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PubMed1996

Identification of promoter in the 5'-flanking region of the E. coli thioredoxin-linked thiol peroxidase gene: evidence for the existence of oxygen-related transcriptional regulatory protein.

E. coli thiol peroxidase (Tpx) linked to the thioredoxin as an in vivo thiol regenerating system acts as an antioxidant enzyme removing peroxides and H2O2. In order to elucidate the mechanism regulating tpx gene expression in E. coli in response to oxygen stress, we made 5' progressive deletions of upstream region from tpx gene, and fused to lacZ gene. LacZ activity was increased 6-fold by oxygen stress and inverted repeat sequence located between -47 and -33 nt was proven to be essential for the oxygen response of tpx promoter. Primer extension experiment and analysis of upstream sequence revealed transcription start point, -10, and -35 regions, which are in good agreements with the consensus sequences recognized by E sigma 70. Northern hybridization showed that expression of tpx gene is regulated at the transcriptional level. DNA binding assays using inverted repeat sequence including -35 region provides preliminary evidence that expression of tpx requires additional transcriptional factor in response to oxygen stress.

Amino Acid Sequence↗
PubMed1992

Transcription and autoregulation of the stabilizing functions of broad-host-range plasmid RK2 in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa.

The broad-host-range plasmid RK2 has been shown to encode several proteins important for its maintenance within bacterial populations of a number of Gram-negative bacteria. Their genes are organized into two operons: parCBA and parD. These operons have been proposed to be transcribed from two divergent promoters, p-parCBA and p-parD, located within a sequence of approximately 150 bases. In this report we identify and characterize the sequences required for regulated transcription from these promoters in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa. Both of these promoters are repressed by their own gene products in the same manner in all three bacteria tested, with ParA functioning as the primary repressor of p-parCBA and ParD functioning as the repressor of p-parD. The binding regions of these proteins were determined through deletion analyses, DNA mobility shift assays, and an examination of the effect of mutations in this region. Based on these observations, the ParA protein appears to bind to either two inverted repeat or two direct repeat sequences, one downstream from the transcriptional initiation site and the other upstream of the p-parCBA -35 box. The ParD protein appears to bind to one inverted repeat sequence, located between the -35 and -10 boxes of p-parD.

Agrobacterium tumefaciens↗
PubMed1989

Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences.

In a previous report, we described the presence, in pituitary tissue, of an alternatively processed species of bovine growth hormone mRNA from which the last intron (intron D) has not been removed by splicing (R. K. Hampson and F. M. Rottman, Proc. Natl. Acad. Sci. USA 84:2673-2677, 1987). Using transient expression of the bovine growth hormone gene in Cos I cells, we observed that splicing of intron D was affected by sequences within the downstream exon (exon 5). Deletion of a 115-base-pair FspI-PvuII restriction fragment in exon 5 beginning 73 base pairs downstream of the intron 4-exon 5 junction resulted in cytoplasmic bovine growth hormone mRNA, more than 95% of which retained intron D. This contrasted with less than 5% of the growth hormone mRNA retaining intron D observed with expression of the unaltered gene. Insertion of a 10-base-pair inverted repeat sequence, CTTCCGGAAG, which was located in the middle of this deleted segment, partially reversed this pattern, resulting in cytosolic mRNA from which intron D was predominantly removed. More detailed deletion analysis of this region indicated that multiple sequence elements within the exon 5, in addition to the 10-base-pair inverted repeat sequence, are capable of influencing splicing of intron D. The effect of these exon sequences on splicing of bovine growth hormone precursor mRNA appeared to be specific for the growth hormone intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. The results of this study suggest a unique interaction between sequences located near the center of exon 5 and splicing of the adjacent intron D.

Animals↗
PubMed1984

A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1.

Nucleotide sequences were determined for a region essential for autonomous replication and partitioning of pSC101, a plasmid whose replication is dependent on the Escherichia coli dnaA gene product. The essential replication region contains one long coding sequence, rep101 , for a protein composed of 316 amino acids, and a polypeptide approximately 37 X 10(3) Mr in size was identified as the rep101 gene product. rep101 is preceded by two inverted repeat sequences, three directly repeated sequences and a region of high A + T content containing a sequence similar to the E. coli oriC consensus sequence. Because the lesions in seven replication-deficient insertion mutants, four mutants with increased copy number and one temperature-sensitive replication mutant occur within rep101 , the rep101 gene product must control pSC101 replication and copy number. par, a region adjacent to the replication region, which functions in stable plasmid inheritance, contains several inverted repeat sequences.

