Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “downstream”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48Linked to original sources
PubMed1989

Downstream sequences augment transcription from the essential initiation site of a baculovirus polyhedrin gene.

A series of recombinant baculoviruses containing linker-substituted polyhedrin promoters attached to a reporter gene encoding chloramphenicol acetyl transferase (CAT) were constructed and tested for expression of the gene. The major determinant for promoter activity was narrowed to within eight nucleotides, TAAGTATT, at the start point of polyhedrin mRNA transcription. Mutations within TAAGTATT blocked initiation of transcription from this site and resulted in a 2000-fold decrease in CAT activity. Linker mutations from 12 to 22 bases upstream from the TAAGTATT sequence increased the steady-state levels of RNAs initiated within TAAGTATT and increased CAT expression by up to 50%. Mutations downstream from TAAGTATT and within the region specifying the untranslated RNA leader diminished transcriptional initiation at TAAGTATT and decreased CAT activity two- to 20-fold. The half-lives of CAT RNAs were not noticeably affected by mutations in the untranslated RNA leader region and thus RNA turn-over was not responsible for the reduced levels of these CAT RNAs. Nuclear run-on analysis of two mutant viruses showed that these mutations decrease the rate of transcriptional initiation. Transcriptional initiation thus appears to be the major means of polyhedrin gene regulation. The data define promoter-related roles for TAAGTATT and the sequences specifying the untranslated mRNA leader in transcriptional initiation. A model by which the viral-induced RNA polymerase distinguishes late and very late initiation sites is proposed.

Base Sequence↗
PubMed1989

Conserved sequence elements upstream and downstream from the transcription initiation site of the Caulobacter crescentus rrnA gene cluster.

The nucleotide sequence and in vivo transcription start sites for rrnA, one of the two rRNA gene clusters of the eubacterium Caulobacter crescentus, have been determined. Two transcription start sites, a major and minor, for the rRNA gene cluster are located more than 700 nucleotides upstream from the 16 S rRNA gene. Transcription was detected from only the major start site in swarmer cells. But after the swarmer-to-stalked cell transition, transcription was detected from both rRNA start sites and continued throughout the developmental cell cycle when cells were grown in minimal medium. On the other hand, transcription from only the major start site was detected in cells growing in a complex medium. A small open reading frame was found upstream from the rRNA gene transcription start sites and was followed by an inverted repeat sequence. No sequence homology was found between the major rRNA gene transcription start site and the Escherichia coli sigma 70 promoters or the consensus sequence elements reported for C. crescentus fla promoters. However, there were two areas of homology when the major rRNA gene promoter was compared to the nucleotide sequence of the C. crescentus trpFBA promoter. There was a 12 nucleotide sequence centered around the -10 region of both promoters that was closely homologous. In addition, immediately downstream from the transcription start there was a sequence element that was identical in both promoters. These nucleotide sequence elements were not in the temporally expressed fla promoters of C. crescentus.

Base Sequence↗
PubMed1989

Sequences linked to prokaryotic promoters can affect the efficiency of downstream termination sites.

The efficiency of transcription termination at certain well-defined prokaryotic rho-independent terminators depends on the promoter unit from which transcription is initiated. Some promoter units allow substantial readthrough of strong termination signals, a phenomenon we term "factor-independent antitermination". This observation is not easily explained by current models for prokaryotic terminator function that consider the terminator to be a "cassette" involving only sequences and RNA transcript structures in the immediate region of the terminator or directly upstream. When transcription is carried out in vitro employing only purified Escherichia coli RNA polymerase, up to 20 times as many RNA polymerase molecules pass through a particular terminator when transcription is initiated from the E. coli tac promoter unit, as compared to transcription initiated from the T7A1 or rrnB P2 promoters. This effect cannot be attributed to antitermination factors separate from the core RNA polymerase. Similar differences in termination efficiency are found for the same promoters in vivo. These termination differences are affected by sequences just downstream from the start site for transcription, including those in the +25 region of the nascent transcript. These early transcribed sequences can confer factor-independent antitermination onto a heterologous promoter, but only when the sequences are precisely positioned relative to the start site for transcription. We have considered several possible models to explain how early transcribed sequences might affect termination, including those in which the 5' end of the transcript interacts with either the terminator RNA or the polymerase. We favor an alternative model in which these sequences interact with the core RNA polymerase to convert the enzyme from a termination-proficient state (T-state) to a conformation resistant to termination (R-state). Such enzyme conformations may be an important component of factor-dependent antitermination systems.

