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PubMed2006

Inhibitory cis-element-mediated decay of human papillomavirus type 16 L1-transcript in undifferentiated cells.

Production of human papillomavirus type 16 major capsid protein L1 in undifferentiated cells is negatively regulated by several yet unidentified cis-acting inhibitory RNA elements, among which a major element is located within the first 514 nucleotides of the L1-mRNA. By Northern blotting we examined effect of the major element on the steady-state level of mRNA transiently transcribed in 293T cells from the firefly luciferase (Fluc) gene combined with the L1 DNA fragment encoding the major element. As reported previously, the element down-regulated steady-state level of the mRNA. The most efficient down-regulation was achieved by insertion of the element near the 5' end of mRNA, resulting in an undetectable level of the mRNA. The longer the distance from the 5' end of the mRNA to the element, the weaker the down-regulation. The half-life of the mRNA having the element was similar to that of normal Fluc-mRNA. When the element near the 5' end was removed by splicing, the steady-state level of the resultant mRNA was raised to a readily detectable level. The steady-state level of RNA synthesized by RNA polymerase-I was not influenced by the presence of the element. Taken together, it is suggested that DNA region encoding the major inhibitory element does not disturb transcription and that the pre-mRNA is degraded by an RNA element-mediated mechanism after the splicing step in the course of mRNA maturation.

Capsid Proteins↗
PubMed2005

hATpin, a family of MITE-like hAT mobile elements conserved in diverse plant species that forms highly stable secondary structures.

We identified a 178 bp mobile DNA element in lettuce with characteristic CGAGC/GCTCG repeats in the subterminal regions. This element has terminal inverted repeats and 8-bp target site duplications typical of the hAT superfamily of class II mobile elements, but its small size and potential to form a single-stranded stable hairpin-like secondary structure suggest that it is related to MITE elements. In silico searches for related elements identified 252 plant sequences with 8-bp target site duplications and sequence similarity in their terminal and subterminal regions. Some of these sequences were predicted to encode transposases and may be autonomous elements; these constituted a separate clade within the phylogram of hAT transposases. We demonstrate that the CGAGC/GCTCG pentamer maximizes the hairpin stability compared to any other pentamer with the same C + G content, and the secondary structures of these elements are more stable than for most MITEs. We named these elements collectively as hATpin elements because of the hAT similarity and their hairpin structures. The nearly complete rice genome sequence and the highly advanced genome annotation allowed us to localize most rice elements and to deduce insertion preferences. hATpin elements are distributed on all chromosomes, but with significant bias for chromosomes 1 and 10 and in regions of moderate gene density. This family of class II mobile elements is found primarily in monocot species, but is also present in dicot species.

Base Sequence↗
PubMed1992

Ear3/COUP-TF binds most tightly to a response element with tandem repeat separated by one nucleotide.

Ear3/COUP-TF belongs to the steroid/thyroid hormone receptor superfamily of nuclear transcription factors. Previous studies have identified six target response elements to which the factors of the family bind with various affinities. They are palindromic pair of hexameric half site (Thyroid hormone Response Element; TRE, Estrogen Response Element; ERE, Glucocorticoid Response Element; GRE) or directly repeated half sites [Direct Repeat (DR) 3, DR4, DR5]. In this report, these six elements and three additional putative elements, DR0, DR1, and DR2, which are directly repeated half sites separated by 0, 1 and 2 nucleotides, respectively, were tested for binding to Ear3/COUP-TF protein employing gel retardation method. Among the palindromic series of response elements, TRE showed the highest affinity, ERE showed second highest, and no binding was observed for GRE. Among six direct repeat response elements, DR1 showed the highest affinity. Comparison of binding affinities between DR1 and TRE revealed that the DR1 has the highest affinity of all the nine response elements tested. The consensus sequence of the DR1 element resembles well the COUP sequence found in the upstream promoter of chicken ovalbumin gene, which has been shown to serve as a natural response element for Ear3/COUP-TF. Retinoid X receptor (RXR), another member of the receptor family, reportedly utilizes DR1 sequence as its response element, suggesting that Ear3/COUP-TF could regulate the expression of the same subsets of genes as RXR does.

Base Sequence↗
PubMed1990

Disruption of cellular elements and water in neurotoxicity: studies using electron probe X-ray microanalysis.

