Downstream Core Research Articles

Transcriptional activation of c-fos protooncogene by 17beta-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition.

17Beta-estradiol (E2) induced c-fos protooncogene mRNA levels in MCF-7 human breast cancer cells, and maximal induction was observed within 1 h after treatment. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibited the E2-induced response within 2 h. The molecular mechanism of this response was further investigated using pFC2-CAT, a construct containing a -1400 to +41 sequence from the human c-fos protooncogene linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In MCF-7 cells transiently transfected with pFC2-CAT, 10 nM E2 induced an 8.5-fold increase of CAT activity, and cotreatment with 10 nM TCDD decreased this response by more than 45%. Alpha-Naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, blocked the inhibitory effects of TCDD; moreover, the inhibitory response was not observed in variant Ah-nonresponsive MCF-7 cells, suggesting that the AhR complex was required for estrogen receptor cross-talk. The E2-responsive sequence (-1220 to -1155) in the c-fos gene promoter contains two putative core pentanucleotide dioxin-responsive elements (DREs) at -1206 to -1202 and -1163 to -1159. In transient transfection assays using wild-type and core DRE mutant constructs, the downstream core DRE (at -1163 to -1159) was identified as a functional inhibitory DRE. The results of photo-induced cross-linking, gel mobility shift, and in vitro DNA footprinting assays showed that the AhR complex interacted with the core DRE that also overlapped the E2-responsive GC-rich site (-1168 to -1161), suggesting that the mechanism for AhR-mediated inhibitory effects may be due to quenching or masking at the Sp1-binding site.

The DPE, a conserved downstream core promoter element that is functionally analogous to the TATA box.

Transcription by RNA polymerase II is a fundamentaland important biological process. There are tens of thousands of diverse genes that are transcribed in a highly regulated manner by the RNA polymerase II transcriptionalmachinery, and thus the mechanisms by which transcription is controlled are necessarily complex (for reviews,see Conaway and Conaway 1993; Smale 1994; Koleskeand Young 1995; Zawel and Reinberg 1995; Björklundand Kim 1996; Kaiser and Meisterernst 1996; Orphanides et al. 1996; Reines et al. 1996; Roeder 1996;Svejstrup et al. 1996; Verrijzer and Tjian 1996)...

The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila.

We analyzed the function of the downstream promoter element (DPE), a distinct 7-nucleotide core promoter element that is approximately 30 nucleotides downstream of the transcription start site of many TATA-box-deficient (TATA-less) promoters in Drosophila. There is a strict requirement for spacing between the Inr and DPE motifs, as an increase or decrease of 3 nucleotides in the distance between the Inr and DPE causes a seven- to eightfold reduction in transcription as well as a significant reduction in the binding of purified TFIID. These results suggest a specific and somewhat rigid interaction of TFIID with the Inr and DPE sequences. Photo-cross-linking analysis of purified TFIID with a TATA-less DPE-containing promoter revealed specific cross-linking of dTAFII60 and dTAFII40 to the DPE, with a higher efficiency of cross-linking to dTAFII60 than to dTAFII40. These data, combined with the previously well-characterized interactions between the two TAFs and their homology to histones H4 and H3, suggest that a dTAFII60-dTAFII40 heterotetramer binds to the DPE. Human and Drosophila transcription factors exhibit essentially the same requirements for DPE sequence and for Inr-DPE spacing. In addition, the TATA-less promoter of the human interferon regulatory factor-1 (IRF-1) gene contains a DPE that is important for transcriptional activity both in vitro and in cultured cells. Hence, these studies provide evidence for a direct role of TAFs in basal transcription of TATA-less DPE-containing genes and collectively indicate that the DPE is, in many respects, a downstream counterpart to the TATA box that is present in Drosophila to humans.

