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At least 19 recordsLinked to original sources
PubMed1988

Three-dimensional visualization of velocity fields downstream of six mechanical aortic valves in a pulsatile flow model.

Velocity fields downstream of 27 mm Björk-Shiley Standard, Björk-Shiley Convex-Concave, Björk-Shiley Monostrut, Hall-Kaster (Medtronic-Hall), St. Jude Medical and Starr-Edwards Silastic Ball aortic valves were studied in a pulsatile mock circulation. Stroke volume was 70 cm3 and frequency 71 min-1 and 88 min-1. Fluid velocity was measured by a catheter mounted hot-film anemometer probe in a glycerol water mixture one and two diameters downstream of the aortic valve. Velocity fields were dynamically visualized by a three-dimensional technique and revealed qualitative independence of frequency. All profiles were flat in the acceleration phase of systole. From peak systole and throughout the systolic deceleration phase profiles characteristic of the individual valves appeared. The pivoting and tilting disc valves caused a skewed velocity profile with highest velocities downstream of the major orifice and lowest velocities downstream of the minor orifice. The differences between the three investigated Björk-Shiley valves were remarkable. The St. Jude Medical valve generated velocity peaks downstream of the two major orifices and the central slit, and lower velocities in the hinge areas. A rather flat profile with central hollowing was seen downstream of the Starr-Edwards Ball valve. All velocity profiles were more or less dampened two diameters downstream.

Aortic Valve↗
PubMed1985

Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites.

In addition to the highly conserved AATAAA sequence, there is a requirement for specific sequences downstream of polyadenylic acid [poly(A)] cleavage sites to generate correct mRNA 3' termini. Previous experiments demonstrated that 35 nucleotides downstream of the E2A poly(A) site were sufficient but 20 nucleotides were not. The construction and assay of bidirectional deletion mutants in the adenovirus E2A poly(A) site indicates that there may be redundant multiple sequence elements that affect poly(A) site usage. Sequences between the poly(A) site and 31 nucleotides downstream were not essential for efficient cleavage. Further deletion downstream (3' to +31) abolished efficient cleavage in certain constructions but not all. Between +20 and +38 the sequence T(A/G)TTTTT was duplicated. Function was retained when one copy of the sequence was present, suggesting that this sequence represents an essential element. There may also be additional sequences distal to +43 that can function. To establish common features of poly(A) sites, we also analyzed the early simian virus 40 (SV40) poly(A) site for essential sequences. An SV40 poly(A) site deletion that retained 18 nucleotides downstream of the cleavage site was fully functional while one that retained 5 nucleotides downstream was not, thus defining sequences required for cleavage. Comparison of the SV40 sequences with those from E2A did not reveal significant homologies. Nevertheless, normal cleavage and polyadenylation could be restored at the early SV40 poly(A) site by the addition of downstream sequences from the adenovirus E2A poly(A) site to the SV40 +5 mutant. The same sequences that were required in the E2A site for efficient cleavage also restored activity to the SV40 poly(A) site.

Adenovirus Early Proteins↗
PubMed1986

Sequences capable of restoring poly(A) site function define two distinct downstream elements.

Several recent studies have shown that a functional poly(A) site consists of both an AAUAAA element as well as sequences downstream of the cleavage site. Two downstream regions were analyzed in an attempt to accurately locate and define the critical sequences. Chemically synthesized oligonucleotides of sequence from the early SV40 and the adenovirus E2A poly(A) sites were able to restore efficient cleavage to a deleted SV40 poly(A) site. Inversion of the sequence completely abolished poly(A) site function. A series of base substitution mutants were generated in each downstream sequence. Certain single base changes drastically altered poly(A) site function. Thus, it is concluded that a defined downstream sequence of limited complexity is important for efficient processing of the primary transcript at the poly(A) site. The position of the downstream elements relative to the AAUAAA and cleavage site was found to be critical since moving either the E2 element or the SV40 element an additional 40 nucleotides downstream abolished function. There were differences, however, in the effect of spacing on the function of the two elements. This observation, along with the fact that the two sequences are clearly different, indicates that there are at least two distinct genetic elements that direct efficient cleavage at the poly(A) site.

Adenovirus Early Proteins↗
PubMed1990

A downstream initiation element required for efficient TATA box binding and in vitro function of TFIID.

The gfa gene encodes glial fibrillary acidic protein, an intermediate filament protein expressed in glial cells. In vitro transcription analysis has shown that the human gfa promoter contains two initiation elements that can independently specify the transcription startpoint. One of the elements is a TATA box 25 base pairs (bp) upstream from the transcription startpoint; the other is located between 10 and 50 bp downstream from the transcription initiation site. We have now shown by transfection that both elements are required for efficient transcription in cultured cells. A partially purified natural human TATA box-binding factor (TFIID) from HeLa cells gave footprints that extended from upstream of the TATA box through the downstream initiator. Deletion of the downstream initiator inhibited both TFIID binding to the TATA box and transcription in vitro. In contrast to natural human TFIID, clone human and yeast TFIIDs expressed in bacteria gave footprints covering only the TATA box region, although hypersensitive sites were observed in the downstream region. The cloned TFIIDs also showed less dependence than natural human TFIID on the downstream initiator for both TATA box binding and in vitro transcription. These results suggest that natural human TFIID contains an additional component(s) that contribute(s) to stable TFIID binding and effective transcription by interacting with the downstream initiator.

