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PubMed1993

The processing of macronuclear DNA sequences during macronuclear development of the hypotrichous ciliate Stylonychia lemnae.

The organization of two macronuclear DNA sequences in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae and their processing from the micronucleus via the polytene chromosome stage up to the macronucleus was analyzed. The overall organization of these sequences in the polytene chromosomes resembles that described for the micronucleus of other hypotrichous ciliates, i.e. they are interrupted by internal eliminated sequences and not associated with telomeric sequences. The spacer region between the genes is bordered by direct repeats and inverted repeats are found at the termini of macronuclear sequences and in the spacer region. The organization of these macronuclear DNA sequences in the micronucleus was analyzed by polymerase chain reactions. The results obtained show that in the sequences analyzed no DNA reorganization occurs during polytene chromosome formation.

Animals↗
PubMed1992

The 10 S BC-1 ribonucleoprotein particle contains identifier sequence-binding proteins that interact with an array of GCAAG/CTTGC motifs between split promoter sequences for RNA polymerase III.

BC-1 RNA is a brain-specific small RNA transcript of identifier sequences present in the somas and dendrites of neurons. We recently reported that the RNA is complexed with a protein(s) to form a 10 S ribonucleoprotein particle (Kobayashi, S., Goto, S., and Anzai, K. (1991) J. Biol. Chem. 266, 4726-4730). We demonstrate here that this 10 S BC-1 ribonucleoprotein particle contains a DNA-binding protein(s) (Bp-1 protein) capable of interacting with a region between split promoter sequences for RNA polymerase III within the identifier sequences. The region has short inverted repeats: a perfect octanucleotide repeat (GCGCTTGCCTAGCAAGCGC) and an imperfect heptanucleotide repeat (GCCTAGCAAGCGCAAGGC), each of which contains a GCAAG/CTTGC motif. We also demonstrate that the binding of this protein either to the array of pentamer motifs or to BC-1 RNA is mutually exclusive. The molecular masses of photo-cross-linking adducts of Bp-1 protein to a 32P-labeled GCAAG/CTTGC motif-specific probe were estimated to be about 31 and 36 kDa, indicating that two species of Bp-1 proteins may be present in the brain.

Animals↗
PubMed1982

A site-specific, conservative recombination system carried by bacteriophage P1. Mapping the recombinase gene cin and the cross-over sites cix for the inversion of the C segment.

The bacteriophage P1 genome carries an invertible C segment consisting of 3-kb unique sequences flanked by 0.6-kb inverted repeats. With insertion and deletion mutants of P1 derivatives the site-specific recombinase gene cin for C inversion) has been mapped adjacent to the C segment and the cix sites (for C inversion cross-over) have been located at the outside ends of the inverted repeats. Inversion of the C segment functions as a biological switch and controls expression of the gene(s) responsible for phage infectivity carried on the C segment. The cin gene product can promote recombination between a 'quasi- cix ' site on plasmid pBR322 and a cix site on P1 DNA. The junctions formed on the resulting co-integrate can also serve as cix sites. This observation implies a potential evolutionary process to bring genes under the control of a biological switch acting by DNA inversion.

Base Sequence↗
PubMed2006

Positive selection on transposase genes of insertion sequences in the Crocosphaera watsonii genome.

Insertion sequences (ISs) are mobile elements that are commonly found in bacterial genomes. Here, the structural and functional diversity of these mobile elements in the genome of the cyanobacterium Crocosphaera watsonii WH8501 is analyzed. The number, distribution, and diversity of nucleotide and amino acid stretches with similarity to the transposase gene of this IS family suggested that this genome harbors many functional as well as truncated IS fragments. The selection pressure acting on full-length transposase open reading frames of these ISs suggested (i) the occurrence of positive selection and (ii) the presence of one or more positively selected codons. These results were obtained using three data sets of transposase genes from the same IS family that were collected based on the level of amino acid similarity, the presence of an inverted repeat, and the number of sequences in the data sets. Neither recombination nor ribosomal frameshifting, which may interfere with the selection analyses, appeared to be important forces in the transposase gene family. Some positively selected codons were located in a conserved domain, suggesting that these residues are functionally important. The finding that this type of selection acts on IS-carried genes is intriguing, because although ISs have been associated with the adaptation of the bacterial host to new environments, this has typically been attributed to transposition or transformation, thus involving different genomic locations. Intragenic adaptation of IS-carried genes identified here may constitute a novel mechanism associated with bacterial diversification and adaptation.

