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PubMed1992

Speciation of trace elements in biological materials: trends and problems.

In the determination of trace elements in biological materials, speciation is of particular importance as the essential effects or toxicity of an element and its metabolic behaviour depend to a large extent on the chemical forms in which it is present in the organism. Speciation is relatively easy if a property of a particular compound can be measured directly in the sample without interference from the other components of the material, e.g., the enzymic activities of the metallo-enzymes. Another possibility for speciation is immunoassay, which likewise allows direct determination of a particular trace element. At present, however, with most trace elements both fractionation methods and analytical procedures have to be combined and speciation has to be carried out by determining the elemental content in the separated fractions. The methods and apparatus used in taking, storing and preparing the samples can, therefore, not be selected solely according to the requirements of trace element determination, but it is also essential to ensure that the biological structures of the components to be separated remain intact. In this work the need for speciation in the investigation of the toxic and essential effects of trace elements is shown with the help of some examples, and the problems that can occur in the various steps of sampling, storage and sample preparation are discussed.

Animals↗
PubMed2001

Monitoring the distribution and deposition of trace elements associated with a zinc-lead smelter in the Trail area, British Columbia, Canada.

The atmospheric deposition and regional dispersion pattern of the elements As, Cd, Cu, Hg, Pb and Zn, emitted from Pb-Zn smelters at Trail, British Columbia, Canada, were studied for a period of two years using the passive collection moss-monitoring method. Concentrations of these elements in moss bags, exposed to atmospheric deposition for three months, were first corrected for geogenic input and then site-specific local background level to produce common background elemental concentrations for all monitoring stations. The results of this study indicate that the deposition of these elements is greatest in proximity to the smelter and decreases with an increase in distance. The regional dispersion pattern generally varies seasonally, with the magnitude of variation being dependent on the element under study and the location of the monitoring station. Overall, the deposition of these elements (as registered by moss-monitoring stations) appears to be a complex phenomenon involving various factors such as meteorological conditions (prevailing wind direction and precipitation), physiography (topography, valley shape and orientation) and in-process activities at the smelters (production level, emission control efficiency and the geochemistry of feed). However, the results of this study indicate that, on an annual basis, in-process activities, particularly production levels of Pb and Zn and the quantity of emissions from the stack, are more directly related to the observed deposition of the elements than any of the other factors.

Air Pollutants↗
PubMed1996

Characterization of cell-specific modulatory element in the murine ornithine decarboxylase promoter.

The promoter of the murine ornithine decarboxylase (ODC) gene contains, adjacent to the TATA box, a cAMP response element (CRE)-like motif that interacts with specific nuclear proteins. Here we examine the role of this CRE-like element (CREL) in ODC promoter activation in proliferating cells. Mutations that abolished binding of nuclear proteins to CREL influenced only marginally the cAMP induction of the reporter constructs driven by 1.6 kb of the ODC promoter. Instead, these mutations altered the basal promoter function in a cell-specific manner, in that they reduced the promoter activity in CV-1 cells, but increased it in NIH/3T3, CHO and HeLa cells. Thus, depending on the cell type, the CREL motif is able to confer either repression or activation on ODC gene transcription. In contrast with 1.6 kb promoter constructs, the same mutations in the context of a shorter sequence (proximal 133 nt) reduced the promoter strength in all cell types studied. The ability of the CREL element to attenuate transcription seems to be connected with the function of some upstream regulatory elements. Differences in nuclear proteins binding to CREL, as studied by electrophoretic mobility shift assays (EMSAs), did not explain the findings on cell-type specificity in transcriptional activation, as mutations in CREL abrogated formation of specific CREL-protein complexes in all cell lines examined. The protein complexes interacting with CREL were not recognized by antibodies specific for CRE-binding proteins CREB-1 and CREB-2, or activating transcription factors ATF-1, ATF-2 and ATF-3. EMSA experiments also demonstrated co-operative interactions between the CREL motif-binding proteins and other nuclear proteins, such as Sp1, interacting with CG-rich sequences of the promoter. In conclusion, the proximal ODC promoter contains a well-conserved regulatory element, which is clearly different from the CRE/ATF element. This motif acts in concert with other distal and proximal elements in a complex cell-specific manner.