Regulation of elements and water in nerve cells is a complex, multifaceted process which appears to be vulnerable to neurotoxic events. However, much of our knowledge concerning the potential role of elements in nerve cell injury is limited by the relatively gross level of corresponding analyses. If we are to confirm and understand the proposed role, more precise and detailed information is needed. As indicated in this commentary, research employing electron probe microanalysis and digital X-ray imaging has begun to provide this necessary information. Recent EPMA studies of nerve and glial cells in the peripheral and central nervous systems have shown that each cell type and their corresponding morphologic compartments exhibit unique distributions of elements and water. The use of microprobe analysis has allowed us to document precisely how elements and water redistribute in morphological compartments of damaged nerve cells. Accumulating evidence from EPMA studies suggests that, rather than being an epiphenomenon, intracellular changes in diffusible elements might mediate the functional and structural consequences of neurotoxic insult. It is also evident from this research that elements other than Ca might play a pertinent role in the injury response and that changes in intraneuronal elemental composition might develop according to a specific temporal pattern, e.g., transection-induced sequential alterations in axonal K, Na, Cl, and Ca. Therefore, rather than conducting end-point studies, longitudinal investigations are necessary to define the sequential pattern of elemental perturbation associated with a given neurotoxic event. Such research can also help identify the role of individual elements in the injury response. Future microprobe studies should be combined with measurements of ion levels (e.g., using fura-2 or ion selective electrodes) to provide a comprehensive and dynamic view of elemental deregulation. In addition, parallel biochemical studies should be performed to determine mechanisms of elemental disruption and possible biochemical and metabolic consequences of this disruption. Although evidence presented in this commentary suggests that each type of neurotoxic event produces a characteristic pattern of decompartmentalization, further work is necessary to confirm this possibility. Finally, based on a presumed involvement of elements in nerve injury, efforts are currently underway in several laboratories to develop appropriate pharmacological therapies for certain chemical- and trauma-induced neuropathological conditions (Dretchen et al., 1986; El-Fawal et al., 1989; Beattie et al., 1989).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Angiogenesis in carcinosarcomas of the uterus: differences in the microvessel density and expression of vascular endothelial growth factor between the epithelial and mesenchymal elements.

Carcinosarcoma of the uterus is a highly aggressive neoplasm. However, the angiogenesis of this neoplasm is still unknown. This is the first study to examine the differences in angiogenesis between the epithelial and mesenchymal elements of this biphasic neoplasm. Surgical specimens from 21 primary uterine carcinosarcomas were histopathologically evaluated, and then immunohistochemically analyzed for tumor angiogenesis, using an anti-vascular endothelial growth factor (VEGF) antibody. The microvessel density (MVD) was also measured in each element of these neoplasms, using anti-CD34 monoclonal antibody. The MVD in the epithelial element was found to be higher than that of the mesenchymal element in 20 of 21 (95.2%) primary tumors. The epithelial elements showed a higher MVD (mean, 81.6 +/- 41.1) than the mesenchymal elements (mean, 36.7 +/- 23.8) in these primary tumors (P < .0001). Moreover, the epithelial elements showed a higher VEGF expression (mean, 0.78 +/- 0.23) than the mesenchymal elements (mean, 0.37 +/- 0.20) (P < .0001). The tumors with lymph-vascular invasion showed a higher VEGF expression (n = 17; mean, 0.85 +/- 0.17) than the tumors without lymph-vascular invasion (n = 4, mean, 0.47 +/- 0.12) (P < .01). Microscopically, neither lymph-vascular space invasion nor metastatic tumors consisted of sarcoma alone in this series. In addition, a decrease in the VEGF expression was found in the transitional areas between carcinomatous and sarcomatous elements in all 10 homologous and 4 heterologous tumors evaluated. These results suggest that the tumor angiogenesis in the epithelial element may be more active than that of the mesenchymal element and also substantiated the high metastatic potential of the epithelial element in uterine carcinosarcoma. Based on these findings, carcinoma cells thus may play a key role in the angiogenesis of this biphasic neoplasm.

Aged↗
PubMed2001

Elemental release from dental casting alloys into biological media with and without protein.