Autoradiography↗
PubMed1988

Plasmid pIJ699, a multi-copy positive-selection vector for Streptomyces.

A plasmid vector, pIJ699, which provides positive selection for cloned DNA, was constructed using the replication functions of the Streptomyces wide-host-range multi-copy plasmid pIJ101. The selection for inserts is based on the principle that plasmids with long uninterrupted perfect palindromes (inverted repeats) are 'not viable' in bacteria. For cloning, pIJ699 is digested with BglII. This produces two fragments, one of which is the linearized vector, with two arms of the palindrome at its ends, and the other is a 'spacer' which is needed to keep the inverted repeat sequences apart. The vector fragment is separated from the 'spacer' fragment and ligated with the DNA to be cloned. Plasmids with a fragment of cloned DNA, but not the circularized vector, give rise to thiostrepton-resistant transformants in Streptomyces lividans. The inverted repeat sequences contain a strong transcription terminator which reduces transcriptional read-through both in and out of the cloned fragment. This improves the stability of many hybrid plasmids and facilitates the study of the regulation of cloned genes.

Cloning, Molecular↗
PubMed2002

Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences, short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing.

In genetically transformed plants, transgene silencing has been correlated with multiple and complex insertions of foreign DNA, e.g. T-DNA and vector backbone sequences. Occasionally, single-copy transgenes also suffer transgene silencing. We have compared integration patterns and T-DNA/plant DNA junctions in a collection of 37 single-copy T-DNA-transformed Arabidopsis lines, of which 13 displayed silencing. Vector sequences were found integrated in five lines, but only one of these displayed silencing. Truncated T-DNA copies, positioned in inverse orientation to an intact T-DNA copy, were discovered in three lines. The whole nptII gene with pnos promoter was present in the truncated copy of one such line in which heavy silencing has been observed. In the two other lines no silencing has been observed over five generations. Thus, vector sequences and short additional T-DNA sequences are not sufficient or necessary to induce transgene silencing. DNA methylation of selected restriction endonuclease sites could not be correlated with silencing. Our collection of T-DNA/plant DNA junctions has also been used to evaluate current models of T-DNA integration. Data for some of our lines are compatible with T-DNA integration in double-strand breaks, while for others initial invasion of plant DNA by the left or by the right T-DNA end seem important.

Arabidopsis↗
PubMed2000

FARE, a new family of foldback transposons in Arabidopsis.

A new family of transposons, FARE, has been identified in Arabidopsis. The structure of these elements is typical of foldback transposons, a distinct subset of mobile DNA elements found in both plants and animals. The ends of FARE elements are long, conserved inverted repeat sequences typically 550 bp in length. These inverted repeats are modular in organization and are predicted to confer extensive secondary structure to the elements. FARE elements are present in high copy number, are heterogeneous in size, and can be divided into two subgroups. FARE1's average 1.1 kb in length and are composed entirely of the long inverted repeats. FARE2's are larger, up to 16.7 kb in length, and contain a large internal region in addition to the inverted repeat ends. The internal region is predicted to encode three proteins, one of which bears homology to a known transposase. FARE1.1 was isolated as an insertion polymorphism between the ecotypes Columbia and Nossen. This, coupled with the presence of 9-bp target-site duplications, strongly suggests that FARE elements have transposed recently. The termini of FARE elements and other foldback transposons are imperfect palindromic sequences, a unique organization that further distinguishes these elements from other mobile DNAs.

Amino Acid Sequence↗
PubMed1993

Insertion and deletion mutations in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance.