Adaptation, Biological↗
PubMed1989

Characterization and nucleotide sequence determination of a repeat element isolated from a 2,4,5-T degrading strain of Pseudomonas cepacia.

Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T- strain PT88 by a ColE1::Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) was identified which was repeated several times on the wild-type chromosome and was also present on AC1100 plasmid DNA. Various chromosomal fragments containing this sequence were cloned and their nucleotide sequence was determined. Examination of RS1100 revealed the presence of 38-39-bp terminal inverted repeats immediately flanked by 8-bp direct repeats. The translated sequence of the single large open reading frame of RS1100 showed structural similarity to the phage Mu transposase and other DNA-binding proteins. Thus the AC1100 repeated sequence has several structural features in common with insertion sequence elements. Three copies of RS1100 were mapped near 2,4,5-t genes encoding degradation of 5-chloro-1,2,4-trihydroxybenzene, an intermediate in 2,4,5-T degradation. Neither RS1100 nor the 2,4,5-t genes hybridized to DNA isolated from Pseudomonas strains, including P. cepacia, suggesting that both gene fragments may be of foreign origin recruited in strain AC1100. The origin of these two DNA segments as well as the role played by RS1100 in the recruitment of 2,4,5-t genes in AC1100 are presently under investigation.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed2004

Heat-shock treatment-mediated increase in transduction by recombinant adeno-associated virus 2 vectors is independent of the cellular heat-shock protein 90.

Recombinant adeno-associated virus 2 (AAV) vectors transduction efficiency varies greatly in different cell types. We have described that a cellular protein, FKBP52, in its phosphorylated form interacts with the D-sequence in the viral inverted terminal repeat, inhibits viral second strand DNA synthesis, and limits transgene expression. Here we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV transduction because FKBP52 forms a complex with HSP90, and because heat-shock treatment augments AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosine dephosphorylation of FKBP52, led to stabilization of the FKBP52-HSP90 complex, and resulted in approximately 6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor tyrosine kinase, which phosphorylates FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. HSP90 was shown to be a part of the FKBP52-AAV D-sequence complex, but HSP90 by itself did not bind to the D-sequence. Geldanamycin treatment, which disrupts the HSP90-FKBP52 complex, resulted in >22-fold increase in AAV transduction in heat-shock-treated cells compared with heat shock alone. Deliberate overexpression of the human HSP90 gene resulted in a significant decrease in AAV-mediated transduction in tyrphostin 23-treated cells, whereas down-modulation of HSP90 levels led to a decrease in HSP90-FKBP52-AAV D-sequence complex formation, resulting in a significant increase in AAV transduction following pre-treatment with tyrphostin 23. These studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment is unlikely to be mediated by HSP90 alone and that increased levels of HSP90, in the absence of heat shock, facilitate binding of FKBP52 to the AAV D-sequence, thereby leading to inhibition of AAV-mediated transgene expression. These studies have implications in the optimal use of recombinant AAV vectors in human gene therapy.

Benzoquinones↗
PubMed1995

S elements: a family of Tc1-like transposons in the genome of Drosophila melanogaster.

The S elements form a diverse family of long-inverted-repeat transposons within the genome of Drosophila melanogaster. These elements vary in size and sequence, the longest consisting of 1736 bp with 234-bp inverted terminal repeats. The longest open reading frame in an intact S element could encode a 345-amino acid polypeptide. This polypeptide is homologous to the transposases of the mariner-Tc1 superfamily of transposable elements. S elements are ubiquitous in D. melanogaster populations and also appear to be present in the genomes of two sibling species; however, they seem to be absent from 17 other Drosophila species that were examined. Within D. melanogaster strains, there are, on average, 37.4 cytologically detectable S elements per diploid genome. These elements are scattered throughout the chromosomes, but several sites in both the euchromatin and beta heterochromatin are consistently occupied. The discovery of an S-element-insertion mutation and a reversion of this mutation indicates that S elements are at least occasionally mobile in the D. melanogaster genome. These elements seem to insert at an AT dinucleotide within a short palindrome and apparently duplicate that dinucleotide upon insertion.

Amino Acid Sequence↗
PubMed1990

Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family.

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).

Amino Acid Sequence↗
PubMed1987

Transposition of IS91 does not generate a target duplication.

We determined the DNA sequences surrounding the junctions of IS91 in two insertion derivatives: pSU234 (pACYC184::IS91) and pSU240 (pBR322::IS91). The termini of IS91 consist of two imperfect inverted repeats eight base pairs long. Their sequence is 5'-TCGAGTAGG. . . CCTATCGA-3'. Insertion of IS91 did not generate direct repetitions in the target DNAs.