OBJECTIVE: The objective of this study was to determine the role of proteins in affecting elemental release from a variety of clinically available dental casting alloys. An important role for proteins was suspected based on previous reports about the corrosion of stainless steel and the cytotoxicity of alloys after exposure to a saline-protein solution. METHODS: Clinically available alloys with compositions ranging from 0 to 94at.% noble elements were exposed for 1 week to either saline, saline with 3% bovine serum albumin (BSA), or complete cell-culture medium with 3% serum. Atomic absorption spectrophotometry was used to measure the release of elements from the alloys. Elemental release was normalized for the exposed surface area of the alloys. RESULTS: In general, more elemental release occurred into the saline-BSA solution compared to saline alone for all released elements (Ag, Cu, Pd, and Zn) except for Ni. Ni release from the NiCr alloy was lower in the presence of BSA. Each element responded somewhat differently with Pd being the least predictable in its behavior. Elemental release was less in the cell-culture medium than in the saline-BSA solution for most elements. For alloys which released multiple elements, all elements responded similarly but not identically to the presence of protein. A high elemental release during exposure to the saline-BSA solution correlated with a low alloy cytotoxicity post-exposure to the saline-BSA. SIGNIFICANCE: This study demonstrates the importance of defining exactly the composition of biological solutions used to assess in vitro corrosion and biocompatibility of dental casting alloys. Other molecules in addition to proteins appeared to be critical to the corrosion of these alloys in vitro.

Analysis of Variance↗
PubMed1999

Intimate association of microsatellite repeats with retrotransposons and other dispersed repetitive elements in barley.

Simple sequence repeat (SSR)-based genetic markers are being actively developed for the majority of crop plant species. In barley, characterization of 290 dinucleotide repeat-containing clones from SSR-enriched libraries has revealed that a high percentage are associated with cereal retrotransposon-like and other dispersed repetitive elements. Associations found were with BARE-1, WIS2-1A, PREM1 and the dispersed repetitive element R173. Additional similarities between different SSR clones, which have no matches in DNA sequence databases, indicate that this phenomenon is probably widespread in the barley genome. Sequence homologies to the non-coding regions of several cereal genes were also explained by homology to mobile genetic elements. The SSRs found can therefore be classified into two types: (1) those with unique sequences on either flank, and (2) those which are intimately associated with retro-transposons and other dispersed repetitive elements. As the cereal genome is thought to consist largely of this type of DNA, some random association would be expected. However, the conserved positions of the SSRs, relative to repetitive elements, indicate that they have arisen non-randomly. Furthermore, this class of SSRs can be classified into three subtypes: (1) those which are positioned 3' of a transposable element with unique sequence on the other flank, (2) those positioned 5' of a transposable element, and (3) those which have arisen from an internal sequence and so have transposable element sequence on both flanks. The first appear to be analogous to the class of SSRs in mammalian systems which are associated with Alu elements and SINEs (short interspersed elements) and which have been postulated to arise following integration of an extended and polyadenylated retro-transcript into the host genome, followed by mutation of the poly(A) tract and expansion into an SSR. For the second, we postulate that a proto-SSR (A-rich sequence) has acted as a 'landing pad' for transposable element insertion (rather than being the result of insertion), while the third includes those which have evolved as a component of an active transposable element which has spread throughout the genome during bursts of transposition activity. The implications of these associations for genome and SSR evolution in barley are discussed.

Base Sequence↗
PubMed1998

[Voice prostheses with sound-producing metal reed element--an experimental study and initial clinical results].

BACKGROUND: Following total laryngectomy the voice is produced by esophageal speech as well as with voice prostheses by vibrations of pharyngeal mucosal folds. This pharyngeal sound normally has a significantly lower fundamental frequency than the healthy voice (men about 120 Hz, women about 240 Hz, pharyngeal voice about 70 Hz), which is a handicap especially for female laryngectomy patients. In order to improve the postlaryngectomy voice, a new type of voice prostheses containing an integrated sound-producing metallic reed element was developed (ADEVA Company, Lübeck, Germany). METHODS/PATIENTS: Thirty-five of these new sound-producing voice prostheses were tested in vitro for different prosthesis-specific physical parameters (pressure, flow, sound pressure, flow resistance, frequency range). In 15 voice prosthesis speakers, a sound-producing prosthesis was introduced during a routine outpatient visit. Besides measurement of the above mentioned physical parameters in patients with conventional and sound-producing prostheses, the resulting voice as also evaluated by means of a video recording. RESULTS: In vitro all prostheses with the metallic reed element produced a clear sound. Flow resistance of the prostheses was slightly elevated by the reed element. Insertion of the prostheses was hindered by the reed element. Period of uninterrupted sound production was prolonged after insertion of a sound-producing prosthesis and patients could speak on a lower pressure level, but the sound of the reed element was permanently distinguishable only in 6 of 15 patients. CONCLUSIONS: In principle a variation of the pharyngeal voice by means of a sound producing element, which is integrated into a voice prosthesis, is possible. The current design of the metallic reed element tested is not yet suitable for routine clinical use: 1. The reed element is too sensitive and is easily damaged during insertion, so the insertion device has to be improved. 2. The sound producing element is blocked by small amounts of tracheal secretions, so that this element should be replaceable separately without requiring removal of the silicone value (if possible by the patient himself). Prior to insertion of the sound producing voice prosthesis the maximum air flow through the shunt should be measured to determine if the patient can produce the necessary air flow for activation of the reed element. A further improvement for these special types of voice prostheses would be a sound producing element, which generates a variable frequency of sound. Limiting the patient to only one fundamental frequency creates a monotone, which does not sound naturally. Initial progress toward a sound-producing voice prostheses has been made. This should be followed by the necessary improvements in order to improve the feasibility of this design for routine clinical use.

