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PubMed1992

Tourist: a large family of small inverted repeat elements frequently associated with maize genes.

The wx-B2 mutation results from a 128-bp transposable element-like insertion in exon 11 of the maize Waxy gene. Surprisingly, 11 maize genes and one barley gene in the GenBank and EMBL data bases were found to contain similar elements in flanking or intron sequences. Members of this previously undescribed family of elements, designated Tourist, are short (133 bp on average), have conserved terminal inverted repeats, are flanked by a 3-bp direct repeat, and display target site specificity. Based on estimates of repetitiveness of three Tourist elements in maize genomic DNA, the copy number of the Tourist element family may exceed that of all previously reported eukaryotic inverted repeat elements. Taken together, our data suggest that Tourist may be the maize equivalent of the human Alu family of elements with respect to copy number, genomic dispersion, and the high frequency of association with genes.

Base Sequence↗

PubMed1994

Stowaway: a new family of inverted repeat elements associated with the genes of both monocotyledonous and dicotyledonous plants.

Members of a new inverted repeat element family, named Stowaway, have been found in close association with more than 40 monocotyledonous and dicotyledonous plant genes listed in the GenBank and EMBL nucleic acid data bases. Stowaway elements are characterized by a conserved terminal inverted repeat, small size, target site specificity (TA), and potential form stable DNA secondary structures. Some elements are located at the extreme 3' ends of sequenced cDNAs and supply polyadenylation signals to their host genes. Other elements are in the 5' upstream regions of several genes and appear to contain previously identified cis-acting regulatory domains. Although the Stowaway elements share many structural features with the recently discovered Tourist elements, the two families share no significant sequence similarity. Together, the Stowaway and Tourist families serve to define an important new class of short inverted repeat elements found in possibly all flowering plant genomes.

Base Sequence↗

PubMed1993

Identification and characterization of IS1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family.

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS1138, and the target site into which it inserted were determined. IS1138 consists of 1288 bp with 18 bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3 bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS1138 are present in most, if not all, strains of M. pulmonis, but IS1138-like sequences were not detected in other mycoplasmal species.

Amino Acid Sequence↗

PubMed1994

Characterization and occurrence of two repeated palindromic DNA elements of Brucella spp.: Bru-RS1 and Bru-RS2.

Two repeated DNA elements of 103 bp and 105 bp were discovered in brucellae and designated Bru-RS1 and Bru-RS2, respectively. The two elements are palindromic, are 65% similar in sequence, form two families of elements that are slightly divergent in sequence, appear to be intergenic, and are found, collectively, in more than 35 copies in brucellae. These elements are bounded by perfect or nearly perfect inverted repeats. A third copy of the terminal repeat is found within the elements and is the terminus for several truncated copies of the Bru-RS1 family. Hybridization patterns for the elements among brucellae were unique. The elements are dispersed, highly conserved among brucellae, and hot-spots for insertion by IS711.

Base Sequence↗

PubMed2006

An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice.

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.

Alleles↗

PubMed1996

A (G+C)-rich motif in the aldolase C promoter functions as a constitutive transcriptional enhancer element.

The enzyme fructose-1,6-bisphosphate aldolase consists of three isozymes that are expressed in a tissue-specific manner. Using antibodies against aldolase B and C, it is shown that aldolase C is expressed in virtually all neuronal cell lines derived from the central and peripheral nervous system. Recently, experiments with transgenic mice indicated that a (G+C)-rich region of the aldolase C promoter might function as a neuron-specific control element of the rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H. (1993) Eur. J. Biochem. 218, 143-151). To functionally analyse this element, a plasmid consisting of four copies of this (G+C)-rich sequence, a TATA box, and the rabbit beta-globin gene as reporter was constructed. This plasmid was transfected into neuronal and nonneuronal cell lines and transcription was monitored by RNase protection mapping of the beta-globin mRNA. It is shown that the (G+C)-rich element of the aldolase C promoter directs transcription in neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter, used as a control for neuron-specific gene expression, directed transcription only in neuronal cells. In gel-retardation assays, two major DNA-protein complexes were detected with the (G+C)-rich element of the aldolase C promoter used as a DNA probe and nuclear extracts from brain and liver as a source for DNA-binding proteins. These DNA-proteins interactions could be impaired by a DNA probe that contained an Sp1-binding site, indicating that Sp1 or an Sp1-related factor binds to the aldolase C promoter (G+C)-rich element. This was confirmed by supershift analysis with antibodies specific for Sp1. The zinc finger transcription factor zif268/egr-1, also known to recognize a (G+C)-rich consensus site, did not, however, bind to the (G+C)-rich motif of the aldolase C promoter, nor could it stimulate transcription in transactivation assays from this control region. From these data, we conclude that the (G+C)-rich element of the aldolase C promoter functions as a constitutive transcriptional response element mediated by Sp1 and Sp1-related transcription factors.

