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PubMed2002

Characterization of a novel insertion sequence, IS Bp1, in Burkholderia pseudomallei.

During screening for antigenic proteins in Burkholderia pseudomallei, a novel insertion sequence, IS Bp1, was found by sequence similarity searches. IS Bp1 contains two overlapping ORFs of 261 bp ( orfA) and 852 bp ( orfB), encoding 87 and 284 amino acid residues, respectively, and an imperfect inverted repeat. The putative protein encoded by orfA (OrfA) is similar to the OrfA in insertion sequences of the IS 3 family in other bacteria, showing 49% and 76% amino acid identity and similarity, respectively, with the transposase encoded by IS D1 of Desulfovibrio vulgaris vulgaris. The putative protein encoded by orfB (OrfB) is similar to the OrfB in insertion sequences of the IS 3 family in other bacteria, showing 43% and 62% amino acid identity and similarity, respectively, with the transposase encoded by IS 1222 of Enterobacter agglomerans. Sequence analysis of OrfA showed the presence of an alpha-helix-turn-alpha-helix motif, as well as the putative leucine zipper at its 3' end, for possible DNA binding to the terminal inverted repeats. Sequence analysis of OrfB showed the presence of a DDE motif of aspartic acid, aspartic acid, and glutamic acid, a highly conserved motif present in OrfB of other members of the IS 3 family. Furthermore, several other conserved amino acid residues, including the arginine residue located seven amino acids downstream from the glutamic acid residue, were observed. PCR amplification of the IS Bp1 gene showed a specific band in 65% of the 26 B. pseudomallei strains tested. Southern blot hybridization after XhoI or SacI digestion showed nine different patterns of hybridization. The number of copies of IS Bp1 in those strains that possessed the insertion sequence ranged from three to 12. Using several insertion sequences and a combination of insertion-sequence-based and non-insertion-sequence-based methods such as ribotyping will probably increase the discriminatory power of molecular typing in B. pseudomallei.

Bacterial Proteins↗
PubMed1996

Fate of direct and inverted repeats in the RNA hypermutagenesis reaction.

RNA hypermutagenesis results from cDNA synthesis in the presence of highly biased dNTP precursor concentrations and preferentially exploits human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Such reaction conditions slow down DNA synthesis, which might be conducive to strand transfer and deletion. This has been investigated. A 6 bp inverted repeat nested between 10 bp repeats was efficiently deleted at dCTP concentrations typically used. Inter- or intramolecular strand transfer between 10 bp repeated sequences separated by runs of templated G residues occurred, but at lower concentrations. If RNA hypermutagenesis of a sequence containing direct and inverted repeats is unavoidable, avian myeloblastosis virus (AMV) reverse transcriptase could be used, as strand transfer occurs with much diminished dCTP substrate dependence.

Animals↗
PubMed1989

Human thymidylate synthase gene: isolation of phage clones which cover a functionally active gene and structural analysis of the region upstream from the translation initiation codon.

Two genomic DNA fragments partially encoding human thymidylate synthase (TS) [EC 2.1.1.45] were previously cloned in lambda phage from the mouse cell transformant, but had no transforming activity on mouse TS-negative mutant cells. In this study, an additional genomic DNA for human TS was cloned and demonstrated to have the transforming activity in combination with one of the two previously cloned DNAs and to produce human TS mRNA. The two transforming genomic DNAs overlapped and covered a region of 23 kb in total. Using fragments from one of these DNAs, the structure of the 1.2-kb region around the ATG initiator codon of the TS gene was analyzed in relation to regulatory sequences of the gene. Sequence determination demonstrated the presence of an unusual inverted repeat consisting of a triple tandem repeat of a 28-bp sequence and an inverted sequence of the same length. These sequences can form three possible, stable, stem-loop structures, which may be interconvertible. Based on S1 nuclease mapping data and a line of circumstantial evidence, we deduced two major mRNA cap sites within the inverted sequence. Comparison of the human and mouse sequences upstream from the ATG initiator codon revealed many significant blocks of sequence homology, especially in the regions around the deduced cap sites.

Animals↗
PubMed1992

The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene.