Base Sequence↗
PubMed1990

Transcription pausing by Escherichia coli RNA polymerase is modulated by downstream DNA sequences.

Escherichia coli RNA polymerase pauses immediately after transcription of certain sequences that can form stable secondary structures in the nascent RNA transcript; pausing appears to be essential for several types of bacterial transcription attenuation mechanisms. Because base changes that weaken the RNA secondary structures reduce the half-life of pausing by RNA polymerase, nascent transcript RNA hairpins are thought to cause pausing at these sites. We show here that, for the well characterized trpL pause site, the determinants of transcription pausing are not limited to the RNA hairpin, but include the not-yet-transcribed sequence of DNA immediately downstream from the pause site. We show that this effect extends to bases up to fourteen nucleotides downstream from the pause site, that placement of a oligo(dT) tract in the nontranscribed strand in this region does not convert the pause site to a termination site, and that shifting the position of pausing by one nucleotide downstream almost eliminates pausing. From an analysis of many variants of this downstream sequence, we argue that the effect of downstream sequence is not related simply to its GC content. We suggest that these effects are mediated by altered interactions between RNA polymerase and the DNA template downstream from the enzyme's active site.

Base Sequence↗
PubMed1991

Human immunodeficiency virus type 2 (HIV-2) gene expression: downmodulation by sequence elements downstream of the transcriptional initiation site.

Human immunodeficiency virus type 2 (HIV-2) gene expression is downmodulated by sequence elements downstream of the transcriptional initiation site, corresponding to the U5 region of the long terminal repeat (LTR) and further downstream. This repression appeared to be related more to the length of the sequence intervening the transcriptional initiation site and the coding region than to a particular sequence content. The repressive effect of the downstream segment was not affected by HIV-2 and HIV-1 TAT or by the cytomegalovirus transactivator IE-2 gene. Nor was it affected by T-cell activation signals or by such cytokines as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interferon-gamma (IFN gamma), and interferon-alpha (IFN alpha). In contrast to HIV-1, HIV-2 LTR-directed gene expression was not modulated by TNF-alpha. A specific sequence element, located downstream of the TAR element in the R region, seemed to participate in modulation of gene expression. This element interacted with a nuclear protein with a mobility of about 26 kD. The repressive effect of the downstream sequence was to a certain extent cell type dependent, suggesting the involvement of cell type-specific factors. It was more effective in human lymphocytic CEM cells than in Jurkat cells. This may be relevant to the HIV-2 cell tropism (replication), latency, and virulence.

Animals↗
PubMed1992

Bipartite structure of the downstream element of the mouse beta globin (major) poly(A) signal.

The downstream region of the mouse beta (major) globin poly(A) signal was mutated and analyzed for function in transfected COS cells. From analysis of unidirectional Bal31 deletions, the 3' boundary of the downstream element was defined as +22 (22 nucleotides downstream from the cleavage site). Analysis of cluster mutations, in which 5 or 6 adjacent bases were replaced with a random CA-containing sequence in a manner that did not alter spacing, confirmed +22 as the 3' boundary of the downstream element. The analysis also revealed two short UG-rich sequences, located from +5 to +10 and from +17 to +22, as major functional components. In contrast, a more refined series of mutations, in which clusters of 3 bases were replaced, failed to cause loss of function. We conclude that the downstream element of the mouse beta globin poly(A) signal is bipartite in structure, and that portions of its sequence are functionally redundant.

Animals↗
PubMed1991

Sequences downstream from the transcriptional start site are essential for microaerobic, but not symbiotic, expression of the Rhizobium meliloti nifHDK promoter.

Deletion analysis studies have been carried out on the nifHDK promoter (P1) of R. meliloti in an attempt to determine sequences involved in the expression of this promoter under both free-living microaerobic and symbiotic conditions. Deletion of a region downstream (+17 to +61) from the promoter element resulted in low levels of expression under free-living microaerobic conditions. However, wild-type levels of expression were obtained during symbiosis with Alfalfa plants. The sequences in this region were designated the "downstream sequences'. The pattern of expression observed when the downstream sequences were deleted was similar to that observed when a previously identified upstream activator sequence (UAS) was deleted. Only when both the downstream sequences and the UAS were deleted, did activity from the P1 promoter become significantly decreased during symbiosis. Expression studies of the P1 promoter in a nifA mutant background indicate that nifA is required for symbiotic expression of P1 which is enhanced by the presence of the downstream sequences.

Aerobiosis↗
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↗
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↗
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↗
PubMed1987

Three-dimensional visualization of axial velocity profiles downstream of six different mechanical aortic valve prostheses, measured with a hot-film anemometer in a steady state flow model.

