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PubMed1987

Preparation of a mixed human diet material for the determination of nutrient elements, selected toxic elements and organic nutrients: a preliminary report.

Using 201 foods from the United States Food and Drug Administration's Total Diet Study (FDA TDS), a mixed diet composite (USDIET-I) was prepared to represent the intake of 25-30-year-old males in the United States. Proximate analyses, phytate determination, and assays for nutrient elements and selected toxic elements, as well as organic nutrients were carried out on this composite. As part of a quality control exercise for a coordinated research program, atomic absorption spectrophotometry, inductively coupled atomic emission spectrometry, colorimetry and neutron activation analysis were used to determine up to 30 elements in this diet material. A comparison of the daily intakes of As, Ca, Cd, Cu, Fe, Hg, K, Mg, Mn, Na, P, Se and Zn from the composite USDIET-I shows excellent to good agreement with FDA TDS values calculated from results of single food analyses. These USDIET-I results demonstrate the feasibility of the mixed diet concept as a viable approach for a reliable assessment of daily intakes, especially for a number of elements such as Cd, Cr, Hg and Mo that occur at low concentrations in individual food products. Simultaneously, stability of some organic nutrients during storage was also investigated. Initial findings suggest that this program may also be useful in the development of reference materials for organic nutrients, for which there is a great need. These aspects are discussed.

Adult↗
PubMed2004

A conserved initiator element on the mammalian poly(ADP-ribose) polymerase-1 promoters, in combination with flanking core elements, is necessary to obtain high transcriptional activity.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a conserved nuclear protein present in nearly all eukaryotes. In mammalian cells, its abundant expression and its ability to specifically bind to DNA strand breaks make it an important enzyme in the rapid cellular response to DNA damage. Although the promoter regions of the three known mammalian PARP-1 genes, from human, rat and mouse, are different, they share common features, such as multiple GC-rich regions, lack of a functional TATA box, and presence of a putative initiator element. In this study, we analyzed the core promoter region of the rat PARP-1 gene, and show that it contains a functional initiator element surrounding the transcription start site. This core element lies within an approximately 40-base-pair region that is highly conserved in all three mammalian PARP-1 promoters. Furthermore, we show that other core elements located upstream and downstream of the PARP-1 initiator, including a functional Sp1 target site, synergize to regulate rat PARP-1 transcription. As the initiator region of all three PARP-1 gene promoters is highly conserved, their transcriptional regulation is likely achieved through similar mechanisms.

Animals↗
PubMed2005

Regulation of alpha-smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic CArG element and the transforming growth factor-beta1 control element.

Myofibroblasts are specialized contractile fibroblasts that are critical in wound closure and tissue contracture. Generation of contractile force is correlated with the expression of alpha-smooth muscle actin (alpha-SMA); however, little is known regarding molecular mechanisms that control activation of alpha-SMA in myofibroblasts in granulation tissue. The aims of the present studies were to identify sufficient promoter regions required for alpha-SMA expression in myofibroblasts in vivo and to determine whether activation of alpha-SMA expression in myofibroblasts in vivo is dependent on an intronic CArG [CC(A/T)6GG] and a transforming growth factor-beta1 control element (TCE) that are required for alpha-SMA expression in smooth muscle cells. A Lac Z transgene construct from -2600 through the first intron was expressed in myofibroblasts within granulation tissue of cutaneous wounds in a pattern that closely mimicked endogenous alpha-SMA expression. Mutation of either the intronic CArG element or the TCE completely inhibited transgene expression in myofibroblasts in granulation tissue and responsiveness to transforming growth factor-beta1 in cultured transgenic fibroblasts. These same elements were also critical in regulating alpha-SMA expression during skeletal muscle repair but not during skeletal muscle development. Taken together, these results provide the first in vivo evidence for the importance of the intronic CArG and TCE cis-elements in the regulation of alpha-SMA expression in myofibroblasts in granulation tissue.

Actins↗
PubMed1997

A sucrose repression element in the Phaseolus vulgaris rbcS2 gene promoter resembles elements responsible for sugar stimulation of plant and mammalian genes.