Animals↗

PubMed2004

The pregnane X receptor binds to response elements in a genomic context-dependent manner, and PXR activator rifampicin selectively alters the binding among target genes.

The pregnane X receptor (PXR) is a key regulator of genes encoding several major types of cytochrome P450 enzymes and transporters (e.g., multidrug resistance-1, MDR1); therefore, PXR contributes significantly to drug-drug interactions. PXR binds to response elements and confers transactivation. Several target genes such as CYP3A4 and 3A7 contain two PXR elements (distant and proximal) that are separated by more than 7000 nucleotides in the genome. Disruption of the distant element causes a 73% decrease of the reporter activity, whereas inactivation of the proximal element decreases by only 53%. This study was undertaken to test the hypothesis that PXR differentially binds to the elements with the distant enhancer being bound to a higher extent. To test this hypothesis, a stable transfected line (hPXR-HRE) was prepared to constitutively express human PXR and harbor a chromatinized CYP3A4-ER6 reporter. This line responded to rifampicin and dexamethasone similarly as hepatocytes based on the relative potency and activation kinetics. Contrary to the hypothesis, chromatin immunoprecipitation experiments showed that the genomic fragment harboring the proximal element was preferably precipitated over the fragment containing the distant element in the CYP3A4 gene, but the opposite was true with the CYP3A7 gene. In addition, the promoters from the MDR1 and CYP2B6 genes were abundantly present in the PXR immunocomplexes from the vehicle-treated cells. However, such abundant interactions were markedly diminished when cells were treated with PXR activator rifampicin. These findings suggest that PXR binding is dependent on the genomic context and PXR activators modulate such bindings.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1987

The maize transposable element Ds is spliced from RNA.

In some instances, insertion of maize transposable elements into exons does not result in the total loss of enzymatic activity. In other instances, messenger RNAs of wild-type size are encoded by genes known to contain the maize transposable element Dissociation (Ds) in exons. To understand how Ds is processed from RNA, a study was made of transcripts encoded by two alleles of the maize waxy (wx) gene containing Ds insertions in exon sequences. The analysis was carried out in strains where the Ds element could not excise from the wx gene. Despite insertions of 4.3- and 1.5-Ds elements, the predominant transcripts encoded by these two genes were wild type in size. For both alleles, DNA sequencing of complementary DNAs revealed that the Ds elements had been spliced in a similar manner. Splicing was accomplished by the utilization of multiple 5' donor splice sites in the Ds termini and a 3' acceptor site within the wx gene adjacent to the Ds element. The net effect in both cases was the removal of most of the Ds element from the messenger RNA.

Base Sequence↗

PubMed2006

The locus coding for the 3-nitrobenzoate dioxygenase of Comamonas sp. strain JS46 is flanked by IS1071 elements and is subject to deletion and inversion events.