The complete nucleotide sequence of the inverted repeat component (IR; 12,776 bp each) of the genome of equine herpesvirus type 1 (EHV-1) has been determined. Transcription analyses have revealed that the EHV-1 IR sequence encodes at least 6 genes. In this report, we present the DNA sequence and transcriptional characterization of a gene (IR3) that maps entirely within the IR sequences. The IR3 open reading frame (ORF) is located between nucleotides (nt) 6123-6411 of the IR sequence and possesses an ORF of 95 amino acids. Interestingly, this ORF does not show homology to any known herpesvirus gene, suggesting that the IR3 gene is unique to EHV-1. Moreover, the location of the IR3 gene between the immediate-early (IR1) gene and the origin of replication is unique in comparison to the IR gene arrangement of other alphaherpesviruses such as herpes simplex virus type 1 and varicella zoster virus. Putative cis-acting elements flanking the IR3 ORF include a TATA box (nt 5648-5652), a GC box (nt 5600-5605), and three polyadenylation signals (nt 6533-6538, 6648-6653, and 6663-6668). Northern blot analyses identified a 1.0 kb mRNA that exhibits characteristics of a late gene of the gamma-1 class. Northern blot, S1 nuclease, and primer extension analyses revealed that transcription of IR3 initiates within the intron of the immediate-early gene (IR1) on the opposite stand of the genome. Thus, the 5' end of IR3 transcript is antisense to the 5' end of the IR1 mRNA and promoter, and IR3 transcription may regulate the expression of IR1 during late times of infection.

Animals↗
PubMed1996

Regulatory region with putA gene of proline dehydrogenase that links to the lum and the lux operons in Photobacterium leiognathi.

Nucleotide sequence of regulatory region (R & R) with putA gene (EMBL Accession No. U39227) from Photobacterium leiognathi PL741 has been determined, and the putA gene encoded amino acid sequence of proline dehydrogenase is deduced. Alignment and comparison of proline dehydrogenase of P. leiognathi with the proline dehydrogenase domain in the PutA protein of Escherichia coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that regulatory region with the putA gene is linked to the lum and lux operons in genome; the gene order is <--putA--R & R(I)<--ter-lumQ-lumP-R & R-luxC-luxD-luxA-luxB-luxE--> (R & R: regulatory region; ter:transcriptional terminator), whereas the R & R is the regulatory region for the lum and the lux operons, ter is the transcriptional terminator for the lum operon, and R & R(I) apparently is the regulatory region for the putA and related genes. Nucleotide sequence analysis illustrates the specific inverted repeat (SIR), cAMP-CRP consensus sequence, canonical -10/-35 promoter, putative operator and Shine-Dalgarno (SD) sequence on the regulatory region R & R(I) for the putA and related genes; it suggests that the putA and related genes are simply linked to the lum and the lux operons in genome, the regulatory region R & R(I) is independent for the putA and related genes.

Amino Acid Sequence↗
PubMed1996

Mrs, a new subfamily of Tourist transposable elements.

We have characterised a new family of repetitive sequences that we have named Mrs (maize repetitive sequences). Mrs elements are associated with different maize genes and seem to be specific for the genome of Zea species. Mrs elements are short, AT-rich and contain terminal inverted repeats (TIRs). The sequence of their TIRs, as well as the fact that they are flanked by short repetitions that tend to be TAA, allows us to propose Mrs as a new subfamily of Tourist transposable elements.

Base Sequence↗
PubMed1989

Plasmid-partition functions of the P7 prophage.