A Noncanonical Poly(A) Signal, UAUAAA, and Flanking Elements in Epstein–Barr Virus DNA Polymerase mRNA Function in Cleavage and Polyadenylation Assays

Two forms of the Epstein–Barr virus DNA polymerase (pol) mRNA (3.7 and 5.1 kb) have been detected, neither of which contains a canonical poly(A) signal. The 5.1-kb pol mRNA, which contains a rare poly(A) signal, UAUAAA, studied only in transcripts of Hepadnaviridae and a plant pararetrovirus, was analyzed in cleavage and polyadenylation assays. Incubation of the pol transcript in cell extracts produced relatively low efficiency of cleavage (12 to 14%), which was improved by conversion of the poly(A) signal to AAUAAA. Deletion of the UAUAAA signal abolished cleavage and polyadenylation. An auxiliary element, UUUGUA, 3–8 nt upstream of the poly(A) signal and two downstream core elements, a GU-rich sequence 36–46 nt, and an AUUUGUGU sequence 47–53 nt downstream of the signal (8–19 nt and 20–28 nt downstream of cleavage site) facilitated processing of pol mRNA. Replacement of sequences near the cleavage/poly(A) site affected cleavage accuracy. Binding of the 64-kDa cleavage stimulatory factor to the U-rich as well as the GU-rich elements correlated with cleavage efficiency. Thus the UAUAAA hexanucleotide plus the other cis-acting elements are clearly functional in the native pol mRNA, but are relatively inefficient. Implications of the use of an anomalous poly(A) signal and its elements are discussed.

Evolution of Three-Dimensional Vortex Structure of Coflowing Fuel Jet.

We study the transition mechanism governing the three-dimensional evolution of a coflowing fuel jet by means of HSMAC simulations which can handle the density change. In the purely axisymmetric case without any perturbations, calculations provide only Kelvin-Helmholz-type vortices. The spatial and temporal calculations triggered by an azimuthal perturbation, however, provide a detailed picture of orderly three-dimensional transition processes leading to the concentration. reorientation, and stretching of the vorticity. Furthermore, the azimuthal vorticity shows the helical structure in the braid region of vortex rings. The streamwise vorticity, which indicates azimuthally several pairs of vortices with opposite rotation, is produced in the braid region immediately upstream of the core of vortex ring. The streamwise vorticity is stretched in the braid region under the tension of vortex ring, penetrates inside the downstream core, wraps round the upstream core and finally connects each core. The streamwise vortices with opposite rotation induce the radial ejection flow.

Changes of longitudinal vortex roll structure in a mixed convective air flow through a horizontal plane channel: an experimental study

Flow visualization and temperature measurement were conducted to investigate the buoyancy induced longitudinal vortex flow structure in a mixed convective air flow through a bottom heated horizontal plane channel for the Reynolds number ranging from 20 to 50 and Rayleigh number from 6000 to 30 000. The results suggested that at low buoyancy-to-inertia ratio a longitudinal roll pair with spanwise symmetry is first induced near the duct sides and more rolls are induced adjacent to the existing rolls as the flow moves downstream. Steady vortex flow prevails after the initial transient. At high buoyancy-to-inertia ratio the splitting and merging of the rolls take place frequently causing the flow to become unsteady and highly asymmetric at high Rayleigh numbers. Several types of the roll splitting processes were identified, namely, the splitting of one roll to three rolls and two rolls to four rolls, formation of two new rolls and two successive roll splittings of the above types. The reverse processes were noted during the roll merging. The data from the measured time histories of the air temperature indicated that the flow oscillation is position dependent with the oscillation amplitude being higher in the downstream core regions. Besides, the oscillation amplitude inside a roll is determined largely by the spanwise or axial position adjustment during the roll splitting and merging processes. Finally, the data for the onset of steady and unsteady vortex flows were empirically correlated.

Identification of cell type-dependent enhancer core element located in the 3'-downstream region of the human angiotensinogen gene.

We previously demonstrated that the 3.8-kilobase DNA fragment containing exons 4 and 5 of the human angiotensinogen (hAG) gene enhances the expression of chloramphenicol acetyltransferase gene, under control of the hAG promoter, in human hepatoma HepG2 cells. In the present study, to define regulatory elements of the hAG gene, we have functionally dissected this downstream region and localized a cell type-dependent enhancer to the 832-base pair sequence containing the exon 5 and 3'-flanking region. Further deletion analyses revealed that the 24-base pair core DNA fragment present in the 3'-flanking region was responsible for this enhancement. Electrophoretic mobility shift assay demonstrated that the 3'-enhancer core element interacts specifically with two nuclear factors from the HepG2 cells, one of which is an uncharacterized factor (human angiotensinogen enhancer factor-1: hAEF-1), the other is an AP-3-related factor. Mutation analyses indicated that the disruption of hAEF-1 binding alone completely impaired the enhancer activity of the core element. These results suggested that the downstream enhancer core element interacting with hAEF-1 plays an important role in activating the angiotensinogen promoter in a cell type-dependent manner.

