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PubMed2006

Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition.

The Tol2 element is a naturally occurring active transposable element found in vertebrate genomes. The Tol2 transposon system has been shown to be active from fish to mammals and considered to be a useful gene transfer vector in vertebrates. However, cis-sequences essential for transposition have not been characterized. Here we report the characterization of the minimal cis-sequence of the Tol2 element. We constructed Tol2 vectors containing various lengths of DNA from both the left (5') and the right (3') ends and tested their transpositional activities both by the transient excision assay using zebrafish embryos and by analyzing chromosomal transposition in the zebrafish germ lineage. We demonstrated that Tol2 vectors with 200 bp from the left end and 150 bp from the right end were capable of transposition without reducing the transpositional efficiency and found that these sequences, including the terminal inverted repeats (TIRs) and the subterminal regions, are sufficient and required for transposition. The left and right ends were not interchangeable. The Tol2 vector carrying an insert of >11 kb could transpose, but a certain length of spacer, <276 but >18 bp, between the left and right ends was necessary for excision. Furthermore, we found that a 5-bp sequence, 5'-(A/G)AGTA-3', is repeated 33 times in the essential subterminal region. Mutations in the repeat sequence at 13 different sites in the subterminal region, as well as mutations in TIRs, severely reduced the excision activity, indicating that they play important roles in transposition. The identification of the minimal cis-sequence of the Tol2 element and the construction of mini-Tol2 vectors will facilitate development of useful transposon tools in vertebrates. Also, our study established a basis for further biochemical and molecular biological studies for understanding roles of the repetitive sequence in the subterminal region in transposition.

Animals↗
PubMed1983

The orir to ori+ mutation in spontaneous yeast petites is accompanied by a drastic change in mitochondrial genome replication.

The orir petite mutants of Saccharomyces cerevisiae show a very low level of suppressivity (5-12%; suppressivity is the percentage of diploid petites issued from a cross of the parental haploid petite with a wild-type cell), indicating a poor replication efficiency of their mitochondrial genome. The latter is made up of repeat units containing two inverted ori sequences and arranged as tandem pairs in inverted orientation relative to their nearest neighbors. After subcloning orir petites or crossing with wild-type cells a large number of ori+ petites are found in the progeny. In contrast to the orir petites, from which they are derived, these ori+ petites are characterized by high suppressivity levels (approx. 90%) and contain mitochondrial genomes made up of tandem repeat units containing single ori sequences. The structural changes underlying the orir to ori+ mutation are therefore accompanied by a dramatic increase in suppressivity, indicating that the elimination of inverted ori sequences causes a drastic change from very poor to very good replicative efficiency in the mitochondrial genome. Finally, crosses of ori0 petites with wild-type cells were also studied; the results obtained have clarified the reasons for the high frequency of petites having genomes similar to those of orir petites after mutagenesis with ethidium bromide.

DNA Replication↗
PubMed1992

Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference.

Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.

Animals↗
PubMed1992

Discovery and characterization of a new transposable element, Tn4811, in Streptomyces lividans 66.

Transposition of a new 5.4-kb transposon, Tn4811, of Streptomyces lividans to the melC operon of Streptomyces antibioticus on plasmid pIJ702 was discovered. The nucleotide sequence of this copy of Tn4811, which contained an imperfect (9 of 11 bp) terminal inverted repeat, five putative Streptomyces coding sequences for an oxidoreductase and its transcription regulator, and three transposition-related proteins, was determined. SLP- strains of S. lividans contained one copy (A) of Tn4811, while SLP2+ strains contained an additional copy (B) on the SLP2 plasmid. The nucleotide sequences at three insertion junctions of Tn4811 were determined. Copy B lacked 41 bp from the left end. At the other five junctions the duplication of a putative 3-bp target sequence (TGA) was observed. A sequence of less than 3 kb homologous to Tn4811 was present in S. antibioticus. DNA homologous to Tn4811 was not detected in 14 other Streptomyces species.

Amino Acid Sequence↗
PubMed1996

The trpF nucleotide sequence and its promoter analysis in Pseudomonas aeruginosa.

