upstream tirofiban versus downstream high bolus dose tirofiban or abciximab on tissue-level perfusion and troponin release in high-risk acute coronary syndromes treated with percutaneous coronary interventions: the EVEREST trial.

OBJECTIVES: We aimed to compare the effects of upstream tirofiban versus downstream high-dose bolus (HDB) tirofiban and abciximab on tissue level perfusion and troponin I release in high-risk non-ST-segment elevation acute coronary syndrome (ACS) patients treated with percutaneous coronary intervention (PCI). BACKGROUND: Optimal timing and dosage of glycoprotein IIb/IIIa inhibitors for ACS remain to be explored. METHODS: We randomized 93 high-risk ACS patients undergoing PCI to receive upstream (in the coronary care unit) tirofiban, downstream (just prior to PCI) HDB tirofiban, and downstream abciximab. We evaluated the effects of the three drug regimens on tissue-level perfusion using the corrected Thrombolysis In Myocardial Infarction (TIMI) frame count, the TIMI myocardial perfusion grade (TMPG), and intracoronary myocardial contrast echocardiography (MCE) before and immediately after PCI and after cardiac troponin I (cTnI). RESULTS: The TMPG 0/1 perfusion was significantly less frequent with upstream tirofiban compared with HDB tirofiban and abciximab both before (28.1% vs. 66.7% vs. 71%, respectively; p = 0.0009) and after PCI (6.2% vs. 20% vs. 35.5%, respectively; p = 0.015). Upstream tirofiban was also associated with a significantly higher MCE score index (0.88 +/- 0.18 vs. 0.77 +/- 0.32 vs. 0.71 +/- 0.30, respectively; p < 0.05). Post-procedural cTnI elevation was significantly less frequent among patients in the upstream tirofiban group compared with the HDB tirofiban and abciximab groups (9.4% vs. 30% vs. 38.7%, respectively; p = 0.018). The cTnI levels after PCI were significantly lower with upstream tirofiban compared with HDB tirofiban (3.8 +/- 4.1 vs. 7.2 +/- 12; p = 0.015) and abciximab (3.8 +/- 4.1 vs. 9 +/- 13.8; p = 0.0002) CONCLUSIONS: Among high-risk non-ST-segment-elevation ACS patients treated with an early invasive strategy, upstream tirofiban is associated with improved tissue-level perfusion and attenuated myocardial damage.

Abciximab↗
PubMed2004

Downstream DNA selectively affects a paused conformation of human RNA polymerase II.

Transcriptional pausing by human RNA polymerase II (RNAPII) in the HIV-1 LTR is caused principally by a weak RNA:DNA hybrid that allows rearrangement of reactive or catalytic groups in the enzyme's active site. This rearrangement creates a transiently paused state called the unactivated intermediate that can backtrack into a more long-lived paused species. We report that three different regions of the not-yet-transcribed DNA just downstream of the pause site affect the duration of the HIV-1 pause, and also can influence pause formation. Downstream DNA in at least one region, a T-tract from +5 to +8, increases pause duration by specifically affecting the unactivated intermediate, without corresponding effects on the active or backtracked states. We suggest this effect depends on RNAPII-modulated DNA plasticity and speculate it is mediated by the "trigger loop" thought to participate in RNAP's catalytic cycle. These findings provide a new framework for understanding downstream DNA effects on RNAP.

Base Sequence↗
PubMed2005

Retinal G-substrate, potential downstream component of NO/cGMP/PKG pathway, is located in subtype of retinal ganglion cells and amacrine cells with protein phosphatases.

The aim of this study was to determine the distribution and function of G-substrate, a specific substrate of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-cGMP-dependent protein kinase (PKG) signaling pathway, in normal rat retina and in G-substrate knockout mice. The retinas of adult wild-type rats and mice and G-substrate knockout mice were studied immunohistologically to characterize the upstream and downstream components of the NO-cGMP-PKG pathway. Immunoblot analysis showed that the molecular weight of retinal G-substrate was similar to that of cerebellar G-substrate. In adult rats and mice, retinal G-substrate was located in a subpopulation of amacrine cells and in C38-positive retinal ganglion cells (RGCs) but not in alpha RGCs. In addition, retinal G-substrate was co-expressed with other upstream and downstream signaling components of the NO-cGMP-PKG-G-substrate-phosphatase pathway in the adult retina. Electroretinographic (ERG) analysis demonstrated that there was no significant difference between the ERGs of wild-type and G-substrate knockout mice. These results suggest that retinal G-substrate plays a role as a downstream component of the NO-cGMP-PKG pathway. The co-localization of retinal G-substrate with protein Ser/Thr phosphatases suggests that it acts as an endogenous protein phosphatase inhibitor as in the cerebellum.

Amacrine Cells↗
PubMed2003

Translocations into human chromosome 14 JH region: factors influencing downstream abortive immunoglobulin class switching.

Bcl-2 translocations in follicular lymphomas (FL) are often associated with downstream immunoglobulin class switch recombination (CSR) on the translocated allele. We studied cell lines with different translocations into the IgH locus to gauge any common features associated with downstream CSR events. CSR associated with chromosomal rearrangements was observed in cells (RL) with translocations similar to those frequently observed in FL (bcl-2-JH), and such CSR was also seen with a myc (near exon 1)-JH5 intron translocation (MC116), but not for far 5'-myc-JH5 intron (P3HR-1) or myc-Smu translocations (Ramos). Both MC116 and P3HR-1 myc translocations showed evidence for an origin from somatic hypermutation. Therefore, the association of JH translocations with CSR on the translocated allele is unlikely to be linked with specific translocation mechanisms, and the P3HR-1 configuration indicated that the downstream class switching is not a necessary consequence of (or precondition for) such a translocation event. MC116 and RL, but not P3HR-1 cells, showed constitutive transcription through the translocated IgH alleles, suggesting that transcription through this region or the processing of such transcripts may promote CSR. However, while CSR events clearly occurred in the precursors of MC116 and RL, neither cell line could undergo complete class switching.

Base Sequence↗
PubMed2006

A nerve growth factor-induced retrograde survival signal mediated by mechanisms downstream of TrkA.

Considerable evidence suggests that mammalian neurons are always poised to destroy themselves by apoptosis but are blocked by retrograde survival signals triggered in their axon terminals by neurotrophic factors secreted by the target cells they innervate. Studies with nerve growth factor (NGF) and its receptor, TrkA, form the basis of the prevalent theory of retrograde signaling. According to this theory, retrograde survival signals travel to the cell bodies in the form of endosomes produced at the axon terminals with internalized NGF in their lumens bound to phosphorylated TrkA in their membranes. The inhibition of TrkA phosphorylation in the cell bodies of sympathetic neurons in compartmented cultures by K252a blocked retrograde NGF signaling in some studies in accord with this theory, but other studies do not show a block. We report that local block of TrkA phosphorylation in the cell bodies and proximal axons with another kinase inhibitor, Gö6976 (25nM), did not block the survival signal from NGF at distal axons, while Gö6976 at the distal axons completely blocked the retrograde survival signal. These results suggest that downstream signals activated by phosphorylated TrkA in the distal axons carry the retrograde survival signals to the cell bodies, possibly via a downstream type of signaling endosome not necessarily transporting NGF or phosphorylated TrkA. Unlike Gö6976, K252a exerted a survival effect on its own when applied to cell bodies/proximal axons or distal axons of completely NGF-deprived neurons. The latter effect suggests that downstream retrograde survival signals can arise from alterations in one or more kinase activities in the distal axons without activation of TrkA by NGF.

Animals↗