The sequences responsible for the proper partition of the P7 plasmid prophage to daughter cells lie within a discrete block of non-similarity between P7 and its close relative P1. The DNA sequence of the P7 region was determined. A segment with near identity to the replication (rep) region of P1 is followed by sequences (P7 par) that are clearly related to but very divergent from the P1 partition region. Subcloning was used to define the ends of the functional P7 partition region. It begins with a transcription promoter followed by two large open reading frames, parA and parB, that overlap by a single base and are read in the same direction. The genes direct the synthesis of two proteins, P7 ParA and ParB, with apparent Mr of 44,000 and 37,000. Specific frameshift mutations were introduced into the two genes. Each mutation blocked plasmid partition and both were complemented when the P7 ParA and ParB proteins were supplied in trans. The amino acid sequences of the P7 proteins show strong similarities to the P1 ParA and ParB proteins. However, the DNA sequences of the P7 and P1 open reading frames are remarkably divergent, largely caused by variability at the third positions in the codons. Interspecific complementation tests showed that the P7 proteins are unable to complement P1 parA or parB mutants, and the P1 proteins fail to complement the P7 mutations. Downstream from the P7 parB open reading frame is a sequence that conserves 27 of the 34 base-pairs of the P1 partition site parS. Unlike the P1 parS site, the P7 equivalent does not contain as extensive an inverted repeat. The heptamer sequences that define ParB binding sites within P1 parS are represented in P7 but differ from it by one base. A related sequence that coincides with the secondary ParB binding site within the P1 incB sequences is present nearby. Other sequences within the P7 incB region are rather different from their P1 counterparts. The basis for the major differences in specificity of the P1 and P7 par components is discussed. Comparison of the P1 and P7 sequences, and the nature of the junctions between similar and different sequences, suggest that the phages could have evolved by the pickup of divergent cassettes by recombination.

Amino Acid Sequence↗
PubMed1994

Hypertransposing derivatives of the streptomycete insertion sequence IS493.

Transposons derived from the Streptomyces lividans insertion sequence IS493 are useful for the genetic analysis and manipulation of a number of Streptomyces spp. Tn5099-10, an IS493 derivative that contains a spontaneous deletion terminating in the left inverted repeat (IR-L), transposed at a 1000-fold higher frequency in Streptomyces griseofuscus, and at a tenfold higher frequency in Streptomyces fradiae, than the IS493 derivatives, Tn5096 and Tn5099. The IR-L from Tn5099-10 was used to construct a cassette which hypertransposes from plasmids containing the transposon genes, ORFA and ORFB, outside of the inverted repeats. The target sequences of two Tn5099-10 insertions conformed to the consensus target sequence of the other IS493 derivatives, gNCaNTgNNy (where lower-case letters indicate that other nt have been observed at this position and N is any nt). Transposition mutant libraries of S. griseofuscus and S. fradiae can be easily prepared in broth culture by using the hypertransposing elements and a temperature-sensitive delivery plasmid.

Base Sequence↗
PubMed1986

Enhancer sequences responsible for DNase I hypersensitivity in polyomavirus chromatin.

DNase I preferentially cleaves polyomavirus minichromosomes at two sites in the enhancer, each of which comprises the sequence AAGCAPuPuAAG flanked by short inverted repeats. A tandem duplication of this sequence generates an additional hypersensitive locus. Mutations which alter either the AAGCAPuPuAAG or flanking repeats diminish hypersensitivity. This region must determine the chromatin conformation recognized by DNase I.

Base Sequence↗
PubMed1989

A human muscle adenine nucleotide translocator gene has four exons, is located on chromosome 4, and is differentially expressed.

The human heart-skeletal muscle adenine nucleotide translocator (ANT1) gene was isolated and sequenced. It spans 5.8 kilobases and contains four exons. The 5'-nontranscribed region contains typical CCAAT and TATA sequences, a 22-nucleotide pair inverted repeat and a 13-nucleotide pair sequence homologous to a similar region in the ATP synthase beta subunit gene. The region surrounding the first exon and intron is G+C-region surrounding the first exon and intron is G+C-rich, and the intron contains three Sp1 binding motifs. ANT1 was assigned to chromosome 4 using both flow-sorted chromosomes and segregating human-mouse hybrid cells. Additional ANT sequences were found on at least two other chromosomes. ANT1 transcripts were present at high levels in human heart and skeletal muscle but were almost undetectable in liver, kidney, and brain. By contrast, fibroblast ANT (ANT2) mRNAs were present in all five tissues. The unique nature and arrangement of the ANT1 transcriptional control elements may account for this differential expression.

Adult↗
PubMedDate not supplied

An unusual transposon with long terminal inverted repeats in the sea urchin Strongylocentrotus purpuratus.

A 3-kilobase DNA segment characteristic of a transposable element was found within a histone H2B pseudogene in a higher eukaryote, the sea urchin Stronglyocentrotus purpuratus. The inserted segment (TU1) is flanked by 8-base pair (bp) direct repeats of the H2B sequence. TU1 has long terminal inverted repeats approximately 840 bp long with an outer domain of 15-bp tandem repeats and a non-repeating inner domain, and is a member of a heterogeneous family of transposable elements. TU1 differs from most previously characterized eukaryotic transposable elements with terminal direct repats, but resembles the foldback transposon family in Drosophila.