Base Sequence↗
PubMed1994

Partitioning of plasmid R1. The parA operon is autoregulated by ParR and its transcription is highly stimulated by a downstream activating element.

The parA partitioning system of plasmid R1 mediates efficient stabilization of R1 and F-derived replicons. The parA system is encoded by a continuous DNA segment of approximately 1600 base-pairs and consists of three components. Two adjacent genes, parM and parR, coding for the trans-acting proteins ParM and ParR, and the cis-acting parC site. The centromere-like parC site is located upstream of parM and parR and contains the parA promoter. The parM and parR genes are co-transcribed as an operon from the parA promoter. The 5' end of the parA encoded transcript was mapped to the center of the parC region at +115. The -10 and -35 core promoter sequences are flanked by the two sets of five direct repeats in parC (the ParR boxes). The parA promoter was found to be negatively regulated by the parR gene product, whereas the parM gene product seemingly was not involved in the regulation. Surprisingly, a region downstream of the parA promoter enhanced transcription from the promoter many-fold (30 to 50-fold). The parC site titrated the ParR protein, suggesting that the ParR protein interacts directly with the parC site. Using an engineered parA system we found that the parC site could be complemented in cis by the parM and parR genes. Furthermore, the proper function of the parC site was highly dependent on the expression level of ParM and ParR. The incompatibility associated with the parC site could not be suppressed by overexpression of the ParM and ParR proteins. Based on these results we suggest a novel partition model involving pairing of newly replicated plasmid molecules.

Bacterial Proteins↗
PubMed1988

Cauliflower mosaic virus 35 S RNA leader region inhibits translation of downstream genes.

The cauliflower mosaic virus (CaMV) 35 S RNA is a full-length transcript of the viral genome. It encodes the genes VII and I-V, arranged in tandem along the RNA, preceded by a long leader region (600 bases) containing many short open reading frames. We have examined the effects of the leader and the first gene (gene VII) on downstream gene I translation in vitro and in an in vivo transient expression system (carrot protoplasts). RNAs from constructs containing the intact leader, and from various deletion constructs, were translated in a rabbit reticulocyte system. Gene I was translated efficiently only when the long leader region and the upstream gene VII were deleted. Translational fusions of gene VII or I to the firefly luciferase reporter gene were also constructed, and a similar series of leader sequence deletion mutants were examined in vivo and in vitro. The 600-base leader region was found to repress translation of gene VII 8- to 30-fold as compared to the truncated gene lacking the leader region. Gene I expression as compared to that of gene VII was reduced an additional 7- to 20-fold by the presence of the upstream leader region including gene VII. This represented an overall reduction in gene I expression of greater than 100-fold as compared to expression in the absence of any leader sequence. The reduced translation of gene I in the context of the 35 S RNA leader region was not due to the action of the gene VII protein product but may result from efficient blocking of scanning 40 S ribosomes by translation of upstream open reading frames.

Chromosome Deletion↗
PubMed1983

A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes.

Transcriptional enhancers, originally discovered in viral genomes, are short, cis-acting, regulatory sequences that strongly stimulate transcription from promoters of nearby genes. We demonstrate the existence of an enhancer within a mouse immunoglobulin heavy chain gene. A DNA fragment located between the joining region and the switch recombination region in the intron upstream of the immunoglobulin mu constant region has been linked, in both orientations, to genes coding for rabbit beta-globin or SV40 T antigen. This element enhances the number of correct beta-globin gene transcripts by at least two orders of magnitude and also stimulates production of T antigen. It acts from several hundred to several thousand base pairs up or downstream of a promoter without amplifying template copy number. Of the various cell lines tested, the immunoglobulin gene enhancer functions only in lymphocyte-derived (myeloma) cells. We propose that this tissue-specific enhancer contributes to the activation of somatically rearranged immunoglobulin variable region genes and possibly to abnormal expression of other genes (e.g. c-myc) that become translocated to its domain of influence.