Mutants of IncFII plasmid NR1 that have transposons inserted in the repA4 open reading frame (ORF) are not inherited stably. The repA4 ORF is located immediately downstream from the replication origin (ori). The repA4 coding region contains inverted-repeat sequences that are homologous to the terC inverted repeats located in the replication terminus of the Escherichia coli chromosome. The site of initiation of leading-strand synthesis for replication of NR1 is also located in repA4 near its 3' end. Transposon insertions between ori and the right-hand terC repeat resulted in plasmid instability, whereas transposon insertions farther downstream did not. Derivatives that contained a 35-bp frameshift insertion in the repA4 ORF were all stable, even when the frameshift was located very near the 5' end of the coding region. This finding indicates that repA4 does not specify a protein product that is essential for plasmid stability. Examination of mutants having a nest of deletions with endpoints in or near repA4 indicated that the 3' end of the repA4 coding region and the site of leading-strand initiation could be deleted without appreciable effect on plasmid stability. Deletion of the pemI and pemK genes, located farther downstream from repA4 and reported to affect plasmid stability, also had no detectable effect. In contrast, mutants from which the right-hand terC repeat, or both right- and left-hand repeats, had been deleted were unstable. None of the insertion or deletion mutations in or near repA4 affected plasmid copy number. Alteration of the terC repeats by site-directed mutagenesis had little effect on plasmid stability. Plasmid stability was not affected by a fus mutation known to inactivate the termination function. Therefore, it appears that the overall integrity of the repA4 region is more important for stable maintenance of plasmid NR1 than are any of the individual known features found in this region.

Amino Acid Sequence↗
PubMed1983

Coordinately expressed chorion genes of Bombyx mori: is developmental specificity determined by secondary structure recognition?

Short inverted repeat sequences have been observed in the DNA flanking the 5' and 3' termini of a pair of coordinately expressed chorion structural genes of the silkmoth Bombyx mori. When superhelical cloned DNA containing the two chorion genes is digested with S1 nuclease, several sites are specifically cleaved including those around the centers of the putative cruciform structures resulting from the short inverted repeat sequences. The possible implication of conformational changes in the DNA surrounding the chorion structural genes in the process of determination of the developmental specificity and the coordinate transcriptional regulation of these genes is discussed.

Journal Article↗
PubMed1980

Structure of the 5-S ribosomal RNA gene and its adjacent regions in Torulopsis utilis.

A DNA segment spanning one repeating unit of ribosomal RNA(rRNA) genes in a yeast strain, Torulopsis utilis, has been cloned on a bacterial plasmid pBR322. The size of the cloned segment was about Mr = 8.0 x 10(6). All of the genes for the four species of rRNA were linked on it, and there was a long spacer between the 5-S and 18-S rRNA coding regions. The nucleotide sequences of the regions flanking the 5-S rRNA gene were determined and compared with those in the corresponding regions of Sacharomyces cerevisiae [Valenzuela, P., Bell, G. I., Masiarz, F. R., DeGennaro, L. J., and Rutter, W. J. (1977) Nature, 267, 641-643; Maxam A. M., Tizard, R., Skryabin, K. G., and Gilbert, W. (1977) Nature, 267, 643-645]. The sequence of the T. utilis 5-S rRNA is identical with that of S. cerevisiae except for two residues at positions 18 and 61. However, its upstream region contained a quite different sequence, and a sequence which showed some homology was only found at positions -21 to -28. The sequences were d(T-G-T-A-A-C-C-T) in T. utilis and d(T-A-T-C-A-C-C-T) in S. cerevisiae. Although the presence of various repeat sequences having the same or opposite directions was noted, these repeats occurred at different positions in the two yeast species. In the downstream region, the common sequence was only seven dT deoxynucleotides, which occurred immediately after the 5-S rRNA coding sequence. Significant direct and inverted repeat sequences were found in T. utilis, but such repeats are not seen in S. cerevisiae.

Base Sequence↗
PubMed1977

Sequence organization and expression of a yeast plasmid DNA.