Animals↗
PubMed1980

Nucleotide sequence of the bacterial transposon Tn1681 encoding a heat-stable (ST) toxin and its identification in enterotoxigenic Escherichia coli strains.

the Escherichia coli heat-stable toxin (ST I) is encoded within a transposon (Tn1681) flanked by inverted repeats of insertion sequence 1 (IS1) [So, M., Heffron, F. & McCarthy, B. J. (1979) Nature (London) 277, 453-456]. By subcloning restriction fragments and by insertion mutagenesis, we located precisely the gene for ST I within the transposon. We determined the complete nucleotide sequence of the central portion of Tn1681 (i.e., that part flanked by IS1) and identified the coding sequence of the toxin. From the nucleotide sequence, we deduced a probable amino acid sequence for ST I. The NH2-terminal portion of the amino acid sequence is extremely hydrophobic and bears a striking resemblance to the signal sequence of the fd phage minor coat protein. By using a subcloned restriction fragment containing the gene for ST I but no IS1 sequences, we determined (i) that the ST toxin with activity assayable in suckling mice (ST I) is genetically distinct from the St toxin assayable in ligated ileal loops (ST II) and (ii) that ST I can be responsible for diarrheal disease in different animals.

Amino Acid Sequence↗
PubMed1989

Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257.

Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.

Amino Acid Sequence↗
PubMed1998

Characterization of the DNA-binding and dominant negative activity of v-erbA homodimers.

The oncoprotein v-erbA is a mutated form of thyroid hormone receptor alpha1 that is virtually incapable of binding T3. V-erbA is a dominant repressor of transcription induced by thyroid hormone receptors and retinoic acid receptors; however, the genetic targets of v-erbA that lead to oncogenesis are not known. Although v-erbA can bind as monomers and dimers to DNA containing the consensus sequence AGGTCA arranged as direct, inverted, or everted repeats, it is not known which sequence represents the optimal v-erbA-binding site. Determination of the DNA recognition properties of v-erbA would allow a better understanding of the repressor activity of this oncoprotein. The current studies, by using a random DNA selection strategy, have determined that the imperfect everted repeat 5'-TGACC(T/C)NT(A/G)AGGTCAC is the optimal v-erbA homodimer-binding site, where N represents any di- or trinucleotide. Functional studies show that everted repeats containing this sequence are substantially more potent v-erbA response elements than direct or inverted repeats, even though many classic T3 response elements are direct repeats. Thus, v-erbA represses only a subset of T3 response elements. In a similar fashion, v-erbA was found to repress a subset of vitamin D response elements. Of general interest, the data indicate that the two molecules of a transcription factor homodimer do not necessarily have identical DNA-binding specificities.

Animals↗
PubMed1981

Nucleotide sequence of cloned unintegrated avian sarcoma virus DNA: viral DNA contains direct and inverted repeats similar to those in transposable elements.

We have determined the nucleotide sequence of portions of two circular avian sarcoma virus (ASV) DNA molecules cloned in a prokaryotic host--vector system. The region whose sequence was determined represents the circle junction site--i.e., the site at which the ends of the unintegrated linear DNA are fused to form circular DNA. The sequence from one cloned molecule, SRA-2, shows that the circle junction site is the center of a 330-base-pair (bp) tandem direct repeat, presumably representing the fusion of the long terminal repeat (LTR) units known to be present at the ends of the linear DNA. The circle junction site is also the center of a 15-bp imperfect inverted repeat, which thus appears at the boundaries of the LTR. The structure of ASV DNA--unique coding region flanked by a direct repeat that is, in turn, terminated with a short inverted repeat--is very similar to the structure of certain transposable elements. Several features of the sequence imply that circularization to form the SRA-2 molecule occurred without loss of information from the linear DNA precursor. Circularization of another cloned viral DNA molecule, SRA-1, probably occurred by a different mechanism. The circle junction site of the SRA-1 molecule has a 63-bp deletion, which may have arisen by a mechanism that is analogous to the integration of viral DNA into the host genome. Flanking one side of the tandem direct repeat is the binding site for tRNATrp, the previously described primer for synthesis of the first strand of viral DNA. The other side of the direct repeat is flanked by a polypurine tract, A-G-G-G-A-G-G-G-G-G-A, which may represent the position of the primer for synthesis of the second strand of viral DNA. An A+T-rich region, upstream from the RNA capping site, and the sequence A-A-T-A-A-A are present within the direct repeat sequence. These sequences may serve as a promoter site and poly(A) addition signal, respectively, as proposed for other eukaryotic transcription units.