Animals↗
PubMed1984

Termination of transcription in the mouse alpha-amylase gene Amy-2a occurs at multiple sites downstream of the polyadenylation site.

We have delimited the region of transcription termination in the alpha-amylase gene Amy-2a. Mapping of in vitro elongated nascent transcripts to Amy-2a restriction fragments indicates that transcription terminates in a region between 2.5 and 4 kb downstream of the polyadenylation site. These runoff transcription experiments, combined with S1 nuclease mapping of nuclear transcripts at steady state, suggest that transcription termination occurs at multiple sites.

Animals↗
PubMed1984

Requirement of a downstream sequence for generation of a poly(A) addition site.

The 3' terminus of most, if not all, eucaryotic polyadenylated mRNAs is formed as a result of endonucleolytic cleavage of a larger precursor RNA. That is, transcription does not terminate at the mRNA 3' sequence but rather proceeds through this site, terminating at some distance downstream. Using a plasmid containing the adenovirus E2A transcriptional unit, we have investigated the sequence requirement for the formation of a mature mRNA 3' terminus, focusing on the role of sequences immediately distal to the poly(A) addition site. Deletion mutants were constructed in the region distal to the poly(A) addition site and assayed by transfection into human 293 cells. The results demonstrate that 35 nucleotides distal to the site of poly(A) addition are sufficient for the formation of a mature E2 mRNA. However, removal of an additional 15 nucleotides, leaving 20 nucleotides distal to the poly(A) site, abolished the ability to produce functional E2A mRNA. The defect in the production of functional mRNA from such a mutant appears to be in the proper cleavage of the primary transcript at the poly(A) addition site. It would thus appear that sequences immediately distal to the site of poly(A) addition do not contribute to the mature mRNA but are essential for the formation of mature mRNA.

Adenoviruses, Human↗
PubMed1991

Genetic dissection of a neurodevelopmental pathway: Son of sevenless functions downstream of the sevenless and EGF receptor tyrosine kinases.

We have isolated a dominant mutation in a gene called Son of sevenless (Sos) that is an allele-specific suppressor of the sevenless phenotype. This suppressor function is autonomously required in R7 and is sensitive to the dosage of the Sos and bride of sevenless genes. Loss-of-function alleles of Sos are recessive lethals, but in the eye Sos has a role in R cell development. Mutations in Sos also interact with the Ellipse allele of the Drosophila EGF receptor. We propose a model suggesting that the Sos product is downstream of sevenless and the EGF receptor, and that the dominant suppression results from the overexpression or increased activity of the gene product.

Alleles↗
PubMed1993

A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila.

D-raf, a Drosophila homolog of Raf-1, plays key roles in multiple signal transduction pathways. Dsor1, a putative factor downstream of D-raf, was genetically identified by screening of dominant suppressors of D-raf. Dsor1Su1 mapped on X chromosome significantly suppressed the D-raf mutant phenotypes, and the loss-of-function mutations of Dsor1 showed phenotypes similar to those of the D-raf null mutations. Dsor1Su1 also significantly suppressed the mutations of other terminal class genes acting further upstream of D-raf. Molecular cloning of Dsor1 revealed its product with striking similarity to the microtubule-associated protein (MAP) kinase activator and yeast PBS2, STE7, and byr1. Our genetic results demonstrate the connection between raf and the highly conserved protein kinase cascade involving MAP kinase in vivo.

Amino Acid Sequence↗
PubMed1993

Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal.