3T3 Cells↗
PubMed2004

Shuffling of Sulfolobus genomes by autonomous and non-autonomous mobile elements.

Each of the sequenced Sulfolobus genomes contains large numbers of putatively mobile elements, both IS elements (insertion sequence elements) and MITEs (miniature inverted-repeat transposable elements). There are 344 in the 3.0 Mb genome of Sulfolobus solfataricus P2 and 95 in the 2.7 Mb genome of Sulfolobus tokodaii. In the former they constitute more than 10% of the genome. Experimental data suggest that transposition of IS elements occurs frequently. Moreover, the gene order between the two organisms differs greatly, indicating that multiple rearrangements have occurred. This has also led to considerable speculation as to how the cells are viable. Recently, a third Sulfolobus genome was completed which contains no IS elements or MITEs. This enabled us to compare the gene orders of the three genomes and provide evidence for mobile element-induced rearrangements of sections of the genomes.

DNA Transposable Elements↗
PubMed2003

Lipopolysaccharide represses cholesterol 7-alpha hydroxylase and induces binding activity to the bile acid response element II.

BACKGROUND: Inflammatory states such as hepatitis and sepsis are frequently associated with alterations of bile acid synthesis. These conditions are mediated by bacterial wall products like lipopolysaccharide (LPS). Cholesterol 7-alpha hydroxylase is the rate-limiting enzyme of bile acid synthesis. Hydrophobic bile acids repress cholesterol 7-alpha hydroxylase transcription via binding to the farnesoid X receptor and interaction with the bile acid response element II in the cholesterol 7-alpha hydroxylase promoter. METHODS: We tested the effect of LPS on hepatic expression of cholesterol 7-alpha hydroxylase in C57BL/6 mice and Wistar rats. Further, we analyzed the binding activity of hepatic nuclear extracts to the bile acid response element II and the binding site for farnesoid X receptor heterodimers (ecdysone response element). RESULT: Lipopolysaccharide caused a 100-fold reduction of cholesterol 7-alpha hydroxylase mRNA levels in mice and a 10-fold reduction in rats. Protein levels of cholesterol 7-alpha hydroxylase also decreased in both species. These changes inversely correlated with the increased binding activity of nuclear proteins to the bile acid response element II and the ecdysone response element. CONCLUSION: Lipopolysaccharide-induced repression of cholesterol 7-alpha hydroxylase occurs at the transcriptional level. The underlying mechanism involves an increased binding activity of nuclear proteins to the bile acid response element II and the ecdysone response element. In conclusion, the farnesoid and retinoid X receptors participate in LPS-induced cholesterol 7-alpha hydroxylase repression.

Animals↗
PubMed1997

Structure and function of 59-base element recombination sites associated with mobile gene cassettes.

The integration of gene cassettes into integrons is effected by site-specific recombination catalysed by an integrase, IntI, encoded by the integron. The cassette-associated recombination sites, 59-base elements, are not highly conserved and vary in length from 57 to 141 bp. They can be identified by their location and the relationship of over 20 bp at their outer ends to consensus sequences that are imperfect inverted repeats of one another. The recombination cross-over occurs close to one end of the 59-base element, within a conserved core site with the consensus sequence GTTAGGC or GTTRRRY. By introducing single-base changes at each of these positions in the aadB 59-base element, bases that are critical for site activity were identified. The recombination cross-over was also localized to a unique position between the adjacent G and T residues. Changes introduced in the conserved AAC of the inverse core site (GCCTAAC or RYYYAAC) located at the opposite end of the 59-base element also reduced site activity but to a lesser extent. Sequences of rare recombinants revealed an alternative position for strand exchange and led to the conclusion that 59-base elements comprise two simple sites, analogous to those recognized by other integrases, with each simple site made up of a pair of inversely oriented IntI binding domains separated by a spacer of 7 or 8 bp. Re-examination of the sequences of all known 59-base elements revealed that this simple site configuration was present at both the left and right ends in all 59-base elements. The identity of bases in the spacer is not required for efficient recombination and the cross-over is located at one end of the spacer, suggesting that during IntI1-mediated recombination only one strand exchange occurs.