Equipment Failure Analysis↗
PubMed1984

Genetic properties and chromatin structure of the yeast gal regulatory element: an enhancer-like sequence.

DNA molecules created by fusing a 365-base-pair segment of yeast DNA encoding the galactose-regulated upstream promoter element (gal) to a set of derivatives that systematically delete sequences upstream from the his3 gene are introduced in single copy back into the yeast genome precisely at the his3 locus and then assayed for transcription. Fusions of the gal regulatory element to his3 derivatives containing all normal mRNA coding sequences but lacking essentially the entire promoter region fail to express his3 under any growth conditions. Fusions to derivatives lacking the his3 upstream promoter element but containing the "TATA box" place his3 expression under gal control--i.e., extremely high RNA levels in galactose-containing medium and essentially no his3 RNA in glucose-containing medium. However, of the two normal his3 initiation sites, only the downstream one is activated by the gal element. In fusions of this type, neither the orientation of the gal element nor the distance between the element and the his3 TATA box affects the level or the initiation points of transcription. However, the gal element does not influence transcription when placed 100 or 300 base pairs downstream from the normal mRNA start sites. Fusions to derivatives containing the entire his3 promoter region restore the basal level of his3 transcription in glucose-grown cells, and both transcriptional initiation sites are used. Furthermore, RNA levels in galactose-grown cells, although somewhat higher than in glucose-grown cells, are significantly below the fully induced level. The distance from his3 coding sequences does not affect RNA levels, suggesting that specific sequences, possibly corresponding to the his3 upstream promoter element, reduce the ability of the gal element to activate transcription. Analysis of chromatin from some of these strains indicates a DNase I-hypersensitive site(s) in the middle of the gal element. However, this structural feature is not correlated with transcriptional initiation because it is found when cells are grown in glucose medium and also in derivatives lacking a TATA box. Thus, the gal upstream element possesses most, but not all, of the properties of viral and cellular enhancer sequences of higher eukaryotes. In addition, it appears that the his3 and gal upstream sequences represent two distinct classes of promoter elements, which activate transcription from different initiation sites.

Alleles↗
PubMed1996

Cooperative role of interferon regulatory factor 1 and p91 (STAT1) response elements in interferon-gamma-inducible expression of human indoleamine 2,3-dioxygenase gene.

Interferon (IFN)-gamma induces the expression of the indoleamine 2, 3-dioxygenase (INDO) gene in human cells, which plays a role in the inhibitory effect of IFN-gamma on intracellular pathogens and on cell proliferation. Earlier studies established that the IFN-gamma-inducible expression of the INDO gene was dependent on two upstream elements: (i) a 14-base pair sequence homologous to an interferon-stimulated response element (ISRE) sequence found in IFN-alpha-inducible genes and (ii) a 9-base pair palindromic sequence (palindromic element (PE) II) homologous to an interferon-gamma-activated site (GAS) element found in IFN-gamma-inducible genes. A second GAS element (PE I), between ISRE and PE II, was ineffective in supporting a response to IFN-gamma. Studies were carried out to determine the distinction between the two GAS elements and the relative role of the two elements (ISRE and PE II) required for a response to IFN-gamma. The PE I element was able to form a complex with IFN-gamma-activated p91 (STAT1) factor but with lower efficiency than the complex formed with PE II sequence. However, switching the positions of PE I and II sequences in reporter plasmid constructs (containing chloramphenicol acetyltransferase gene) showed that both PE I and PE II were able to support a response to IFN-gamma if located at the position of PE II but not at the position of PE I. Increasing the distance between the ISRE and PE II also affected the level of response, suggesting that the relative position of the two elements is important for optimal stimulus. To explore whether an interaction between the IFN-gamma-regulated factors (IRF-1 and p91) binding to the ISRE and PE II might be important, we tested whether the ISRE sequence could be replaced by another response element, NF-kappaB. The plasmid construct with NF-kappaB element in place of the ISRE was responsive to IFN-gamma, indicating that an interaction between the IRF-1 and p91 factors was not required. The results indicate that the response of INDO gene to IFN-gamma depends on a cooperative role of IFN-gamma-responsive factors binding to the ISRE and GAS elements.