Upon ligand-induced tyrosine phosphorylation, the platelet-derived growth factor (PDGF) receptor (PDGFR) beta subunit associates with PLC-gamma 1, RasGAP, P13K, and a 64 kd protein. To determine the relative role of each of these associated proteins in PDGFR signaling, we constructed a PDGFR mutant (F5) unable to bind any of them and a panel of "add-back" mutants that could bind only one of the receptor-associated proteins. F5 PDGFR failed to activate PLC-gamma 1, P13K, or Ras and was unable to trigger DNA synthesis. Permitting association of F5 PDGFR with either PLC-gamma 1 or P13K restored Ras activation and a mitogenic response. Surprisingly, even though binding of the 64 kd protein almost fully restored Ras activation, it did not rescue the receptor's ability to trigger DNA synthesis. Thus Ras activation is insufficient to trigger PDGF-dependent DNA synthesis, and PLC-gamma 1 and P13K are independent downstream mediators of PDGF's mitogenic signal.

DNA↗
PubMed1989

Hypothesis: the removal of VH gene downstream sequences is required for its expression in mature B cells.

In immunoglobulin producing cells only the rearranged VH gene is expressed, while all the other germline VH genes are silent. On the other hand, it was shown that unrearranged VH gene segments are expressed at a high level in the early stages of B lymphocyte differentiation. This expression is independent of the Ig heavy chain enhancer element. It seems, therefore, that unrearranged VH gene expression is depressed in mature B cells by a mechanism that will not operate on the rearranged functional heavy chain gene. We have shown that a highly conserved DNA segment is present upstream to several mouse and human VH genes. DNA sequencing downstream to germline genes revealed a DNA element related to the one described at the 5' of the VH genes. A model is proposed in which the 5' and 3' conserved segments are functional in the regulation of VH gene expression. The model suggests the existence of a protein in mature B cells which recognizes the 5' and 3' conserved DNA elements and prevents germline VH gene expression by binding to these segments. VH-D-JH joining results in the deletion of the 3' flanking sequences of the rearranged VH gene but not of other upstream VH genes. This would result in the release of promoter suppression in the case of the rearranged gene, while all other VH genes would remain transcriptionally silent.

Animals↗
PubMed1990

Transition of the mRNA sequence downstream from the initiation codon into a single-stranded conformation is strongly promoted by binding of the initiator tRNA.

Using an RNA footprinting technique, accessible sites on the mRNA initiation region bound to the ribosome have been determined. Chemical probing experiments have been done both in the presence and absence of the initiator tRNA with dimethyl sulfate, kethoxal and carbodiimide as reagent probes. As an mRNA, a mini-mRNA containing the initiation region of bacteriophage lambda gene cro has been used. This region is characterized by a long single-stranded Shine-Dalgarno (SD) sequence followed by two hairpin structures of which the first one comprises in its loop the initiation codon. As compared to a free mRNA, the only nucleotides additionally protected in the binary mRNA-ribosome complex have been those which belong to the S-D sequence and the initiation codon. The protection of other nucleotides has not changed. Addition of the initiator RNA results in the modification of nucleotides in the stems of the downstream hairpin structures of the initiation region. This reflects their transition into a single-stranded conformation promoted by tRNA. A possible implication of these findings for the decoding process is discussed.

Base Sequence↗
PubMed1992

Characterization of transcription and processing from plasmids that use polIII and a yeast tRNA gene as promoter to transcribe promoter-deficient downstream DNA.

Transfer RNA (tRNA) with mutations affecting the internal promoters and thereby causing them to be nontranscribable by polIII (exemplified, e.g., by nematode tDNAPro with large insertions between the two internal promoters) could be transcribed by polIII both in vitro (yeast) and in vivo (oocytes) when cloned behind a yeast tRNAArg gene. PolIII initiated RNA synthesis could also proceed into a downstream structural gene normally read by polIII. the resultant yeast-nematode dimeric tRNA linked in front of mRNA sequences was recognized by processing enzymes to give mature tRNA. Thus, a yeast tRNA gene preceded by its 5' flank can function as a promoter for polIII transcription of any DNA, also of DNA coding for genes that otherwise could not have been expressed either in vitro or in vivo.

Animals↗
PubMed1994

The affinity technology in downstream processing.