Saccharomyces cerevisiae strain A364A D5 contains circular double-stranded DNA molecules of 6230 +/- 30 base pairs (2mu DNA) which are present in 68 copies per cell and make up 2.4% of the haploid genome. About 0.4% of non-poly A containing yeast RNA hybridizes to the yeast DNA circles. When denatured and then self-annealed, the DNA molecules assume a characteristic "dumbbell" shape in the electron microscope indicating that each circle possesses a non-tandem inverted repeat sequence of 630 +/- 10 base pairs. Eco-RI digestion of purified 2mu DNA yields 4 fragments on an agarose gel whose combined molecular mass is twice that of the monomer circle, suggesting that there are 2 populations of circles, each of the same molecular weight. Representatives of each population have been separated by cloning in Escherichia coli via the bacterial plasmid pSC101. Heteroduplex analysis of the cloned circles show that the 2 different populations arise because of intramolecular recombination between the inverted repeat sequences. Acrylamide gel patterns of polypeptides synthesized in bacterial mini-cells containing the hybrid plasmids between 2mu DNA and pSC101 are significantly different than the pattern obtained from mini-cells containing pSC101 alone.

Cell Membrane↗
PubMed1994

Effect of internal direct and inverted Alu repeat sequences on PCR.

We have studied the effect of repeated DNA sequence, especially Alu repeats, on PCR. Alu repeats are sequences that are approximately 300 bp long and interspersed at a very high copy number throughout the human genome. We amplified part of the human low-density lipoprotein receptor gene containing two Alu repeat sequences in the same orientation, approximately 7.8 kb apart, with unique sequence primers outside these repeats. The major PCR product was a DNA fragment with an in vitro deletion between the Alu repeats. The formation of this product depended on the template concentration and the type of polymerase used. Such a product arose apparently as a result of a "jumping reaction" involving a primer whose extension was terminated prematurely within one Alu repeated followed by annealing of such an incompletely extended primer to the other, distant Alu repeat. No such jumping products were seen when a 0.8-kb region containing two nearby inverted Alu repeats within the human alpha-galactosidase A gene was subject to PCR with unique sequence primers annealing just outside these repeats.

Animals↗
PubMed1988

Varicella-zoster virus DNA from persistently infected cells contains novel tandem duplications.

A persistent infection with varicella-zoster virus was established in the Mewo human melanoma cell line. This persistently infected cell line went through periodic crises of virus-induced cell killing and then recovery. Analyses of viral DNA derived from the persistently infected cultures revealed that novel viral nucleic acid rearrangements had been generated. These viral DNA sequences were derived from a specific region of the inverted repeat sequence of the genome flanking the short unique genome segment. The novel DNA was of various lengths, each generated by tandem duplication of an approximately 2760 base pair sub-sequence of the normal viral inverted repeat. These novel sequences were inserted into an otherwise apparently normal genome.

Antigens, Viral↗
PubMed1989

Molecular cloning of alpha-amylase genes from Drosophila melanogaster. III. An inversion at the Amy locus in an amylase-null strain.

Overlapping clones of the structural gene region for alpha-amylase, Amy, were isolated from a lambda EMBL4 library containing genomic DNA fragments from an amylase-null strain of Drosophila melanogaster. Southern blot analysis and restriction endonuclease mapping of the cloned region indicate that it contains an Amy gene duplication within an inverted repeat sequence as is characteristic of the genomic arrangement for this species. Spacing between the cloned gene copies is similar to that commonly found in other strains. Evidence is presented for the presence of an inversion 4 to 9 kb in length within the cloned Amy region of the null strain. We postulate a causal relationship between the presence of the inversion and the failure of individuals from the null strain to express amylase. A model is proposed that suggests the inversion may have arisen through intramolecular (or sister-strand) recombination mediated by homologous pairing of the inverted repeat sequences at the Amy locus.

Animals↗
PubMed2004

Genome plasticity in Streptomyces: identification of 1 Mb TIRs in the S. coelicolor A3(2) chromosome.