In Comamonas sp. strain JS46, 3-nitrobenzoate (3Nba) is initially oxidized at the 3,4 position by a dioxygenase, which results in release of nitrite and production of protocatechuate. The locus coding for the 3Nba dioxygenase (designated mnb, for m-nitrobenzoate) was mobilized from strain JS46 using a plasmid capture method, cloned, and sequenced. The 3Nba dioxygenase (MnbA) is a member of the phthalate family of aromatic oxygenases. An open reading frame designated mnbB that codes for an NAD(P)H-dependent class IA aromatic oxidoreductase is downstream of mnbA. MnbB is tentatively identified as the oxidoreductase that transfers reducing equivalents to MnbA in strain JS46. The mnb locus is flanked by IS1071 elements. The upstream element is interrupted by a novel insertion sequence designated ISCsp1, and the transposase genes of the flanking insertion elements are transcribed in the direction opposite the direction of mnbA transcription. Spontaneous deletion of mnb occurs because of homologous recombination between the directly repeated flanking IS1071 elements. In addition, in approximately 0.007 to 0.2% of any population of JS46 cells growing on 3Nba, alternative orientations of mnb relative to the flanking IS1071 elements are detected. These alternative forms are the result of inversions of mnb and the flanking IS1071 elements. Inversions appear to occur because of homologous recombination between the inverted repeats that flank the IS1071 elements.

Bacterial Proteins↗

PubMed2000

Characterization of Porphyromonas gingivalis insertion sequence-like element ISPg5.

Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions, and its presence in subgingival plaque significantly increases the risk for periodontitis. In contrast to many bacterial pathogens, P. gingivalis strains display considerable variability, which is likely due to genetic exchange and intragenomic changes. To explore the latter possibility, we have studied the occurrence of insertion sequence (IS)-like elements in P. gingivalis W83 by utilizing a convenient and rapid method of capturing IS-like sequences and through analysis of the genome sequence of P. gingivalis strain W83. We adapted the method of Matsutani et al. (S. Matsutani, H. Ohtsubo, Y. Maeda, and E. Ohtsubo, J. Mol. Biol. 196:445-455, 1987) to isolate and clone rapidly annealing DNA sequences characteristic of repetitive regions within a genome. We show that in P. gingivalis strain W83, such sequences include (i) nucleotide sequence with homology to tRNA genes, (ii) a previously described IS element, and (iii) a novel IS-like element. Analysis of the P. gingivalis genome sequence for the distribution of the least used tetranucleotide, CTAG, identified regions in many of the initial 218 contigs which contained CTAG clusters. Examination of these CTAG clusters led to the discovery of 11 copies of the same novel IS-like element identified by the repeated sequence capture method of Matsutani et al. This new 1,512-bp IS-like element, designated ISPg5, has features of the IS3 family of IS elements. When a recombinant plasmid containing much of ISPg5 was used in Southern analysis of several P. gingivalis strains, including clinical isolates, diversity among strains was apparent. This suggests that ISPg5 and other IS elements may contribute to strain diversity and can be used for strain fingerprinting.

Amino Acid Sequence↗

PubMed1987

Identification of transposable elements which activate gene expression in Pseudomonas cepacia.

This study demonstrated that transposable elements in Pseudomonas cepacia could be inserted upstream of a poorly expressed gene and increase its expression more than 30-fold. Five elements, TnPc1, IS402, IS403, IS404, and IS405, were isolated by their ability to increase expression of the beta-lactamase gene of the broad-host-range plasmid pRP1. Increased expression resulted only from insertion of these elements, suggesting that insertional activation is an important means of elevating gene expression in this organism. Four of the elements inserted between a PstI site within the beta-lactamase gene and a BamHI site located 375 base pairs upstream of its promoter. The element IS403 inserted distal to the BamHI site within the coding region for the gene tnpR, suggesting that insertional activation can act over greater than expected distances. In addition, the element IS402 activated the beta-lactamase genes carried on plasmids pRP1 and pMR5 (temperature-sensitive pRP1) equally well in opposite orientations, demonstrating that insertional activation by this element occurs independent of its orientation.

DNA Restriction Enzymes↗

PubMed2002

R391: a conjugative integrating mosaic comprised of phage, plasmid, and transposon elements.

The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South African Providencia rettgeri strain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element from Vibrio cholerae O139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences from Salmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.

Bacteriophages↗

PubMed1964

SPECTROCHEMICAL ANALYSIS OF INORGANIC ELEMENTS IN BACTERIA.