Base Composition↗
PubMed1996

Insertion mutagenesis and study of transposable elements using a new unstable virescent seedling allele for isolation of haploid petunia lines.

The new unstable virescent seedling (vis) allele of a petunia mutant, that has green leaves but white cotyledons with green revertant spots, was used to identify spontaneously occurring haploid petunia lines with active transposable elements. Endogenous transposons were trapped into the single petunia nitrate reductase structural gene (nia) using chlorate selection on haploid protoplasts. In two mutant lines, the dTph1-like transposable element dTph1-3 was inserted at almost the same position but in opposite orientations in the first exon of the nia gene. In a third mutant, a different transposable element was integrated into the fourth exon. This element, called dTph4, is 787 bp long and has 13 bp terminal inverted repeats of which 12 bp are identical to those of dTph1. Insertion of dTph1-3 and dTph4 results in an 8 bp duplication of the target site, as already described for dTph1. In contrast to dTph1-like elements, dTph4 is present at low copy number in the petunia genome. This can facilitate its use for gene tagging in petunia. The dTph1-3 and dTph4 elements excise frequently, as transposon footprints were found in most of the insertion mutants. The data demonstrate that haploid petunia is an excellent system for gene tagging and for the study of transposable elements.

Alleles↗
PubMed2003

Identification and characterization of the Xlsirt cis-acting RNA localization element.

There are many RNAs that are localized to the vegetal cortex of Xenopus laevis oocytes. One family of localized transcripts, Xlsirts (Xenopus laevis short interspersed repeat transcripts), are defined by the presence of non-coding repeat units 79-81 nucleotides long. Endogenous Xlsirt RNAs are localized through the METRO (message transport organizer) pathway that localizes RNAs during stages I and II of oogenesis. Interestingly, exogenous Xlsirt RNAs that are injected into oocytes can utilize both the METRO pathway as well as the Late pathway, which localizes RNAs during the late stages of oogenesis (stages III-VI). In all cases thus far analyzed, the localization process relies on the presence of cis-acting elements on the transcripts that are responsible for directing localization. To better understand the mechanism responsible for the use of the METRO and Late localization pathways, we sought to identify pathway-specific cis-acting localization elements contained in Xlsirts. The results showed that an intact 137 nucleotide element was necessary and sufficient to localize RNAs through the METRO and Late pathways. Further analysis of this element identified putative METRO and Late pathway localization sub-elements. Computer analysis relates the secondary structure of the 137 nt element to its ability to function as a localization element.

Amino Acid Sequence↗
PubMed2000

Positive and negative elements modulate the promoter of the human liver-specific alpha2-HS-glycoprotein gene.

The human alpha2-HS-glycoprotein (AHSG) and the 63-kDa rat phosphoprotein (pp63) are homologous plasma proteins that belong to the fetuin family. AHSG and pp63 are involved in important functions such as inhibition of insulin receptor tyrosine kinase activity, inhibition of protease activities, and regulation of calcium metabolism and osteogenesis. Studies of the AHSG proximal promoter performed in vitro in rat and human cells indicate that several NF-1 and C/EBP binding sites exert a positive effect on its transcriptional activity. However, until now, no distal elements have been examined in this gene, in either species. We report that the human AHSG gene promoter acts in a liver-specific manner and is further controlled by three distal, 5'-flanking elements. The negative elements III and I are, respectively, located 5' and 3' of the positive element II. All three elements require the natural context of the human AHSG gene to fully exert their negative or positive effect. Element I harbours a single binding site for NF-1. This nuclear factor thus appears to be able to up- or downregulate the AHSG gene depending on the site it binds to. Elements I, II and possibly III are absent in the rodent Ahsg gene encoding pp63.