Base Sequence↗
PubMed2001

The DIRS1 group of retrotransposons.

Only three retrotransposons of the DIRS1 group have previously been described: DIRS1 from the slime mold Dictyostelium discoideum, PAT from the nematode Panagrellus redivivus, and Prt1 from the zygomycetous fungus Phycomyces blakesleeanus. Analyses of the reverse transcriptase sequences encoded by these elements suggest that they are related to the long terminal repeat (LTR) retroelements, such as the Ty3/gypsy retrotransposons and the vertebrate retroviruses. The DIRS1-group elements, however, have several unusual structural features which distinguish them from typical LTR elements: (1) they lack the capacity to encode DDE-type integrases or aspartic proteases; (2) they have open reading frames (ORFs) of unknown function; (3) they integrate without creating duplications of their target sites; and (4) although they are bordered by terminal repeats, these sequences differ from typical LTRs in that they are either inverted repeats or "split" direct repeats. Because of the small number of DIRS1-like elements described, and the unusual structures of these elements, little is known about their evolution, distribution, and replication mechanisms. Here, we report the identification of several new DIRS1-like retrotransposons, including elements from nematodes, sea urchins, fish, and amphibia. We also present evidence for the existence of DIRS1-like sequences in the human genome. In addition, we show that the lack of DDE-type integrase genes from elements of the DIRS1 group is explained by the finding that the previously uncharacterized ORFs of these elements encode proteins related to the site-specific recombinase of bacteriophage lambda. The presence of lambda-recombinase-like genes in DIRS1 elements also accounts for the lack of target-site duplications for these elements and may be related to the unusual structures of their terminal repeats.

Amino Acid Sequence↗
PubMed1997

Three insertion sequences from the cyanobacterium Synechocystis PCC6803 support the occurrence of horizontal DNA transfer among bacteria.

Three insertion sequences were characterized from the widely-used cyanobacterium Synechocystis PCC6803. They all harbored a putative transposase sequence flanked by two imperfect inverted repeats, seemed to have duplicated their target insertion site and occurred as multiple copies in the host genome. They exhibited no obvious homology with any other cyanobacterial ISs and were termed IS5S (871 bp), IS4S (1299 bp) and ISS1987 (949 bp) because they were, respectively, homologous to IS5- and IS4-bacterial elements, and to several members of the IS630-Tc1-mariner superfamily of IS elements occurring in a wide range of hosts. This suggests that these IS-elements were spread through horizontal transfer between evolutionary distant organisms. Three IS5S-copies were isolated as a rescue insertion into a replicating plasmid (IS5Sa), or subsequently cloned from a Synechocystis DNA-library probed with IS5Sa (IS5Sb and IS5Sc), and appeared to be almost identical. In the vicinity of IS5Sb, we found the ISS1987 element inserted into the IS4S element. This indicates that the ISS1987 element has been, and could still be, mobile since its transposase sequence is not interrupted with stop codons or translational frameshifts, unlike that which is found in most members of the IS630-Tc1-mariner superfamily of transposable elements.

Amino Acid Sequence↗
PubMed1990

Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences.

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome II behaves as a stable, independent genetic linkage group in S. pombe. The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA. Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology. The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional. Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I. This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences. These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes.

Base Sequence↗
PubMed1999

Cloning, sequencing and molecular analysis of the Campylobacter jejuni groESL bicistronic operon.

The groESL bicistronic operon from the enteric pathogen Campylobacter jejuni was cloned and sequenced. It consists of two ORFs encoding proteins with molecular masses of 9.5 and 57.9 kDa, which showed a high degree of homology to other bacterial GroES and GroEL proteins. Northern blot analysis suggested that the groESL operon is transcribed as a bicistronic mRNA, and its steady-state level was markedly increased after temperature upshift. By primer extension assay, one potential transcription start point preceding the groESL genes could be demonstrated, and a putative promoter region compatible with both Escherichia coli and C. jejuni sigma70 consensus sequences was identified. A conserved inverted repeat, which is believed to be involved in the regulation of the groESL genes, was found between the -10 promoter box and the groES translation start site. The complete coding region of groEL was fused with pET-22b(+) and expressed in E. coli as a His6-tagged recombinant protein (rCjHsp60-His). After purification, the protein was recognized by an anti-HSP60 monoclonal antibody. ELISA and Western immunoblotting experiments showed that IgG and IgA antibody responses against rCjHsp60-His were not significantly increased in sera from 24 patients with sporadic Campylobacter infection when compared to sera from 16 healthy controls.