Base Sequence↗
PubMed1997

RNA ligands selected by cleavage stimulation factor contain distinct sequence motifs that function as downstream elements in 3'-end processing of pre-mRNA.

Critical events in 3'-end processing of pre-mRNA are the recognition of the AAUAAA polyadenylation signal by cleavage and polyadenylation specificity factor (CPSF) and the binding of cleavage stimulation factor (CstF) via its 64-kDa subunit to the downstream element. The stability of this CPSF.CstF.RNA complex is thought to determine the efficiency of 3'-end processing. Since downstream elements reveal high sequence variability, in vitro selection experiments with highly purified CstF were performed to investigate the sequence requirements for CstF-RNA interaction. CstF was purified from calf thymus and from HeLa cells. Surprisingly, calf thymus CstF contained an additional, novel form of the 64-kDa subunit with a molecular mass of 70 kDa. RNA ligands selected by HeLa and calf thymus CstF contained three highly conserved sequence elements as follows: element 1 (AUGCGUUCCUCGUCC) and two closely related elements, element 2a (YGUGUYN0-4UUYAYUGYGU) and element 2b (UUGYUN0-4AUUUACU(U/G)N0-2YCU). All selected sequences tested functioned as downstream elements in 3'-end processing in vitro. A computer survey of the EMBL data library revealed significant homologies to all selected elements in naturally occurring 3'-untranslated regions. The majority of element 2a homologies was found downstream of coding sequences. Therefore, we postulate that this element represents a novel consensus sequence for downstream elements in 3'-end processing of pre-mRNA.

Animals↗
PubMed1999

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element.

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision. In cooperation with the indispensable mooring sequence and spacer element, a 5' efficiency element and a 3' efficiency element enhance editing in vitro. A phylogenetic comparison of 32 species showed minor differences in the apoB mRNA sequence, and the apoB mRNA from 31 species was robustly edited in vitro. However, guinea pig mRNA was poorly edited. Compared with the consensus sequences of these 31 species, guinea pig apoB mRNA has three variations in the 3' efficiency element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence and the 3' efficiency element form a double-stranded stem. Thirty-one mammalian apoB mRNA sequences are predicted to form this stem positioning C(6666) two nucleotides upstream of the stem. However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C(6743) to U) that predicts an extension of the stem and hence the lower editing efficiency. A test of this model demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutations in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA, to a 58-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence and the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.

Animals↗
PubMed1993

Two essential regulatory elements in the human interferon gamma promoter confer activation specific expression in T cells.