Animals↗
PubMed1996

The roles of CRE, TRE, and TRE-adjacent S1 nuclease sensitive element in the regulation of tyrosine hydroxylase gene promoter activity by angiotensin II.

The cis elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were examined by transfecting tyrosine hydroxylase promoter-luciferase constructs into cultured bone adrenal medullary cells. Angiotensin II-responsive elements are located within -54/+25-bp and -269/-55-bp promoter regions and were identified, respectively, as cyclic AMP (CRE)- and 12-O-tetradecanoylphorbol 13-acetate responsive element (TRE)-like sequences. Unlike CRE, TRE also supports basal promoter activity. Mutations of TRE or CRE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, suggesting that proteins forming CRE- and TRE-inducible complexes may mediate angiotensin II stimulation. The TRE is adjacent to a dyad symmetry element. Those two sites form a common regulatory unit in which the dyad symmetry element acts as a repressor of the TRE site. Isolated dyad symmetry element did not bind nuclear proteins in vitro. In supercoiled DNA it exhibited S1 nuclease sensitivity and was recognized by a DNA cruciform-specific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abolished formation of the cruciform correlated with a loss of repressor activity. We propose a novel model of tyrosine hydroxylase gene regulation in which functions of the TRE are modulated via structural transition in the adjacent DNA.

Angiotensin II↗
PubMed2003

Characterization of a palindromic enhancer element in the promoters of IL4, IL5, and IL13 cytokine genes.

BACKGROUND: The genes encoding the cytokines IL-4, IL-5, IL-13, and GM-CSF are located in close proximity on human chromosome 5. We have previously described a motif in the promoters of these genes with a common palindromic sequence: CCAAG em leader CTTGG. These half sites are variably spaced and, within GMCSF, flank a second internal palindromic site. The GMCSF palindrome was shown to act as a strong enhancer of transcription when linked to a heterologous promoter. OBJECTIVE: We sought to determine whether the related palindromic elements from IL4, IL5, and IL13 also act as enhancers of gene transcription. METHODS: Reporter plasmids driven by palindromic elements were transfected into Jurkat T and HeLa cells to determine enhancer activity, and T-cell extracts were used in electrophoretic mobility shift and methylation interference assays to determine the DNA-binding characteristics of palindrome-binding proteins. RESULTS: Enhancer activity was observed in unactivated T cells and HeLa cells, whereas in T cells the IL4 palindrome mediated an activation-specific response. Mutational analysis of this element revealed that both halves of the CCAAG em leader CTTGG palindrome and part of the intervening sequence were essential for mediating interaction with protein complexes. Electrophoretic mobility shift assays suggested that the 4 palindromes bound similar factors because complexes formed between Jurkat nuclear extracts and each palindromic element showed identical mobility, and these elements were able to cross-compete for binding. CONCLUSIONS: These data suggest that constitutively expressed factors are involved in mediating the enhancer function of these elements in T cells and that these factors might either be present or have closely related homologues in other cell types. Also, an activation-dependent factor might be recruited to modulate the function of the IL4 element.

Base Sequence↗
PubMed2006

Active retrotransposition by a synthetic L1 element in mice.

Long interspersed element type 1 (L1) retrotransposons are ubiquitous mammalian mobile elements and potential tools for in vivo mutagenesis; however, native L1 elements are relatively inactive in mice when introduced as transgenes. We have previously described a synthetic L1 element, ORFeus, containing two synonymously recoded ORFs relative to mouse L1. It is significantly more active for retrotransposition in cell culture than all native L1 elements tested. To study its activity in vivo, we developed a transgenic mouse model in which ORFeus expression was controlled by a constitutive heterologous promoter, and we established definitive evidence for ORFeus retrotransposition activity both in germ line and somatic tissues. Germ line retrotransposition frequencies resulting in 0.33 insertions per animal are seen among progeny of ORFeus donor element heterozygotes derived from a single founder, representing a >20-fold increase over native L1 elements. We observe somatic transposition events in 100% of the ORFeus donor-containing animals, and an average of 17 different insertions are easily recovered from each animal; modeling suggests that the number of somatic insertions per animal exceeds this number by perhaps several orders of magnitude. Nearly 200 insertions were precisely mapped, and their distribution in the mouse genome appears random relative to transcription units and guanine-cytosine content. The results suggest that ORFeus may be developed into useful tools for in vivo mutagenesis.