Avian Sarcoma Viruses↗
PubMed1996

Analysis of the negative transcriptional regulatory element in the angiotensin-converting enzyme gene.

We have characterized the sequence requirements and the protein binding properties of the previously identified transcriptional negative element present in the rabbit angiotensin-converting enzyme (ACE) gene. DNase footprinting experiments revealed that within the negative element (-715 to -610) several regions interact with proteins present in the nuclear extracts of ACE-expressing and -nonexpressing cell lines. Transfection analysis using the heterologous beta-actin promoter and mutated negative elements demonstrated that the SP1 site, the collagen-silencer-like sequence, and the inverted repeat elements are dispensable for their functioning. Deletion of the region between -692 to -668, however, completely eliminated the activity of the negative element, and mutation of the synapsin-silencer-like sequence present within this region vastly reduced it. This region (-692 to -668) by itself, when present in two copies, could effectively repress the activity of the beta-actin promoter. The same point mutations in the silencer element that destroyed its action on the beta-actin promoter greatly increased the transcriptional efficiency of the native ACE promoter. Electrophoretic mobility shift assay using the -692 to -668 ACE silencer sequence demonstrated the formation of a DNA/protein complex. UV cross-linking of the components of this complex revealed the presence of one prominent protein of approximately 21.5 kDa. This protein may be responsible for mediating the transcriptional-repressing activity of the ACE negative element. Homology between the ACE silencer and neuronal silencer consensus sequence, together with the promoter- and tissue-independent function of the the ACE silencer, suggests this element may bind a member of a large family of common negative regulatory transcription factors.

Animals↗
PubMed2002

Genome-wide analysis of the Emigrant family of MITEs of Arabidopsis thaliana.

Miniature inverted-repeat transposable elements (MITEs) are structurally similar to defective class II elements, but their high copy number and the size and sequence conservation of most MITE families suggest that they can be amplified by a replicative mechanism. Here we present a genome-wide analysis of the Emigrant family of MITEs from Arabidopsis thaliana. In order to be able to detect divergent ancient copies, and low copy number subfamilies with a different internal sequence we have developed a computer program to look for Emigrant elements based solely on the terminal inverted-repeat sequence. We have detected 151 Emigrant elements of different subfamilies. Our results show that different bursts of amplification, probably of few active, or master, elements, have occurred at different times during Arabidopsis evolution. The analysis of the insertion sites of the Emigrant elements shows that recently inserted Emigrant elements tend to be located far from open reading frames, whereas more ancient Emigrant subfamilies are preferentially found associated to genes.

Amino Acid Sequence↗
PubMed2005

Assembly of the mariner Mos1 synaptic complex.

The mobility of transposable elements via a cut-and-paste mechanism depends on the elaboration of a nucleoprotein complex known as the synaptic complex. We show here that the Mos1 synaptic complex consists of the two inverted terminal repeats of the element brought together by a transposase tetramer and is designated paired-end complex 2 (PEC2). The assembly of PEC2 requires the formation of a simpler complex, containing one terminal repeat and two transposase molecules and designated single-end complex 2 (SEC2). In light of the formation of SEC2 and PEC2, we demonstrate the presence of two binding sites for the transposase within a single terminal repeat. We have found that the sequence of the Mos1 inverted terminal repeats contains overlapping palindromic and mirror motifs, which could account for the binding of two transposase molecules "side by side" on the same inverted terminal repeat. We provide data indicating that the Mos1 transposase dimer is formed within a single terminal repeat through a cooperative pathway. Finally, the concept of a tetrameric synaptic complex may simply account for the inability of a single mariner transposase molecule to interact at the same time with two kinds of DNA: the inverted repeat and the target DNA.

Animals↗