Like interleukin 2 (IL-2), interferon gamma (IFN-gamma) is an early response gene in T cells and both are prototypical T helper cell type 1 (Th-1) lymphokines. Yet IL-2 and IFN-gamma production are independently regulated, as demonstrated by their differential expression in certain T cell subsets, suggesting that the regulatory elements in these two genes must differ. To explore this possibility, the 5' flank of the human IFN-gamma gene was analyzed. Expression of IFN-gamma promoter-driven beta-galactosidase reporter constructs containing 538 bp of 5' flank was similar to that by constructs driven by the IL-2 promoter in activated Jurkat T cells; expression nearly as great was observed with the construct containing only 108 bp of IFN-gamma 5' flank. These IFN-gamma promoter constructs faithfully mirrored expression of the endogenous gene, in that expression required activation both with ionomycin and PMA, was inhibited by cyclosporin A, and was not observed in U937 or THP-1 cells. The region between -108 and -40 bp in the IFN-gamma promoter was required for promoter function and contained two elements that are conserved across species. Deletion of 10 bp within either element reduced promoter function by 70%, whereas deletions in nonconserved portions of this region had little effect on promoter function. The distal conserved element (-96 to -80 bp) contained a consensus GATA motif and a potential regulatory motif found in the promoter regions of the GM-CSF and macrophage inflammatory protein (MIP) genes. Factors binding to this element, including GATA-3, were found in Jurkat nuclear extracts by electromobility shift assays and two of the three complexes observed were altered in response to activation. One or both of these motifs are present in the 5' flank of multiple, other lymphokine genes, including IL-3, IL-4, IL-5, and GM-CSF, but neither is present in the promoter of the IL-2 gene. The proximal conserved element (-73 to -48 bp) shares homology with the NFIL-2A element in the IL-2 promoter; these elements compete for binding of factors in Jurkat nuclear extracts, although the NFIL-2A element but not the IFN-gamma element binds Oct-1. Factors binding to this element in the IFN-gamma gene were present in extracts from resting and activated Jurkat T cells. However, by in vivo footprinting of intact cells, this element was protected from methylation only with activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1996

Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome.

A yeast autonomously replicating sequence, ARS305, shares essential components with a chromosome III replicator, ORI305. Known components include an ARS consensus sequence (ACS) element, presumed to bind the origin recognition complex (ORC), and a broad 3'-flanking sequence which contains a DNA unwinding element. Here linker substitution mutagenesis of ARS305 and analysis of plasmid mitotic stability identified three short sequence elements within the broad 3'-flanking sequence. The major functional element resides directly 3' of the ACS and the two remaining elements reside further downstream, all within non-conserved ARS sequences. To determine the contribution of the elements to replication origin function in the chromosome, selected linker mutations were transplaced into the ORI305 locus and two-dimensional gel electrophoresis was used to analyze replication bubble formation and fork directions. Mutation of the major functional element identified in the plasmid mitotic stability assay inactivated replication origin function in the chromosome. Mutation of each of the two remaining elements diminished both plasmid ARS and chromosomal origin activities to similar levels. Thus multiple DNA elements identified in the plasmid ARS are determinants of replication origin function in the natural context of the chromosome. Comparison with two other genetically defined chromosomal replicators reveals a conservation of functional elements known to bind ORC, but no two replicators are identical in the arrangement of elements downstream of ORC binding elements or in the extent of functional sequences adjacent to the ACS.

Base Sequence↗
PubMed1995

Composite structure of auxin response elements.

The auxin-responsive soybean GH3 gene promoter is composed of multiple auxin response elements (AuxREs), and each AuxRE contributes incrementally to the strong auxin inducibility to the promoter. Two independent AuxREs of 25 bp (D1) and 32 bp (D4) contain the sequence TGTCTC. Results presented here show that the TGTCTC element in D1 and D4 is required but not sufficient for auxin inducibility in carrot protoplast transient expression assays. Additional nucleotides upstream of TGTCTC are also required for auxin inducibility. These upstream sequences showed constitutive activity and no auxin inducibility when part or all of the TGTCTC element was mutated or deleted. In D1, the constitutive element overlaps the 5' portion of TGTCTC; in D4, the constitutive element is separated from TGTCTC. An 11-bp element in D1, CCTCGTGTCTC, conferred auxin inducibility to a minimal cauliflower mosaic virus 35S promoter in transgenic tobacco seedlings as well as in carrot protoplasts (i.e., transient expression assays). Both constitutive elements bound specifically to plant nuclear proteins, and the constitutive element in D1 bound to a recombinant soybean basic leucine zipper transcription factor with G-box specificity. To demonstrate further the composite nature of AuxREs and the ability of the TGTCTC element to confer auxin inducibility, we created a novel AuxRE by placing a yeast GAL4 DNA binding site adjacent to the TGTCTC element. Expression of a GAL4-c-Rel transactivator in the presence of this novel AuxRE resulted in auxin-inducible expression. Our results indicate that at least some AuxREs have a composite structure consisting of a constitutive element adjacent to a conserved TGTCTC element that confers auxin inducibility.

Animals↗
PubMed1997

The adeno-associated virus type 2 p40 promoter requires a proximal Sp1 interaction and a p19 CArG-like element to facilitate Rep transactivation.