Animals↗
PubMed2001

Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea).

Mobile element dynamics in seven alleles of the chalcone synthase D locus (CHS-D) of the common morning glory (Ipomoea purpurea) are analyzed in the context of synonymous nucleotide sequence distances for CHS-D exons. By using a nucleotide sequence of CHS-D from the sister species Ipomoea nil (Japanese morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. & Iida, S. (1999) Genes Genet. Syst. 74, 141-147], it is also possible to determine the relative frequency of insertion and loss of elements within the CHS-D locus between these two species. At least four different types of transposable elements exist upstream of the coding region, or within the single intron of the CHS-D locus in I. purpurea. There are three distinct families of miniature inverted-repeat transposable elements (MITES), and some recent transpositions of Activator/Dissociation (Ac/Ds)-like elements (Tip100), of some short interspersed repetitive elements (SINEs), and of an insertion sequence (InsIpCHSD) found in the neighborhood of this locus. The data provide no compelling evidence of the transposition of the mites since the separation of I. nil and I. purpurea roughly 8 million years ago. Finally, it is shown that the number and frequency of mobile elements are highly heterogeneous among different duplicate CHS loci, suggesting that the dynamics observed at CHS-D are locus-specific.

Acyltransferases↗
PubMed1988

Transposable and nontransposable elements similar to the I factor involved in inducer-reactive (IR) hybrid dysgenesis in Drosophila melanogaster coexist in various Drosophila species.

The I factor is a transposable element controlling inducer-reactive (IR) hybrid dysgenesis in Drosophila melanogaster, which occurs when males from the class of inducer strains are crossed with females from the class of reactive strains. Inducer strains contain several copies of the complete 5.4-kilobase (kb) I factor at various sites on the chromosomal arms; reactive strains contain no complete I factor. Incomplete and defective I elements occur at constant locations in pericentromeric heterochromatin of both types of strains. The 5.4-kb I factors transpose, whereas incomplete I elements do not transpose. The constant location of defective I elements in all strains indicates that they were in the genome before the spread of D. melanogaster throughout the world. Sequences homologous to I occur in other Drosophila species, and their distribution correlates with the phylogenetic relationships between species. We have studied the organization of I homologues in Drosophila simulans and Drosophila teissieri. These species seem to contain both transposable I elements, even though their structure may differ from that of the 5.4-kb I factors of the inducer strains of D. melanogaster, and nontransposable I elements, which are always at the same place in the genome when different stocks of the same species are compared. These results suggest that both mobile and nonmobile I elements are very old components of the Drosophilidae genome.

Animals↗
PubMed1989

Separate cis-regulatory elements confer expression of phosphoenolpyruvate carboxykinase (GTP) gene in different cell lines.

The gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) [PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32], a key enzyme in gluconeogenesis and glyceroneogenesis, is expressed in tissues that arise from different embryonal origins: the gluconeogenic liver arises from endoderm, whereas the gluconeogenic kidney cortex and glyceroneogenic adipose tissue arise from the mesoderm. To identify the cis-regulatory elements conferring the differential gene expression, PEPCK chimeric genes were transfected into two rat hepatoma cell lines (H4IIEC3 and HTC-M1.1) and mouse adipocytes (3T3F442A), which express the endogenous gene, and into myoblasts and preadipocytes, which do not express it. The results demonstrate that 597 base pairs of the 5' flanking region of the PEPCK gene are sufficient to confer cell-specific gene expression in the PEPCK-expressing hepatoma cells and adipocytes. However, different elements within this 597-base-pair region enhance the gene expression in the hepatoma cells (endoderm) and adipocytes (mesoderm). In the hepatocytes, expression is conferred by two elements--one 5' of position -362 and the other 3' of position -98 with respect to the transcription start site. The region in between these two elements (from -362 to -98), which seems to inhibit the gene expression in the hepatocytes, confers enhanced expression in the adipocytes. Moreover, the distal positive regulatory element of the hepatocytes seems to be orientation and PEPCK promoter dependent. In contrast, the positive regulatory element of the adipocytes seems to act as a more typical enhancer. These results suggest that separate cis-regulatory elements confer cell-specific expression of the PEPCK gene.