The nucleotide sequence of the gene for the phosphoribosyl anthranilate isomerase (PRAI) (trpF) in Pseudomonas aeruginosa was determined. The gene encoded a polypeptide of 211 amino acid residues and its initiation codon was UUG. Northern hybridization analysis showed only one transcript for trpF (about 700 nucleotides). Primer extension analysis revealed three transcription start sites which were at positions 52, 55 and 57 bases upstream of the initiation codon. A highly GC-rich 9-base-pair inverted repeat with 5-base-pair spacing was found upstream of a putative Shine-Dalgarno sequence. This inverted repeat could form a 23-nucleotide stem-loop structure in the transcript. Deletion of the inverted repeat slightly decreased the promoter activity of trpF. The presence of tryptophan had no effect on transcription of trpF.

Aldose-Ketose Isomerases↗
PubMed2003

Specific RNA interference in psbP genes encoded by a multigene family in Nicotiana tabacum with a short 3'-untranslated sequence.

RNA interference with double-stranded RNA is a new method for the study of gene function in various organisms. In this report, we show that an inverted repeat of a short (103-bp) 3'-untranslated sequence of an isogene, 1A, of psbP genes, encoded by a small multigene family of four genes (1A, 2AF, 3F, and 5B) in Nicotiana tabacum, can specifically suppress the expression of psbP isogenes 1A and 5B with a 3'-untranslated sequence similar to a transcribed double-stranded RNA. The expression of other psbP isogenes, 2AF and 3F, was not affected, although the coding sequences of the psbP family genes are highly conserved. Consistent with this observation, small interfering RNAs were detected for the 3'-untranslated sequence used for the inverted-repeat transgene, and not for the coding sequence. These results suggest that double-stranded RNA having a 3'-untranslated sequence could be useful for an isogene-specific RNA interference of the family genes in Nicotiana tabacum.

3' Untranslated Regions↗
PubMed1989

Olfactory marker protein gene: its structure and olfactory neuron-specific expression in transgenic mice.

Olfactory marker protein (OMP) genomic clones were isolated from a Charon 4A phage lambda rat genomic library. A 16.5-kilobase (kb) fragment of the rat genome containing the gene was isolated and characterized. Sequence analysis of the gene showed the absence of introns and the lack of CAAT and TATA boxes in the 5' flanking region. The transcription initiation site was mapped, and two sites 55 and 58 base pairs upstream of the ATG were observed. The 5' flanking region is rich in G+C residues and contains a G+C-rich motif as well as direct and inverted repeats. Functional OMP regulatory sequences were demonstrated in transgenic mice. An 11-kb chimeric gene was constructed in which the coding region for OMP was replaced with that for Thy-1.1. In Thy-1.2 mice carrying this transgene, Thy-1.1 was expressed solely by olfactory receptor neurons and their axons and terminals in the olfactory bulb.

Animals↗
PubMed1980

Deletions and DNA rearrangements within the transposable DNA element IS2. A model for the creation of palindromic DNA by DNA repair synthesis.

Three derivatives of mutant ga10P-308::IS2-I of Escherichia coli were characterized by DNA sequence analysis. Deletions and DNA sequence rearrangements were observed which apparently were initiated at short A-T rich inverted repeats within IS2. Two of the mutants carried newly synthesized DNA sequences which were inverted copies of already existing IS2 sequences. Thus long stretches with twofold symmetry were formed. It is discussed whether these inverted repeats were formed by DNA repair synthesis which was initiated at the A-T rich palindromes of IS2.

Base Sequence↗
PubMed1983

[beta-Lactamase-positive Neisseria gonorrhoeae. II. Localization of homologous regions between the 3.2 mdalton and 4.6 mdalton plasmids by DNA-DNA heteroduplex studies].

The difference between the 4.6 MDalton and 3.2 MDalton N. gonorrhoeae plasmids, both inducing penicillinase production, is examined by restriction endonuclease analysis, transcription-/translation studies and DNA-DNA heteroduplex analysis. One difference between those plasmids is that the 4.6 MDalton one carries a region with inverted repeats on both ends between coordinates 0.9 and 2.3. This region is an insertion sequence most probably. The length of the inverted repeats is 0.15 to 0.19 MDalton. An other difference between both the plasmids is that the 4.6 MDalton one, but not the 3.2 MDalton one, induces a protein of 10800 molecular weight.