Rouf, M. A. (Washington State University, Pullman). Spectrochemical analysis of inorganic elements in bacteria. J. Bacteriol. 88:1545-1549. 1964.-Quantitative spectrochemical analyses of inorganic elements in the vegetative cells of Escherichia coli, Sphaerotilus natans, Micrococcus roseus, Bacillus cereus, and the spores of B. cereus were made. The following elements were found to be present in the ash samples: B, Na, Mg, Al, Si, P, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, Sr, S, Ag, Sn, Ba, Pb, V, and Mo. These could be divided into major, minor, and trace elements, depending on the relative amounts in the cells. Mg, P, K, and S were considered as the major elements; Ca, Fe, Zn, and, perhaps, Cu and Mn as the minor elements, and the rest as trace elements. Mg concentrations were higher in the cells of the gram-positive M. roseus and B. cereus than in the gram-negative E. coli and S. natans. The latter organism contained 2.6% Fe(2)O(3) (dry weight basis). The vegetative cells of B. cereus were higher in Mg, P, K, Na, Ag, and lower in Si, Ca, Zn, Mn, and Cu than were its spores.

Bacillus cereus↗

PubMed1993

Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis.

Five different genetic elements have been found to be associated with genetic rearrangements in Mycobacterium tuberculosis complex strains. Of these elements, the insertion sequence IS6110 is presently the most frequently used genetic marker for strain differentiation of M. tuberculosis. In the present study we compared five genetic elements for their potentials to differentiate a given cluster of M. tuberculosis strains. Because of the presence of only a single copy of IS6110 or two IS6110 copies at the same chromosomal locus, a large number of strains could not be differentiated by IS6110 fingerprinting. Most strains, including the low-copy-number IS6110 strains, could be differentiated by fingerprinting with the 36-bp direct repeat or the polymorphic GC-rich repetitive DNA element. Less discriminative power was obtained with the major polymorphic tandem repeat and the insertion element IS1081. One strain which did not contain IS6110 DNA was encountered. Until now, this element has invariantly been found to be present in all M. tuberculosis complex strains. On the basis of classical taxonomic criteria and sequencing of the 16S rRNA gene, this strain was shown to be a genuine M. tuberculosis strain. Therefore, the use of this element as a target for polymerase chain reaction-facilitated detection of M. tuberculosis should be reconsidered.

Bacterial Typing Techniques↗

PubMed1996

cis-acting elements involved in transcriptional regulation of the herpes simplex virus type 1 latency-associated promoter 1 (LAP1) in vitro and in vivo.

Latency-associated promoter 1 (LAP1) of herpes simplex virus type 1 is required to generate a series of latency-associated transcripts (LATs) in sensory neurons of latently infected animals. Sequence analysis and DNA binding studies have suggested the existence of several cis-acting elements within LAP1 that are potentially important for promoter function, although their role in LAT gene expression during latency is largely unexplored. In this report, we present evidence that the LAP1 TATA box is essential for transcription initiation in vitro. A reduction in LAT synthesis measured by in situ hybridization and reverse transcription-PCR (RT-PCR) of rat brain tissue latently infected with a LAP1 TATA substitution virus demonstrated that this sequence was required for full LAP1 activity in vivo. Analysis of additional site-directed and 5'-deletion mutants of LAP1 by in vitro transcription-primer extension assays showed that upstream elements including the USF and cyclic AMP response element (CRE) site specifically contributed to LAP1 function and that sequences beginning at position -620 relative to the transcription start site were essential for full promoter activity. The combination of deleting USF, CRE, and TATA completely abolished LAT expression in the brain, identifying these as essential elements for the neuron-specific functioning of LAP1 during latency. Mutation of the transcription start site did not abolish transcription, suggesting the absence of an initiator element. However, one of the most exciting findings from this study is that the region downstream of the TATA box appears to contain a true enhancer that is not only essential for transcription, but also functional when positioned 1.6 kb downstream of the start site of transcription. It was concluded that (i) the TATA box was essential for full transcriptional activity from LAP1 both in vitro and in vivo, (ii) the USF element and CRE contribute to LAP1 function during latency in combination with the TATA element, (iii) multiple trans-acting factors besides the USF- and CRE-binding proteins were required for full promoter activity in vitro, and (iv) sequences downstream of the TATA box enhanced promoter activity in vitro.