The quality criteria imposed on several biochemicals are stringent, thus, high-separation purification technology is important to downstream processing. Affinity-based purification technologies are regarded as the finest available, and each one differs in its purifying ability, economy, processing speed and capacity. The most widely used affinity technology is classical affinity chromatography, however, other chromatography-based approaches have also been developed, for example, perfusion affinity chromatography, hyperdiffusion affinity chromatography, high-performance affinity chromatography, centrifugal affinity chromatography, affinity repulsion chromatography, heterobifunctional ligand affinity chromatography and the various chromatographic applications of 'affinity tails'. On the other hand, non-chromatographic affinity technologies aim at high throughput and seek to circumvent problems associated with diffusion limitations experienced with most chromatographic packings. Continuous affinity recycle extraction, aqueous two-phase affinity partitioning, membrane affinity filtration, affinity cross-flow ultrafiltration, reversible soluble affinity polymer separation and affinity precipitation are all non-chromatographic technologies. Several types of affinity ligands are used to different extents; antibodies and their fragments, receptors and their binding substances, avidin/biotin systems, textile and biomimetic dyes, (oligo)peptides, antisense peptides, chelated metal cations, lectins and phenylboronates, protein A and G, calmodulin, DNA, sequence-specific DNA, (oligo)nucleotides and heparin. Likewise, there are several support types developed and used; natural, synthetic, inorganic and composite materials.

Affinity Labels↗
PubMed1995

Doppler ultrasound spectral shape downstream of significant arterial stenosis in vivo.

Peak analysis was applied to pulsed Doppler ultrasound spectra from in vivo poststenotic velocity fields of canine common carotid arteries. Turbulent spectra from a region of jet breakup, five diameters (5 D) down from a 68% diameter reducing stenosis, were closely approximated by a single Gaussian peak. Multiple peaks resulting from the analysis of complex spectra from the shear layers between jet and recirculation zones, 1.5 D downstream of a 40% stenosis, correlated well with the known features of the poststenotic velocity field. It is concluded that peak analysis can be used to separate the spectral components of distinct flow regimes from the complex spectra of regions of complicated flow. This may provide a method for the diagnosis of mild degrees of arterial stenosis using pulsed Doppler ultrasound.

Acceleration↗
PubMed1996

Downstream determinants of pulsatility of the mean velocity waveform in the umbilical artery as predicted by a computer model.

An electrical analog model of the umbilicoplacental circulation that was based on hemodynamic measurements in fetal sheep was used to predict the mean blood velocity waveform in the umbilical artery. A 300% increase in placental resistance reduced the normally forward diastolic blood velocity in the umbilical artery nearly to zero. When resistance was normal, variations in umbilical arterial wall thickness (h) and elastic modulus (E) had little effect on the velocity waveform, whereas a 20% increase in radius (a) halved the ratio of the diastolic-to-systolic blood velocity (D/S). When placental resistance was high, abnormal umbilical arterial wall properties (a large, or h or E small) further reduced D/S. We conclude that high placental resistance, low umbilical arterial resistance, or a combination of high placental resistance with abnormal umbilical arterial wall properties are the primary downstream determinants of elevated pulsatility in umbilical arterial blood velocity.

Animals↗
PubMed1987

Procedures for enhancing the utility of the metallothionein promoter for the regulated expression of downstream open reading frames.

Procedures which enhance the inducibility of the mouse metallothionein I (mMT-I) transcriptional promoter in mouse C127 cells transformed by bovine papilloma virus have been investigated. These include: (i) induction with Zn2+ at low serum concentration, and (ii) use of a 'superinduction' protocol (presence of 1 microgram/ml of cycloheximide during induction with Zn2+, followed by 2 micrograms/ml of actinomycin D). Use of procedure (i) alone gave a 15- to 20-fold induction of expression of a downstream open reading frame (ORF), which is comparable to the maximum inducibility achieved with mMT-I in other systems. Use of procedures (i) and (ii) in combination allowed a 50-fold induction. Three different reporter ORFs (rabbit ferritin L subunit, human chorionic gonadotropin alpha subunit, and human lutropin beta subunit), in three different chromosomal contexts, responded to these procedures. The maximum rate of expression achieved was estimated at over 10(9) molecules per cell per day, which is 20% of the transformed cell's protein synthetic capacity. At these extremely high levels some of the induced products were cytotoxic.

Animals↗