DNA, Bacterial↗
PubMed1992

Gene expression in the Streptomyces temperate phage phi C31.

The repressor gene, c, of the temperate Streptomyces phage, phi C31 was previously cloned and sequenced, and predicted to encode a 74-kDa protein. The c gene actually produces three in-frame, N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa. The repressor proteins are translated from a corresponding nest of transcripts. Genetic and biochemical evidence suggests that the transcription of the c locus is autoregulated possibly by the 42-kDa protein binding to a highly conserved 16-bp perfect inverted repeat. The 16-bp sequence is present at at least twelve loci throughout the phi C31 genome. Transcription of the 'early' region is complex, possibly involving phage-specific promoters. The phi C31 terminators display sequence conservation and may be regulated. The phi C31 gene 'k' may encode a nucleotide kinase-encoding gene.

Bacteriophages↗
PubMed1996

Interferon-gamma induction of the human leukocyte antigen-E gene is mediated through binding of a complex containing STAT1alpha to a distinct interferon-gamma-responsive element.

Expression of the human major histocompatibility complex (MHC) class I genes has been shown previously to increase at the transcriptional level following exposure to interferon-gamma (IFN-gamma). In this report we have examined the molecular mechanisms involved in the IFN-gamma-induced transcription of the human MHC class I gene, HLA-E. Functional analysis of CAT reporter gene constructs under the control of the HLA-E promoter transfected into U937 cells revealed the presence of a distinct IFN-gamma-responsive element, termed the interferon response region (IRR), that was necessary and sufficient to mediate the response to IFN-gamma. This cis-acting regulatory sequence contains an imperfect inverted repeat; the 5'-half of the IRR resembles the IFN-gamma activation site (GAS), and the 3'-half of the IRR resembles the interferon-stimulated response element (ISRE). Gel mobility shift assays demonstrated that the IRR bound a single, specific, IFN-gamma-induced complex (IRR-AC), which was formed rapidly following treatment with IFN-gamma and was independent of protein synthesis. Competition experiments with GAS and ISRE sequences from other IFN-inducible genes showed that GAS sequences competed for the IRR-AC, whereas ISRE sequences did not compete. Mutational analysis demonstrated that point mutations in either the 5'-half or 3'-half of the IRR prevented binding of the complex and abrogated or markedly reduced the IFN-gamma responsiveness of reporter gene constructs. Supershift analysis revealed that the IRR-AC contains a factor that was recognized by antibodies specific for the protein STAT1alpha (signal transducer and activator of transcription). Taken together, these findings suggest that the mechanism of IFN-gamma-induced transcription of the HLA-E gene is distinct from that of other MHC class I genes.

Base Sequence↗
PubMed2004

Mutational analysis of the helix-turn-helix region of Bacillus subtilis response regulator DegU, and identification of cis-acting sequences for DegU in the aprE and comK promoters.

The DegS-DegU two-component system in Bacillus subtilis regulates exoprotease production and competence development. Phosphorylated and unphosphorylated forms of DegU are required for activation of aprE and comK, respectively. Alanine-scanning mutagenesis of the helix-turn-helix region of DegU and in vivo examination of 27 DegU variants revealed five common mutants that showed severe reduction of gene expression of both aprE and comK because of reduced DNA-binding activity. This observation suggested that the DegU-recognized cis-sequences might not be considerably changed for either promoter. We identified a DegU-recognized inverted repeat in the comK promoter using various mutant comK-lacZ fusions. Inspection of the aprE promoter sequence revealed a tandem repeat consisting of short AT-rich sequences containing a consensus one, 5'-TAAAT-3', which was found in the downstream half of the inverted repeat involved in comK activation. Oligonucleotide-directed replacement of the short AT-rich sequences located in the center of each motif decreased DegU-dependent aprE expression, implying that the repeat is required for the activation of aprE. Based on these results, it was concluded that DegU would function through the inverted repeat in the comK promoter and the tandem repeat in the aprE promoter.

Alanine↗
PubMed1990

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1.

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.

Base Sequence↗
PubMed1984

The primary structure of the larval beta 1-globin gene of Xenopus laevis and its flanking regions.