Hot-film anemometry was used for in vitro steady-state measurements downstream of six mechanical aortic valve prostheses at flow rates 10, 20 and 30 l.min-1. Three-dimensional visualizations of velocity profiles at two downstream levels were made with the valves rotated 0 and 60 degrees in relation to the sinuses of valsalvae. The velocity fields downstream of the disc valves were generally skew with increasing velocity gradients and laminar shear stresses with increasing flow rates. Furthermore, increased skewness of the velocity profiles was noticed when the major orifices of the disc valves were towards the commissure than when approaching a sinus of valsalvae. The velocity profiles downstream of the ball valve were generally flat but with considerably more disturbed flow, consistent with the findings in turbulent flow.

Aortic Valve↗
PubMed1988

Nucleosome loss activates yeast downstream promoters in vivo.

Nucleosome depletion can be made to occur in yeast by addition of glucose to strains containing the histone H4 gene under GAL promoter control. This leads to the activation of downstream promoter elements (TATA box and initiation, I, region) of three different regulated yeast promoters fused to the E. coli lacZ gene. Nucleosome loss activates the PHO5 downstream element in the presence or absence of the upstream activator sequences (UAS) through which PHO5 induction is normally mediated. The cytochrome C (CYC1) and galactokinase (GAL1) promoters are normally repressed by glucose through their UAS elements. However, when these UAS are deleted, the remaining downstream promoters are also activated by glucose-mediated nucleosome loss. These data suggest that nucleosome loss increases transcription initiation and subsequent elongation in vivo. They also indicate that the proteins which recognize the downstream promoter are activated and functional, at least in part, even in the absence of the UAS complex.

Cytochrome c Group↗
PubMed1988

Intron sequences and the length of the downstream second exon affect the binding of hnRNP C proteins in an in vitro splicing reaction.

The proteins that are in direct contact with the pre-mRNA in an in vitro splicing reaction were analyzed by UV cross-linking experiments. Six major proteins (120, 55, 44, 42, 39 and 38 KD) and three minor polypeptides (84, 72 and 63 KD) were detected. The predominant proteins 44, 42 KD belong to the class of hnRNP C proteins since they were immunoprecipitated by monoclonal antibodies directed against hnRNP C proteins. The cross-linked proteins were not detected in the absence of Mg2+, ATP or when RNA lacking introns were used as substrates in the splicing reactions. The effect of exon sequences on the binding efficiency for the photocrosslinked proteins was investigated. Transcripts containing a second exon of 24 nucleotides for the beta-globin or 107 nucleotides for the mouse insulin, yielded a reduced amount of cross-linked proteins when compared with "full length" pre-mRNAs. Sequences within the first exon of the beta-globin pre-mRNA did not affect the binding efficiency of these proteins. The reduced binding efficiency of the cross-linked proteins for the truncated beta-globin or mouse insulin pre-mRNAs correlated with the lower efficiency for in vitro splicing. Substitutions with unrelated sequences in the beta-globin second exon restore the binding of the cross-linked proteins indicating that the length of the second exon and not specific sequences are relevant for the binding efficiency of these proteins. The SP6/mouse insulin oligonucleotides cross-linked to the hnRNP C proteins were isolated and sequenced. A 17-mer was located in the second exon (134 nucleotides downstream from the 3' splice site) and a 14-mer in the intron region (25 nucleotides downstream the 5' splice site). The beta-globin oligonucleotides cross-linked to the hnRNP C proteins were a 13-mer in the second exon (28 nucleotides downstream the 3' splice site) and an 8-mer in the first exon (81 nucleotides downstream the 5' end of the pre-mRNA). Our results indicate that the hnRNP C proteins interact with those oligonucleotides located in different regions of the pre-mRNA. The binding efficiency of those proteins, however, depends on the length of the second exon and the presence of intron sequences (secondary and/or tertiary pre-mRNA structure).

Adenosine Triphosphate↗
PubMed1986

Two-component laser velocimeter measurements downstream of heart valve prostheses in pulsatile flow.

Elevated turbulent shear stresses resulting from disturbed blood flow through prosthetic heart valves can cause damage to red blood cells and platelets. The purpose of this study was to measure the turbulent shear stresses occurring downstream of aortic prosthetic valves during in-vitro pulsatile flow. By matching the indices of refraction of the blood analog fluid and model aorta, correlated, simultaneous two-component laser velocimeter measurements of the axial and radial velocity components were made immediately downstream of two aortic prosthetic valves. Velocity data were ensemble averaged over 200 or more cycles for a 15-ms window opened at peak systolic flow. The systolic duration for cardiac flows of 8.4 L/min was 200 ms. Ensemble-averaged total shear stress levels of 2820 dynes/cm2 and 2070 dynes/cm2 were found downstream of a trileaflet valve and a tilting disk valve, respectively. These shear stress levels decreased with axial distance downstream much faster for the tilting disk valve than for the trileaflet valve.

Blood Flow Velocity↗