We have identified the sequence elements that are required for adeno-associated virus type 2 p40 promoter activity. Mutation of specific promoter elements showed that two Sp1 sites at approximately -50 (Sp1-50) and -70 (GGT-70) bp upstream of the start of the p40 messages were necessary for maximal promoter activity. As expected, the TATA site at -30 was also essential. In vitro DNA binding experiments confirmed that the Sp1-50 and GGT-70 sites were bound by Sp1 or Sp1-like proteins. Two other transcription elements, the ATF-80 and AP1-40 sites, may play a role in p40 activity. Mutation of these elements resulted in a modest decrease in p40 transcription, but DNA binding experiments did not clearly demonstrate binding of transcription factors to these sites. In contrast, a major late transcription factor site at -110 was shown to bind the transcription factor, but mutation of this site had no effect on p40 activity. In a previous report, we have shown that transactivation of the p40 promoter by the viral Rep proteins required an upstream Rep binding element (in the terminal repeat or the p5 promoter), an unidentified p19 promoter element, and a p40 promoter element (D. J. Pereira and N. Muzyczka, J. Virol. 71:1747-1756, 1997). Here we demonstrate that the CArG-140 element in the p19 promoter and the Sp1-50 element in the p40 promoter are the specific p19 and p40 elements required for Rep induction of p40. As in the case of the p19 promoter, Sp1 facilitates interaction of Rep with the p40 promoter by interaction of the two proteins. Furthermore, electron microscopy experiments demonstrated that when Rep is bound to an upstream Rep binding element, it can interact with a proximal Sp1 site by protein contacts and create a loop in the intervening DNA. This finding suggests a common mechanism whereby the Rep binding element in the TR or the p5 promoter induces p19 and p40 activity by interaction with their respective Sp1 sites.

Base Sequence↗
PubMed1998

Xenopus TFIIIA gene transcription is dependent on cis-element positioning and chromatin structure.

The Xenopus TFIIIA gene is transcribed very efficiently in oocytes. In addition to a TATA element at -30, we show that from -425 to +7 the TFIIIA gene contains only two positive cis elements centered at -267 (element 1) and -230 (element 2). This arrangement of the cis elements in the TFIIIA gene is striking because these two elements are positioned very close to each other yet separated from the TATA element by approximately 190 nucleotides. We show that the 190-nucleotide spacing between the TATA element and the upstream cis elements (elements 1 and 2) is critical for efficient transcription of the gene in oocytes and that a nucleosome is positioned in this intervening region. This nucleosome may act positively on TFIIIA transcription in oocytes by placing transcription factors bound at elements 1 and 2 in a favorable position relative to the transcription complex at the TATA element.

Animals↗
PubMed2003

Optimization of eight-element multi-detector row helical CT technology for evaluation of the abdomen.

PURPOSE: To evaluate protocols for abdominal imaging with an eight-element multi-detector row computed tomographic (CT) scanner. MATERIALS AND METHODS: An eight-element helical CT scanner was used to acquire data in two phantoms with four-element (pitch, 0.75 and 1.5; section thickness, 1.25, 2.5, and 5.0 mm) and eight-element (pitch, 0.625, 0.875, 1.35 and 1.675; section thickness, 1.25 and 2.5 mm) protocols. One phantom was used for low-contrast detectability and streak artifact; the other, for high-contrast performance. Protocols included near constant radiation dose (140 kV and varied tube current, confirmed by using the above protocols to scan a dedicated radiation dose phantom). Data were analyzed by three blinded readers for streak artifacts, contrast-to-noise ratio, and z-axis resolution (contrast-transfer function). Statistical analysis included studentized range tests. RESULTS: Contrast-to-noise ratios for four and eight elements were not consistently different. Qualitative evaluation for streak artifacts revealed fewer artifacts for all eight-element 1.25-mm-thick section protocols, as compared with eight-element 2.5-mm protocols. All eight-element 2.5-mm protocols except that with 27.0 mm per rotation had fewer streak artifacts than did four-element protocols (P =.02-.04). Contrast-transfer functions along the z axis for eight-element protocols were better than those for four-element protocols, demonstrating improved z-axis resolution (P <.05). CONCLUSION: Images acquired at eight sections per rotation demonstrated no sacrifice of contrast-to-noise ratio, improved z-axis resolution, and fewer streak artifacts, even when radiation dose was similar to that for four-element CT.

Artifacts↗