Protoplasts isolated from the primary leaves of Phaseolus vulgaris L. were used in transient expression experiments to identify promoter sequences of the P. vulgaris rbcS2 gene, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit, concerned with sucrose repression. The protoplasts supported high rates of expression of the chloramphenicol acetyl transferase reporter gene fused to 1433 bp of the rbcS2 5' flanking sequences. Expression was repressed by 50 mM sucrose whereas that driven by control promoters was not. Assays of promoter deletions revealed that 203 bp 5' to the transcription start site were sufficient for high rates of sucrose-repressible expression. A -187 bp deletion supported much lower rates of expression and was not subject to sucrose repression. The -203 to -187 bp region contains sequences resembling elements involved in the sugar stimulation of transcription of other genes: the SURE (sucrose response element) of plant genes and the ChoRE (carbohydrate response element) of mammalian genes. A G-box (CACGTG) located at -200 to -205 was important for high levels of sucrose-repressible expression, since deletion of a nucleotide from this element in the context of the 1433 bp promoter gave much reduced expression. However, a modified G-box (CcCGTG) in the -203 bp fusion and adjacent vector sequences remained functional. Measurements of rbcS and chalcone synthase (CHS) transcript levels in the protoplasts indicated that 4 mM sucrose was sufficient to repress or stimulate the respective genes. Further experiments suggested that metabolism of 6-carbon sugars is the signal for rbcS repression and CHS stimulation.

Acyltransferases↗
PubMed2001

Involvement of multiple cis-elements in the regulation of GA responsive promoters: Definition of a new cis-element in the Amy32b gene promoter of barley (Hordeum vulgare).

The Amy32b gene is a member of the low-pI alpha-amylase gene family of barley, whose expression is tightly regulated by hormones in the aleurone layer. Four cis-elements are known to be important for the GA induction of this gene: GARE, amylase box, pyrimidine box, and O2S. These sequences are located between -101 and -149 relative to the transcription start site. In the present work, we have created a series of Amy32b promoter-GUS reporter constructs introducing mutations in the -79 to -93 region. Using a transient expression system, we have functionally defined an additional region (-81 to -89) essential for the GA activation of the Amy32b promoter. This region is highly conserved among barley, wheat, and wild oat low-pI alpha-amylase promoters. Interestingly, in contrast with the variability in the relative distances among other cis-elements, this region maintains a nearly constant distance to GARE, which suggests that the function of these elements might be coupled. The involvement of this and other sequences in the transactivation of Amy32b by a transcription factor, GAMyb, has also been studied. Our results indicate that the only indispensable element for the GAMyb transactivation of the alpha-amylase promoter is GARE. The present work brings new evidence to the proposed model that considers the GAMyb-GARE interaction as a critical point for the GA induction of alpha-amylase genes, but also strengthens the notion that multiple sequences are required for full regulation of alpha-amylase promoters.

Journal Article↗
PubMed1997

The highest affinity DNA element bound by Pbx complexes in t(1;19) leukemic cells fails to mediate cooperative DNA-binding or cooperative transactivation by E2a-Pbx1 and class I Hox proteins - evidence for selective targetting of E2a-Pbx1 to a subset of Pbx-recognition elements.

Oncoprotein E2a-Pbx1 contains the N-terminal transactivation domains of E2a and the majority of the homeodomain protein, Pbx1. Using recombinant proteins, both Pbx1 and E2a-Pbx1 heterodimerize with Hox proteins on bipartite elements, Pbx1 binding a 5' TGAT core and Class I Hox proteins binding adjacent 3' TAAT, TTAT, or TGAT cores. In contrast to these in vitro results, nuclear extracts from E2a-Pbx1-transformed cells assemble an abundant Pbx-containing complex on TGATTGAT that excludes E2a-Pbx1, suggesting that an uncharacterized in vivo partner discriminates between E2a-Pbx1 and Pbx proteins, distinguishing it from Hox proteins. Here, we describe the DNA-binding properties of this complex, and identify TGATTGAC (PCE; Pbx Consensus Element) as its optimal recognition motif. In vitro, the PCE fails to bind heterodimers of Class I Hox proteins plus either Pbx1 or E2a-Pbx1. Likewise, in vivo, the PCE fails to mediate cooperative transactivation by E2a-Pbx1 plus Class I Hox proteins. Thus, the PCE binds a Pbx dimer partner that behaves unlike Class I Hox proteins. Competition analysis indicates that the Pbx-containing complex that binds the PCE also binds the TGATTGAT Pbx-Hox element and binds promoter elements required for tissue-specific expression of a number of cellular genes. Thus, different Pbx partners dictate targetting of Pbx heterodimers to related DNA motifs that differ in the sequence of their 3' half-sites, and E2a-Pbx1 heterodimerizes with only a subset of Pbx partners, restricting its potential DNA targets.

Base Sequence↗
PubMed1988

In vivo "photofootprint" changes at sequences between the yeast GAL1 upstream activating sequence and "TATA" element require activated GAL4 protein but not a functional TATA element.