The chromosomes of several widely used laboratory derivatives of Streptomyces coelicolor A3(2) were found to have 1.06 Mb inverted repeat sequences at their termini (i.e. long-terminal inverted repeats; L-TIRs), which are 50 times the length of the 22 kb TIRs of the sequenced S. coelicolor strain M145. The L-TIRs include 1005 annotated genes and increase the overall chromosome size to 9.7 Mb. The 1.06 Mb L-TIRs are the longest reported thus far for an actinomycete, and are proposed to represent the chromosomal state of the original soil isolate of S. coelicolor A3(2). S. coelicolor A3(2), M600 and J1501 possess L-TIRs, whereas approximately half the examined early mutants of A3(2) generated by ultraviolet (UV) or X-ray mutagenesis have truncated their TIRs to the 22 kb length. UV radiation was found to stimulate L-TIR truncation. Two copies of a transposase gene (SCO0020) flank 1.04 Mb of DNA in the right L-TIR, and recombination between them appears to generate strains containing short TIRs. This TIR reduction mechanism may represent a general strategy by which transposable elements can modulate the structure of chromosome ends. The presence of L-TIRs in certain S. coelicolor strains represents a major chromosomal alteration in strains previously thought to be genetically similar.

Amino Acid Sequence↗
PubMed2002

Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence.

Ferric uptake repressor (Fur) proteins regulate the expression of iron homeostasis genes in response to intracellular iron levels. In general, Fur proteins bind with high affinity to a 19-bp inverted repeat sequence known as the Fur box. An alignment of 19 operator sites recognized by Bacillus subtilis Fur revealed a different conserved 15-bp (7-1-7) inverted repeat present twice within this 19-bp consensus sequence. We demonstrated using electrophoretic mobility shift assays that this 7-1-7 inverted repeat comprises a minimal recognition site for high-affinity binding by Fur. The resulting revised consensus sequence is remarkably similar to a related 7-1-7 inverted repeat sequence recognized by PerR, a Fur paralog. Our analysis of the affinity and stoichiometry of DNA binding by B. subtilis Fur, together with a reinterpretation of previously described studies of Escherichia coli Fur, supports a model in which the 19-bp Fur box represents overlapping recognition sites for two Fur dimers bound to opposite faces of the DNA helix. The resulting recognition complex is reminiscent of that observed for the functionally related protein DtxR. Like Fur, DtxR contains a helix-turn-helix DNA-binding motif, recognizes a 19-bp inverted repeat sequence, and has a typical DNase I footprint of approximately 30 bp. By envisioning a similar mode of DNA recognition for Fur, we can account for the internal symmetries noted previously within the Fur box, the tendency of Fur to extend into adjacent regions of DNA in a sequence-selective manner, and the observed patterns of DNA protection against enzymatic and chemical probes.

Bacterial Proteins↗
PubMed1999

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1/ISRm7 and ISRm220-13-5 belong to a new family of insertion sequence elements.

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1 and ISRm220-13-5 are 1481 and 1550 base pairs (bp) in size, respectively. ISRm102F34-1 is bordered by 15 bp imperfect terminal inverted repeat sequences (two mismatches), whereas the terminal inverted repeat of ISRm220-13-5 has a length of 16 bp (two mismatches). Both insertion sequence elements generate a 6-bp target duplication upon transposition. The putative transposase enzymes of ISRm102F34-1 and ISRm220-13-5 consist of 449 or 448 amino acid residues with predicted molecular weights of 50.7 or 51.3 kDa and theoretical isoelectric points of 10.8 or 11.1, respectively. ISRm102F34-1 is identical in 98.9% of its nucleotide sequence to an apparently inactive copy of an insertion sequence element, designated ISRm7, which flanks the left-end of the nodule formation efficiency (nfe) region of plasmid pRmeGR4b of S. meliloti strain GR4. ISRm102F34-1 and ISRm220-13-5 are closely related since they show an overall identity of 57.0% at the nucleotide sequence level and of 47.3% at the deduced amino acid level of their putative transposases. Both insertion sequence elements displayed significant similarity to the Xanthomonas campestris ISXc6 and its homolog IS1478a. Since none of these insertion sequence elements could be allocated to existing families of insertion sequence elements, a new family is proposed. Analysis of the distribution of ISRm102F34-1/ISRm7 in various local S. meliloti populations sampled from Medicago sativa, Medicago sphaerocarpa and Melilotus alba host plants at different locations in Spain revealed its presence in 35% of the isolates with a copy number ranging from 1 to 5. Furthermore, ISRm102F34-1/ISRm7 homologs were identified in other rhizobial species.

Amino Acid Sequence↗
PubMed1990

Bacteriophage T7 DNA packaging. III. A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction.

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.

Base Sequence↗