Animals↗
PubMed1991

Retroviral-like element in a marine invertebrate.

Retroviral-like elements (RL elements) include retroviruses and long terminal repeat (LTR)-containing retrotransposons. We report the presence of sea urchin RL elements (termed SURL) in eight species of sea urchins and find that these RL elements belong to several subfamilies. The complete DNA sequence of one SURL element in Tripneustes gratilla is 5266 base pairs long, including 254-nucleotide-long identical long terminal repeats (LTRs). It contains a single open reading frame nearly 4 kilobases long including the gag and pol genes. Comparison of conserved DNA sequences of RL elements from different sea urchin species indicates that active elements have been inserting copies into echinoid genomes for at least 200 million years.

Animals↗
PubMed1998

Transposition of the mariner element from Drosophila mauritiana in zebrafish.

With the increased popularity of zebrafish (Danio rerio) for mutagenesis studies, efficient methods for manipulation of its genome are needed. One approach is the use of a transposable element as a vector for gene transfer in this species. We report here the transformation of zebrafish and germ-line transmission of the mariner element from Drosophila mauritiana. The mariner element was selected because its transposition is independent of host-specific factors. One- to two-cell-stage zebrafish embryos were coinjected with a supercoiled plasmid carrying the nonautonomous mariner element peach and mRNA encoding the transposase. Surviving larvae were reared to adulthood, and the transmission of peach to the F1 generation was tested by PCR. Four of the 12 founders, following plasmid injections on 2 different days, transmitted the element to their progeny. Inheritance of the transgene from the F1 to the F2 generation showed a Mendelian pattern. No plasmid sequences were detected by PCR or Southern blot analysis, indicating transposition of peach rather than random integration of the plasmid DNA. These data provide evidence of transformation of a vertebrate with a transposable element and support the host-independent mechanism for transposition of the mariner element. We suggest this system could be used for insertional mutagenesis or for identifying active regions of the genome in the zebrafish.

Animals↗
PubMed1995

Elements controlling the expression and induction of the skin hyperproliferation-associated keratin K6.

The suprabasal keratin 6 (K6) is remarkable among the keratins as, in addition to being constitutively expressed in different stratified epithelia, it is induced in epidermis under hyperproliferative conditions, such as benign or malignant tumors, psoriasis, and wound healing. In addition, this keratin is also induced in skin treated with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid (RA). These characteristics make the study of K6 regulatory elements an especially interesting issue, in particular because these elements could be useful in designing gene constructs for the therapy of skin diseases. We have analyzed by mobility shift and footprinting experiments the cell type-specific enhancer of the bovine K6 beta gene (Blessing, M., Jorcano, J. L., and Franke, W. W. (1989) EMBO J. 8, 117-126) and have identified an AP-2-like element, two AP-1 elements (one of them composite), and a retinoic acid-responsive element (RARE). Mutagenesis experiments and cotransfections with retinoic acid receptors show that the RARE mediates enhancer activation by RA. Chloramphenicol acetyltransferase assays show that under normal culture conditions, the AP-1 element retains most of the enhancer transcriptional activity, while the RARE and AP-2 are weakly active. However, following RA treatment, the AP-1 element is repressed and the RARE is activated, resulting in an overall stimulation of the enhancer by RA in the BMGE+H cells used in our study. These results explain in part the complex and sometimes contradictory response of keratin 6 to hyperproliferative stimuli.

Animals↗