Transcription of the yeast GAL1 and GAL10 genes is induced by growth on galactose. Using the technique of photofootprinting in vivo, we previously documented equivalent transcription-dependent footprints within the putative "TATA" elements of both genes. To explore the functional significance of these observations, we created a 3-base-pair substitution mutation within the GAL1 promoter TATA element, which disrupted the ATATAA consensus sequence but left intact the photomodification targets. The mutation reduced galactose-induced RNA levels by a factor of 100. The mutant promoter no longer displayed the characteristic TATA sequence footprint, supporting the hypothesis that transcription activation involves the binding of a TATA box factor. We also observed a collection of transcription-correlated alterations in the modification pattern at sites between the UASG and the GAL1 TATA element, within sequences that are not required for inducible transcription. These patterns, characteristic of the induced wild-type GAL1 gene, were still galactose inducible with the TATA mutant GAl1 promoter, despite the low level of transcription from this promoter. We conclude that the GAL4-dependent protein/DNA structure responsible for the altered pattern within nonessential sequences is therefore not strictly coupled to an active TATA element or to high levels of expression. Nonetheless, the patterns probably reflect a stable protein-dependent structure that accompanies assembly of the transcription initiation complex.

DNA, Fungal↗
PubMed1990

NICER elements: a family of nerve growth factor-inducible cAMP-extinguishable retrovirus-like elements.

We have shown previously that the transcription of the gene designated d5 is induced by nerve growth factor (NGF) in rat adrenal pheochromocytoma PC-12 cells and that this NGF induction is repressed by cAMP. In this paper we demonstrate that d5 is a member of a gene family that contains several hundred members, which is closely related to retroviruses and retrotransposons, as demonstrated by the following observations: (i) the original d5 cDNA hybridized to numerous restriction fragments in genomic DNA; (ii) d5 cDNA hybridized to genomic clones with various intensities, and genomic clones can be isolated with a frequency suggesting that this family includes several hundred members; and (iii) there were minor sequence variations in four independently isolated cDNA clones that were homologous to d5 cDNA. Primer extension studies show that initiation of the 5.7-kilobase d5 mRNA(s) occurs at a unique site relative to a synthetic primer. The 5' end of the cDNA sequence was homologous to Rasheed rat sarcoma virus; and a genomic clone contained several elements that are typical of a long terminal repeat (LTR), including a CCAAT box, a TATA box, a primer binding site, a poly(A) addition signal, and a poly(A) addition site. Furthermore, there is a LTR at the 3' end of at least one of the genes in this family, and there appeared to be a four-base duplication at the probable site of integration into host DNA. Since several members of this family retain responses to NGF and cAMP, we conclude that the regulatory elements present in the LTR have been conserved in many members of this family. We have named this family of genes the NICER elements because they are a family of NGF-inducible cAMP-extinguishable retrovirus-like elements.

Adrenal Gland Neoplasms↗
PubMed1999

A novel element and a TEF-2-like element activate the major histocompatibility complex class II transactivator in B-lymphocytes.

Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly.

B-Lymphocytes↗
PubMed2000

An ordered array of cold shock domain repressor elements across tumor necrosis factor-responsive elements of the granulocyte-macrophage colony-stimulating factor promoter.

The tumor necrosis factor-alpha-responsive region of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter (-114 to -31) encompasses binding sites for NF-kappaB, CBF, AP-1, ETS, and NFAT families of transcription factors. We show both here and previously that mutation of any one of these binding sites greatly reduces tumor necrosis factor-alpha induction of the GM-CSF promoter. Interspersed between these elements are sequences that when mutated lead to an increase in GM-CSF promoter activity. We have previously shown that two of these repressor elements bind proteins known as cold shock domain (CSD) factors and that overexpression of CSD proteins leads to repression of GM-CSF promoter activity in fibroblasts. CSD proteins are single strand DNA- and RNA-binding proteins that contact 5'-CCTG-3' sequences in the GM-CSF repressor elements. We show here that two newly identified repressor sequences in the proximal promoter can also bind CSD proteins. We have characterized the CSD-containing protein complexes that bind to the GM-CSF promoter and identified a novel protein related to mitochondrial single strand binding protein that forms part of one of these complexes. The four CSD-binding sites on the promoter occur in pairs on opposite strands of the DNA and appear to form an ordered array of binding elements. A similar ordered array of CSD sites are present in the promoters of the granulocyte colony-stimulating factor and interleukin-3 genes, implying a common mechanism for negative regulation of these myeloid growth factors.

