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PubMed1989

Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.

The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treated with iron chelators, which decrease ferritin biosynthesis, a four- to fivefold increase in the binding activity is seen and this increase is entirely caused by an increase in high-affinity binding sites. In desferrioxamine-treated cells, the high-affinity form makes up about 50% of the total IRE-BP, whereas in hemin-treated cells, the high-affinity form makes up less than 1%. The total amount of IRE-BP in the cytosol of cells is the same regardless of the prior iron treatment of the cell. Furthermore, a mutated IRE is not able to interact with the IRE-BP in a high-affinity form but only at a single lower affinity Kd of 0.7 nM. Its interaction with the IRE-BP is insensitive to the sulfhydryl status of the protein.

Base Sequence↗

PubMed2002

Holographic optical elements recorded in silver halide sensitized gelatin emulsions. Part 2. Reflection holographic optical elements.

Silver halide sensitized gelatin (SHSG) holograms are similar to holograms recorded in dichromated gelatin (DCG), the main recording material for holographic optical elements (HOEs). The drawback of DCG is its low energetic sensitivity and limited spectral response. Silver halide materials can be processed in such away that the final hologram will have properties like a DCG hologram. Recently this technique has become more interesting since the introduction of new ultra-fine-grain silver halide (AgHal) emulsions. In particular, high spatial-frequency fringes associated with HOEs of the reflection type are difficult to construct when SHSG processing methods are employed. Therefore an optimized processing technique for reflection HOEs recorded in the new AgHal materials is introduced. Diffraction efficiencies over 90% can be obtained repeatably for reflection diffraction gratings. Understanding the importance of a selective hardening process has made it possible to obtain results similar to conventional DCG processing. The main advantage of the SHSG process is that high-sensitivity recording can be performed with laser wavelengths anywhere within the visible spectrum. This simplifies the manufacturing of high-quality, large-format HOEs, also including high-quality display holograms of the reflection type in both monochrome and full color.

Journal Article↗

PubMed1993

A human metal responsive element-binding protein interacts with a homologous element of the mouse metallothionein-I gene.

Metallothionein (MT) is thought to play a central role in the detoxification of heavy metals, and thus studies on its regulation are toxicologically important. Heavy metal-dependent induction of MT genes is mediated by metal responsive elements (MREs) located upstream of the genes. Zinc regulatory factor (ZRF) is a zinc-dependent MRE-binding protein that was originally detected in HeLa cell nuclear extracts using the most proximal MRE of the human MT-IIA gene (hMREa) as a probe. We show that ZRF in HeLa cell nuclear extracts can also bind to the most potent MRE of the mouse MT-I gene (mMREd). This finding was further confirmed by using partially purified ZRF. Moreover, cadmium could not promote complex formation between ZRF and mMREd at any concentration tested, as is also the case with ZRF and hMREa. These observations suggest that the transcriptional regulatory system of MT genes by zinc is conserved beyond species.

Animals↗

PubMed1992

Regulation of transcription of the germ-line Ig alpha constant region gene by an ATF element and by novel transforming growth factor-beta 1-responsive elements.

Inasmuch as transcription of unrearranged, or germ-line, Ig CH genes appears to direct switch recombination, understanding the regulation of this transcription is essential for understanding the regulation of class switching. Transforming growth factor-beta 1 (TGF-beta 1) induces germ-line alpha transcripts and increases class switching to IgA in the I.29 mu B lymphoma and in Peyer's patch and splenic B cells. It has been previously demonstrated that induction of germ-line alpha transcripts by TGF-beta occurs at the transcriptional level in I.29 mu cells. We now demonstrate that the DNA segment located 5' to the initiation sites of germ-line alpha RNA drives expression of a luciferase reporter gene construct in transient transfection experiments. Full constitutive expression requires no more than 106 bp of the 5' flanking segment. By creating a series of deletion and substitution mutations, we have demonstrated that an ATF/CRE site residing within this region is very important for constitutive expression of the germ-line alpha promoter, but mutation of this motif does not diminish TGF-beta induction. Inducibility by TGF-beta requires additional sequences residing between -128 to -106 relative to the first RNA initiation site. Two copies of a tandemly repeated sequence 5' CA-CAG(G)CCAGAC 3' (termed Ig alpha TGF-beta-RE) are located in the region from -127 to -105. An oligonucleotide containing multimers of these repeats confers TGF-beta inducibility to a heterologous promoter. An additional copy of the TGF-beta-RE was identified at -41/-30 and its deletion reduces the TGF-beta response. Thus, we conclude that tandem repeats of a novel TGF-beta-RE are the positive regulatory element for the TGF-beta response. Our study provides further evidence that TGF-beta directs class switching to IgA through induction of transcription of the germ-line C alpha gene and demonstrates that TGF-beta can activate the promoter for the germ-line alpha gene.