We present the complete nucleotide sequence of the larval beta I-globin gene of Xenopus laevis including 240 nucleotides of the 5' flanking region and 594 nucleotides beyond the polyadenylation site. The site of transcription initiation was mapped by S1 nuclease, and the site of polyadenylation was determined by comparison with corresponding cDNA clones. The larval Xenopus beta I-gene shows the same internal structure as the beta-globin genes of higher vertebrates, viz. 3 exons interrupted by 2 intervening sequences. The first intervening sequence, which is of exceptional length, spans over 564 nucleotides and interrupts the coding sequence at amino acid 30, whereas the second one comprises 968 nucleotides and is located between the amino acids 104 and 105. The second intervening sequence contains a long inverted repeat of almost perfect homology. The 5' flanking region contains a TATA- and a CAAT-box at positions -33 and -58, respectively. An additional TATA-box is located at -197 and two more CAAT-boxes occur at positions -105 and -237.

Amino Acid Sequence↗
PubMed1999

Characterization of the chalcone synthase genes expressed in flowers of the common and Japanese morning glories.

The CHS genes encoding chalcone synthase for flavonoid biosynthesis in the common and Japanese morning glories comprise a multigene family. Among these Ipomoea CHS genes, the CHS-D gene is the most abundantly expressed in the pigmented young flower buds and is primarily responsible for flower pigmentation. Majority of the remaining CHS transcripts in the flower buds are produced from the CHS-E gene. We characterized the genomic DNA segments of these CHS-D and CHS-E genes. Both genes have two exons with identical intron positions and carry several copies of two mobile element-like sequences with short terminal inverted repeats, MELS3 and MELS6 of around 200-300 bp. Small tandem repeats were also found in these CHS gene regions. The CHS-D and CHS-E genes are expressed predominantly in flower limbs and tubes, respectively. These structural and functional features and their evolutionary implications are discussed.

Acyltransferases↗
PubMed2003

Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles.

We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2 rep gene contains a cis-acting element responsible for the Rep-dependent replication of a transiently transfected rep-cap plasmid. In this study, we further report that replicated rep-cap sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.

Capsid↗
PubMed1993

A variable DNA region of Entamoeba histolytica is expressed in several transcripts which differ in genetically related clones.

A highly variable DNA region (EhVR1), isolated from Entamoeba histolytica clone A, strain HM1:IMSS, is transcribed into several transcripts, which differ in genetically related clones. EhVR1 (3.5 kb) is composed of two contiguous fragments; one of these 1.9 kb long, at the 3' end, identified similar transcripts in clones A, L6 and C2 (all derived from strain HM1:IMMS), the other of 1.6 kb, at the 5' end, detected 0.5, 0.6 and 0.7 kb transcripts only in clone A. Variability of the 1.6 kb fragment was found even within the same clone maintained under different conditions. EhVR1 was localized to 1.3 and 1.4 Mb linear chromosomes and also found in circular molecules. The sequence of the 1.6 kb fragment revealed the presence of a large number of different repeats, including inverted and palindromic repeats. A p145 sequence, previously detected in episomal DNA of the amoeba, was found at the 5' end of EhVR1. The presence of EhVR1 in linear and circular molecules, its high number of repeats, and its variability in genetically related clones suggest the existence of DNA regions that undergo dynamic non-reciprocal recombination between circular episomes and linear chromosomes, and may thus contribute to variability in the trophozoite genome.

Animals↗
PubMed1993

In-vivo-generated fusion promoters in Pseudomonas putida.

Plasmid pEST1463 carrying the promoterless pheBA operon was cloned into Pseudomonas putida PaW85, and phenol-utilizing colonies were isolated on minimal plates containing phenol as the only carbon and energy source. In these clones, chromosomally located Tn4652 was transposed upstream from the coding sequencing of pheA (encoding phenol monooxygenase). Sequence analysis together with mapping of the transcription start point of the pheBA operon in the recombinant plasmids revealed that fusions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated functional promoters under selective pressure in P. putida cells. These promoter sequences show similarity to the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. In three of the six fusion promoters studied, the generation combined two distinct events: transposition of Tn4652 into DNA containing potential -10 sequences and point mutations in these sequences. These mutations made the -10 sequences more like the sigma 70 promoter consensus sequences.

Base Sequence↗