Base Sequence↗
PubMed2000

Characterization of a repressor element and a juxtaposed tissue-restricted activator element located on the distal neu gene promoter.

The proto-oncogene neu (HER2 or c-erbB2) is overexpressed with or without gene amplification in 20-30% of breast cancers. In patients, neu amplification or overexpression in breast and ovarian cancer correlates with poor prognosis and tumor resistance to chemotherapy. neu-induced transformation can be reversed by the suppression of neu gene transcription. To further understand how neu gene transcription is regulated and to identify a possible transcriptional repressor(s) of neu, we identified a negative regulatory element known previously to be located within a 1-kilobase (kb) DNA fragment of an unknown sequence, upstream of the proximal neu gene promoter. One of several DNA fragments subcloned from this region suppressed transcriptional activity of the proximal neu gene promoter. Sequencing of the 1-kb fragment confirmed the location of the repressor element to be between an AluI and a RsaI sites, around 1.4 kb upstream to the translation start site. Various deletions were introduced into the AluI-RsaI fragment and subcloned into both the native neu promoter and a heterologous thymidine kinase promoter. Subsequent transfections and reporter gene assays in cell lines of various tissues of origin confirmed and narrowed the repressor activity to a 120-base pair NlaIV-MslI fragment located between -1385 and -1266. Importantly, specific protein binding activity to this element could be detected with nuclear extracts isolated from these cell lines. In contrast, a 28-base pair MslI-RsaI fragment (-1265 to -1238), located immediately 3' of the putative repressor element, was found to form protein-DNA complexes with only nuclear extracts isolated from a colon carcinoma cell line. This specific protein binding activity correlated with a previously unknown transcriptional stimulatory activity only in this cell line.

Base Sequence↗
PubMed1998

The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.

Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences.

Bromodeoxyuridine↗
PubMed1979

Organization of the rosy locus in Drosophila melanogaster: further evidence in support of a cis-acting control element adjacent to the xanthine dehydrogenase structural element.

The present report summarizes our recent progress in the genetic dissection of an elementary genetic unit in a higher organism, the rosy locus (ry:3--52.0) in Drosophila melanogaster. Pursuing the hypothesis that the rosy locus includes a noncoding control region, as well as a structural element coding for the xanthine dehydrogenase (XDH) peptide, experiments are described that characterize and map a rosy locus variant associated with much lower than normal levels of XDH activity. Experiments are described that fail to relate this phenotype to alteration in the structure of the XDH peptide, but clearly associate this character with variation in number of molecules of XDH per fly. Large-scale fine-structure recombination experiments locate the genetic basis for this variation in the number of molecules of XDH per fly to a site immediately to the left of the XDH structural element within a region previously designated as the XDH control element. Moreover, experiments clearly separate this "underproducer" variant site from a previously described "overproducer" site within the control region. Examination of enzyme activity in electrophoretic gels of appropriate heterozygous genotypes demonstrates the cis-acting nature of this variation in the number of molecules of XDH. A revision of the map of the rosy locus, structural and control elements is presented in the light of the additional mapping data now available.

Crosses, Genetic↗
PubMed1992

Identification of regulatory elements within the minimal promoter region of the human endogenous ERV9 proviruses: accurate transcription initiation is controlled by an Inr-like element.

ERV9 is a low repeated family of human endogenous retroviral elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. In this report we have analyzed the minimal promoter region located within the ERV9 LTR. Using the transient CAT expression assay we have identified the minimal promoter region, which includes sequences spanning from -70 to +6 relative to the major transcription start site. Deletion analysis, primer extension mapping of the transcription start sites and DNA-protein interactions assays have allowed us to define two important regions within the ERV9 minimal promoter. One region located between -70 to -39 acts as a transcriptional activating sequence and contains an Sp 1 binding site. The second region from -7 to +6, which resembles an initiator element (Inr), was necessary for the correct transcription start site utilization, and binds to a regulatory protein. Cross-competition experiments using various Inr elements have indicated that the protein that binds to the ERV9 Inr element can be competed by the HIV-1 and TdT Inr sequences.

Base Sequence↗
PubMed2003

HY5, Circadian Clock-Associated 1, and a cis-element, DET1 dark response element, mediate DET1 regulation of chlorophyll a/b-binding protein 2 expression.