Activating Transcription Factors↗

PubMedDate not supplied

[Dynamic characteristics of element-by-element reproduction of visual matrix images: phenomenology and pharmaco-testing].

An outfit "Mnemotest" was employed to examine some regularities of the operative memory of man. The dynamic characteristics are provided of reproduction-storage of the elements of visual matrix images: the curves of "fading" and the "reproduction tempo". It has been shown that oral administration in therapeutic doses of the psychostimulant sydnocarb optimizes the mnemonic processes. Under different conditions the drug either increases the "reproduction tempo" or minimizes the rate of the "fading" of the mnemonic trace.

Humans↗

PubMed1989

Cell-specific expression of mouse albumin promoter. Evidence for cell-specific DNA elements within the proximal promoter region and cis-acting DNA elements upstream of -160.

Regulation of albumin gene expression is believed to be mediated by multiple nuclear factors that interact with cis-acting DNA sequences within the first 160 base pairs (bp) of the promoter. The minimal promoter sequence required to generate tissue-specific expression has not been clearly defined. We have constructed a series of transient expression vectors containing progressive deletions of the mouse albumin gene 5'-flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and include the Moloney murine leukemia viral (Mo-MuLV) enhancer. Promoter activity was determined in mouse hepatoma and fibroblast cell lines by chloramphenicol acetyltransferase and S1 nuclease analyses. All constructions were compared with -623 Albcat-Mo-MuLV which contains all the sequence homology between the rat and mouse promoters. Low levels of expression were observed with -60 Albcat-Mo-MuLV (10%) in hepatoma but not fibroblast cells. Addition of promoter sequence to -208 bp progressively increased activity to 190% in the hepatoma cells, while -308 and -1612 Albcat-Mo-MuLV had activity similar to the -623 Albcat-Mo-MuLV level, and -3000 Albcat-Mo-MuLV showed a 2-fold reduction in transcriptional activity. The inclusion of promoter sequences upstream of -60 generated low levels of expression in the fibroblasts. We also show that factors from mouse liver nuclear extracts protect at least five regions of the albumin promoter upstream of -160. Our results indicate that tissue specificity is established within the proximal promoter region and that additional cis-acting elements that may have a functional role in the efficiency of albumin gene expression are located upstream of -160 bp.

Albumins↗

PubMed1988

The iron-responsive element is the single element responsible for iron-dependent translational regulation of ferritin biosynthesis. Evidence for function as the binding site for a translational repressor.

Ferritin, a cytoplasmic protein critical in iron metabolism, displays iron-dependent regulation of its biosynthetic rate with no corresponding changes in mRNA levels. An iron-responsive element (IRE) has been identified in the 5'-untranslated region (UTR) of the human ferritin heavy chain mRNA which, when placed in the 5'-UTR of heterologous reporter genes, confers iron-dependent translational regulation to the hybrid mRNAs. However, whereas the biosynthetic rate of ferritin in response to changes in iron status exhibits a 30-80-fold range, the apparent ranges observed for reporter gene constructs utilizing chloramphenicol acetyltransferase assays or human growth hormone radioimmunoassays have been much less. A deletion and reconstitution study was undertaken to address the possibility that regions of the ferritin gene and mRNA other than the IRE may be necessary for the production of the full range of iron regulation. Data are presented that demonstrate that the IRE alone is capable of conferring iron-dependent translational regulation of biosynthesis to downstream encoded proteins that is both qualitatively and quantitatively similar to that observed with expression of ferritin itself. Thus, the complete range of iron-dependent translational regulation conferred by the IRE occurs independently of the presence of the ferritin promoter, other regions of the ferritin 5'-UTR, the ferritin coding region, and the ferritin 3'-UTR. Additionally, experiments addressing the translatability in vivo of various ferritin construct mRNAs support the theory that the IRE functions as the binding site for a translational repressor.

Binding Sites↗

PubMed1994

Isolation of an X-ray-responsive element in the promoter region of tissue-type plasminogen activator: potential uses of X-ray-responsive elements for gene therapy.