Regulation of brain-specific transcription of the mouse myelin basic protein gene: Function of the NFI-binding site in the distal promoter

We investigated brain-specific transcription elements in the distal region (−253 to −54) of the mouse myelin basic protein (MBP) promoter by in vitro transcription using mouse brain nuclear extracts. Using deletion and base substitution mutants, we identified one tissue-specific transcription element at the downstream core of the NFI-site between −115 and −111. Footprinting assay demonstrated that three transcription factors bind around this element; NFI-related (−130 to −111), M1 (−110 to −97) and Sp1 (−92 to −84). NFI-related factor(s) in brain extracts consisted of a characteristic population which was different from that in other tissues. Methylation interference experiments revealed that this factor(s) interacted with the NFI-downstream core in a brain-specific manner. We suggest the existence of a brain-specific NFI-related factor(s).

Ion reflection and downstream thermalization at the quasi‐parallel bow shock

It is well known that for sufficiently high Mach numbers (typically Mf>2) ion reflection is the primary process by which ion energy dissipation is initiated at quasi‐perpendicular, collisionless shocks in space. In this paper we present evidence from ISEE 2 measurements that coherent ion reflection is also often an important element of the ion energy dissipation process at high Mach number, quasi‐parallel shocks. Such coherent reflection is not evident in a number of published hybrid simulations of quasi‐parallel shocks wherein the dissipation process appears to be associated primarily with incoherent scattering of solar wind ions in the upstream and downstream wave fields. However, such reflection is present in other simulation results. The main evidence for coherent ion reflection as an important element in achieving dissipation at quasi‐parallel shocks is the following: (1) A cold secondary (to the solar wind) beam of ions is often observed within the ramp of the shock where the magnetic field intensity rises precipitously and where the major electron heating occurs. Typically this secondary beam of ions has a velocity space position roughly consistent with specular reflection of a portion of the solar wind ions incident on the shock. (2) Downstream from the shock, a relatively dense and cool core ion population is typically observed, accompanied by either multiple ion bunches or, more commonly, a suprathermal ion shell. (These populations are in addition to the extremely hot, tenuous diffuse ions which appear nearly ubiquitously both upstream and downstream from the shock.) The downstream core and shell (or bunches) are believed to represent directly transmitted solar wind ions and reflected and later scattered ions, respectively, with the motions of the latter population accounting for a large fraction of the ion energy dissipated at the shock.

Seepage Charts for Homogeneous and Zoned Embankments

The writer presents seepage charts for calculating the quantity of flow Q and critical points on the phreatic surface, A and B, for homogeneous and zoned embankments on an impervious foundation. The charts are basically a practitioner's tool for rapidly estimating seepage quantities for both homogeneous and zoned embankments. The charts will also aid in the actual flownet construction. The charts were developed from a computer program developed by the writer using the method of fragments. In comparison to flownet solutions, the charts give an average error for seepage quantity of +5%. For points on the phreatic surface A and B), an average error of -8 and +4%, respectively, are attained. Input variables are upstream and downstream core slopes, downstream shell slope, permeability of core and shell, pool elevation, and crest width. A method is also presented to estimate the seepage quantity for upstream sloping core embankments.

The problem of design of transition zones in earthfill dams

1. The conditions of operation of the transition zone at the upstream slope side of the dam's core, between its foundation and the minimum operating level, are more favorable than those at the downstream slope. There-fore, the design and grain-size selection methods for the transition zone for the dam's core section under consideration should be varied as applicable to any given conditions and not by analogy with the downstream core slope. 2. The heretofore indicated practical recommendations as to the upstream transition zone design and material selection would ensure strength and stability of the dam core. They are economically efficient.