DET1 is a pleiotropic regulator of Arabidopsis development and controls the expression of many light-regulated genes. To gain a better understanding of the mechanism by which DET1 controls transcription from light-regulated promoters, we identified elements in the chlorophyll a/b-binding protein 2 (CAB2) promoter that are required for DET1-mediated expression. Using a series of reporter constructs in which the luciferase gene is controlled by CAB2 promoter fragments, we defined two DET1-responsive elements in the CAB2 promoter that are essential for proper CAB2 transcription. A 40-bp DET1 dark-response element (DtRE) is required for both dark and root-specific repression of CAB2, whereas the known CAB upstream factor-1 element is required for DET1 activation-associated effects in the light and repression in the roots. HY5, a factor that binds CAB upstream factor-1, is also required for DET1 effects in the light. DtRE binds two distinct activities in Arabidopsis seedling extracts: a novel activity with binding site CAAAACGC that we have named CAB2 DET1-associated factor 1 plus an activity that is likely to be the myb transcription factor Circadian Clock-Associated 1. Both activities are altered in dark-grown det1 extracts as compared with wild type, correlating a change in extractable DNA binding activity with a major change in CAB2 expression. We conclude that DET1 represses the CAB2 promoter in the dark by regulating the binding of two factors, CAB2 DET1-associated factor 1 and Circadian Clock-Associated 1, to the DtRE.

Animals↗
PubMed2001

Extension of a tuned log spiral of revolution fluorescence XAFS detector, designed for optimal detection of a particular element Z, to XAFS of elements other than Z.

Recently, it has been demonstrated that an x-ray detector in the form of a log spiral of revolution, covered with highly oriented pyrolytic graphite, is an excellent device for obtaining the fluorescence XAFS of an element of interest in the presence of competing fluorescence from other elements. In the present work we investigate the capabilities of a log spiral of revolution (LSR) detector, with a geometry optimized for one element (in this case Cr), if used for XAFS of other elements.

Journal Article↗
PubMed1999

A novel positive regulatory element that enhances hamster CYP2A8 gene expression mediated by xenobiotic responsive element.

CYP2A8 is a major form of cytochrome P-450 inducible by 3-methylcholanthrene in Syrian hamster liver. To identify DNA elements necessary for the transcriptional activation of the CYP2A8 gene, we analyzed the regulatory region of the CYP2A8 gene and conducted transient transfection experiments of CYP2A8-luciferase fusion plasmids in primary cultures of hamster hepatocytes. We analyzed up to -5 kb of the 5'-flanking region and found the region sufficient for the 3-methylcholanthrene-inducible gene expression. This region contained a consensus sequence for xenobiotic responsive element (XRE) between -2366 and -2349, which was shown to be essential for induction of the gene expression. Furthermore, we found a novel positive regulatory element for XRE-mediated gene expression (PREX) located upstream of the XRE. This element is not identified in any genes inducible by 3-methylcholanthrene so far reported. Without PREX, the XRE-mediated promoter activity was enhanced nearly 10-fold, whereas with PREX, the activity was enhanced 20-fold over the basal level. Gel mobility shift assays revealed specific binding of nuclear proteins to PREX. Mutations and deletions of PREX caused a loss of the binding and promoter-enhancing activities, respectively. Moreover, transient expression experiments showed that the enhancing activity of PREX was not observed in Drosophila Schneider's line 2 cells, which were shown to lack the PREX binding proteins.

Animals↗
PubMed2002

pS2 Gene expression in HepG2 cells: complex regulation through crosstalk between the estrogen receptor alpha, an estrogen-responsive element, and the activator protein 1 response element.

The pS2 promoter is complex with binding sites for a number of protein factors that may participate in modulating its activity. The pS2 gene was transcriptionally activated by estrogens in HepG2 cells transformed (HepER3) to express the estrogen receptor alpha (ERalpha). The phorbol ester phorbol 12-myristate 13-acetate (PMA) stimulated pS2 expression in both HepER3 and the parental, non-ER-expressing HepG2 cells, although its activity was substantially less in HepG2 cells. The use of selective protein kinase inhibitors suggested that the MAPK pathway contributes substantially to estrogen stimulation of the pS2 promoter. The activator protein 1 (AP1) site at -332 to -338 in the pS2 promoter had a dominant role in the response to both estrogens and PMA, although the estrogen response element at -393 to -405 was essential to mediate the response to estrogen. The potentiation of pS2 promoter activity by the AP1 motif in response to estrogen was dependent on the ligand binding domain of ERalpha. Furthermore, the presence of an intact AP1 element in the pS2 promoter sustained suppression of pS2 promoter activity by an LXXLL peptide. In summary, the data suggest that the effect of estrogen is mediated through a cross-talk between the estrogen-responsive element and the AP1 response element and that ERalpha plays a crucial role in mediating the effect of both estrogen and PMA.

Amino Acid Motifs↗