Tissue-type plasminogen activator (t-PA) was induced over 50-fold after X irradiation in radioresistant human melanoma cells (Boothman et al., Cancer Res. 51, 5587-5595, 1991). Activities of t-PA were induced 14-fold in ataxia telangiectasia, 9-fold in Bloom's syndrome and 6-fold in Fanconi's anemia cells, compared to normal human fibroblasts (Fukunaga et al., Int. J. Radiat. Oncol. Biol. Phys. 24, 949-957, 1992). X-ray-inducible synthesis of the protease, t-PA, may play a role(s) in damage-inducible repair processes in mammalian cells, similar to the SOS repair systems in lower eukaryotes and prokaryotes. DNA band shift and DNase I footprinting assays were used to determine binding if transcription factors to a previously unknown X-ray-responsive element (XRE) in the t-PA promoter. The major goals of our research with XREs are to understand (a) which transcription factor(s) regulates t-PA induction after X rays, and (b) the role(s) of t-PA in DNA repair, apoptosis or other responses to X rays. The purpose of this paper is to discuss the potential use of an XRE, such as the one in the t-PA promoter, for gene radiotherapy. Several gene therapy strategies are proposed.

Cell Nucleus↗

PubMedDate not supplied

Methods of analysis for toxic elements in foods. Part IV. General method of ashing for the determination of toxic elements.

As a result of the request of the Codex Alimentarius Committee on Methods of Analysis and Sampling, a general method for ashing of foods for the determination of toxic elements was assembled. This method consolidates the results of more than a decade of interlaboratory work of numerous USSR food laboratories and specifies the details of wet and dry ashing procedures.

Food Analysis↗

PubMed1996

Purification of a novel MHC class I element binding activity from thymus nuclear extracts reveals that thymic RBP-Jkappa/CBF1 binds to NF-kappaB-like elements.

We purified a DNA binding protein that recognizes a portion of the MHC class I regulatory element region 1/NF-kappaB binding site whose expression correlates with the expression of a MHC class I transgene in the thymus. The N-terminal amino acid sequence and the molecular size matched the RBP-Jkappa protein, also known as the EBV C-promoter binding factor, CBF1. Anti-peptide sera reactive with RBP-Jkappa/CBF1 also reacted with this protein in gel mobility shift assays. Although RBP-Jkappa/CBF1 is ubiquitously expressed, binding to the MHC class Ia NF-kappaB site was limited to the thymus. Comparison of the DNA binding specificities of RBP-Jkappa/CBF1 in thymic and splenic nuclear extracts revealed strong binding from both extracts to an IFN-beta kappaB site containing the RBP-Jkappa/CBF1 consensus sequence (CGTGGGAA). In contrast, only the thymic nuclear extract showed strong DNA binding activity with probes containing the NF-kappaB recognition sequences present in the MHC class Ia, IL-2Ralpha, and granulocyte-macrophage CSF promoters. Thus, RBP-Jkappa/CBF1 in thymic extracts demonstrates a clearly distinguishable DNA binding specificity that correlates with tissue-specific expression of a class I transgene. This, coupled with the fact that our previous study showed enhanced expression of the transgene in CD4+CD8+ thymocytes, suggests that RBP-Jkappa/CBF1 may play a role in the development of the immune system.

Amino Acid Sequence↗

PubMed1996

Ethanol exposure alters the phosphorylation of cyclic AMP responsive element binding protein and cyclic AMP responsive element binding activity in rat cerebellum.

The purpose of the present study was to track the acute effects of ethanol on the cerebellar adenylyl cyclase cascade from membrane to nucleus and to determine how this important signaling pathway neuroadapts during chronic ethanol exposure. An acute ethanol challenge increased cyclic AMP content and protein kinase A activity by 80% compared to control rats. In the nucleus the phosphorylated form of cAMP responsive element binding protein (CREB) increased 500%. Gel retardation assays with an oligomer encoding the rat proenkephalin cyclic AMP (CRE)1 were performed. Assays from protein derived from rats acutely exposed to ethanol identified three CRE-protein complexes also observed in assays of protein from saline-treated animals. However, after acute ethanol exposure, the intensity of the upper and middle CRE-protein complexes increased by 3-fold (280 +/- 10 vs. 70 +/- 3 arbitrary units; P < .01) compared to the sham treatment. Intensity of complex formation was still elevated 1 and 6 hr after ethanol exposure compared to sham conditions. In contrast, chronic ethanol treatment as well as pair-fed treatment did not alter the phosphorylation state of CREB or the intensity of the specific CRE-protein complexes on gel retardation assays. In summary, acute ethanol exposure resulted in the activation of the adenylyl cyclase signal transduction cascade from membrane to nucleus. In contrast, chronic ethanol exposure did not alter the phosphorylation of CREB or CRE binding activity. The behavioral significance of these events remain unclear, but may be related to the development of ethanol-induced tolerance in specific cerebellar functions.

Adenylyl Cyclases↗

PubMed1952