Animals↗

PubMed1997

Cell and viral regulatory elements enhance the expression and function of a human immunodeficiency virus inhibitory gene.

Regulated expression of recombinant genes in CD4+ cells is an important objective for gene therapy of AIDS, as these cells represent the principal target for viral replication of human immunodeficiency virus (HIV). We report here that specific combinations of CD4 cell-specific and viral regulatory elements can enhance expression of an antiviral gene product. Different viral regulatory elements were incorporated into a previously reported CD4 locus control region to increase the expression of reporter genes in T and monocytic cell lines. The CD4-specific regulatory elements were included to enhance expression in CD4 cells, and viral regulatory regions, including the cytomegalovirus immediate-early (CMV IE) upstream enhancer, which contains the kappa B and Ap1 regulatory elements and a Tat-responsive element of the HIV type 1 long terminal repeat, were used to increase gene expression and modulate its activity in response to viral infection. In transient transfection assays, this vector was 100- to 1,000-fold more active than the original CD4 regulatory elements alone. Expression of an inhibitory form of the Rev protein, Rev M10, was more effective than previously described vectors and protected against productive viral replication in CD4+ peripheral blood mononuclear cells. The combination of CD4 lineage-specific and viral regulatory elements will facilitate the development of more effective antiviral genetic strategies for AIDS.

CD4 Antigens↗

PubMed2004

Three novel cis-acting elements required for efficient plus-strand DNA synthesis of the hepatitis B virus genome.

Synthesis of the relaxed-circular (RC) DNA genomes of hepadnaviruses by reverse transcriptase involves two template switches during plus-strand DNA synthesis. These template switches require repeat sequences (so-called donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. To determine cis-acting elements apart from the donor and acceptor sites that are required for plus-strand RC DNA synthesis by hepatitis B virus (HBV), a series of mutants bearing a small deletion were made and analyzed for their impact on the viral genome synthesis. We found three novel cis-acting elements in the HBV genome: one element, located in the middle of the minus strand, is indispensable, whereas the other two elements, located near either end of the minus strand, contribute modestly to the plus-strand RC DNA synthesis. The data indicated that the first element facilitates plus-strand RNA primer translocation or subsequent elongation during plus-strand RC DNA synthesis, while the last two elements, although distantly located on the minus strand, act at multiple steps to promote plus-strand RC DNA synthesis. The necessity of multiple cis-acting elements on the minus-strand template reflects the complex nature of hepadnavirus reverse transcription.

Cell Line↗

PubMed2004

The DNA element controlling expression of the varicella-zoster virus open reading frame 28 and 29 genes consists of two divergent unidirectional promoters which have a common USF site.

The mechanism of the divergent expression of the varicella-zoster virus (VZV) ORF 28 and ORF 29 genes from a common intergenic DNA element, the ORF 28/29 promoter, is of interest based on the observation that both genes are expressed during VZV lytic infection but only the ORF 29 gene is expressed in latently infected neurons. In the work presented here, expression driven by the ORF 28/29 intergenic region was examined. We found that the promoter activity towards the ORF 29 direction is more responsive to activation by the major viral transactivator IE62 than that towards the ORF 28 direction in the context of our experimental system. Analysis of the functional DNA elements involved in IE62 activation of the bidirectional ORF 28/29 regulatory element revealed that in both transfected and VZV-superinfected cells it is a fusion of two unidirectional promoters overlapping an essential USF binding site but with distinct TATA elements. A single TATA element directs expression in the ORF 28 direction, whereas the two TATA elements directing ORF 29 gene expression are alternatively and differentially utilized for transcription initiation. We also identified an Sp1 site localized proximal to the ORF 28 gene which functions as an activator element for expression in both directions. These results indicate that the ORF 28 and ORF 29 genes can be expressed either coordinately or independently and that the observed expression of only the ORF 29 gene during VZV latency may involve neuron-specific cellular factors and/or structural aspects of the latent viral genome.

Base Sequence↗