Amino Acid Sequence↗
PubMed1999

Characterization of repetitive DNA elements in Arabidopsis.

We have applied computational methods to the available database and identified several families of repetitive DNA elements in the Arabidopsis thaliana genome. While some of the elements have features expected of either miniature inverted-repeat transposable elements (MITEs) or retrotransposons, the most abundant class of repetitive elements, the AthE1 family, is structurally related to neither. The AthE1 family members are defined by conserved 5' and 3' sequences, but these terminal sequences do not represent either inverted or direct repeats. AthE1 family members with greater than 98% identity are easily identified on different Arabidopsis chromosomes. Similar to nonautonomous DNA-based transposon families, the AthE1 family contains members in which the conserved terminal domains flank unrelated sequences. The primary utility of characterizing repetitive sequences is in defining, at least in part, the evolutionary architecture of specific Arabidopsis loci. The repetitive elements described here make up approximately 1% of the available Arabidopsis thaliana genomic sequence.

Algorithms↗
PubMed1995

Characterization of a dispersed repetitive DNA sequence associated with the CCDD genome of wild rice.

A HindII repetitive fragment (pOD3) was isolated and cloned from the genomic DNA of an accession of Oryza latifolia, a wild rice species that possesses a tetraploid CCDD genome. Southern blot analysis using this clone as a probe demonstrated that this repetitive DNA sequence had a dispersed organization in the CCDD genome and seemed to be highly specific for this genome type. This fragment is the first CCDD-specific repeated DNA sequence to be described. The hybridization pattern is similar for most CCDD accessions tested, although a few showed no hybridization signal. The nucleotide sequence of the element cloned in pOD3 was determined and analysed. The 1783 base pair long repeated sequence shows no homology with other known nucleotide sequences. In addition, none of the amino acid sequences deduced from the potential open reading frames contained in the pOD3 repeat is homologous to any known protein. The nucleotide sequence presents several internal repeats, direct or inverted, but their significance remains unknown.

Base Sequence↗
PubMed1986

Site-specific and illegitimate recombination in the oriV1 region of the F factor. DNA sequences involved in recombination and resolution.

We have defined some of the sequences involved in high frequency recA-independent recombination at the oriV1 region of the F factor. Using a mobilization assay, we determined that plasmid pMB080, a pBR322 derivative bearing the PvuII-BamHI (F factor co-ordinates 45.43 to 46.0) fragment from the oriV1 region of F, contained all sequences necessary to undergo efficient site-specific recombination with the F derivative pOX38, which retains the oriV1 region. We constructed a series of pMB080 deletions in vitro using exonucleases S1 and Bal31. Deletions removing a ten base-pair sequence, which forms part of an inverted repeat segment located 62 base-pairs to the left of the NcoI site (45.87) within the cloned fragment, totally eliminated the recA-independent recombination reaction. Other deletions differentially affected both the frequency and stability of cointegrate molecules formed by the site-specific recombination system. The F factor oriV1 region is involved also in low-frequency recombination with several sites on pBR322 and related plasmids. We have determined the precise location of these recombination sites within oriV1 by DNA sequencing. These studies revealed that recombination always took place within an eight base-pair spacer region between the ten base-pair inverted repeats found to be important for oriV1-oriV1 interactions. We propose that the low-efficiency recombination between pBR322 and pOX38 results from the ability of the F site-specific recombination apparatus to weakly recognize and interact with sequences that bear some resemblance to the normal oriV1 recognition elements. Furthermore, we suggest, by analogy with the lambda paradigm, that the nucleotide sequences at the junctions of secondary site recombinants define at least one crossover site used during the normal site-specific recombination process.

Base Sequence↗
PubMed2004

Identification of two mariner-like elements in the genome of the mosquito Ochlerotatus atropalpus.

Two distinct mariner-like elements, Atmar-1 and Atmar-2, were isolated from the genome of the mosquito Ochlerotatus atropalpus. Full-sized Atmar-1 elements, obtained by screening a genomic library, have a 1293-bp consensus sequence with 27-bp inverted terminal repeats and a 1047-bp open reading frame (ORF) encoding the transposase. The Atmar-2 elements were amplified by polymerase chain reaction from genomic DNA and contain the central part of the transposase ORF. Individual clones of both mariner elements contain deletions, frameshifts, and stop codons. The Atmar-1 elements are present in 370-1200 copies, while the Atmar-2 elements are present in approximately 100-300 copies per haploid genome. One of the Atmar-1 elements, Atmar-1.33, could be mobilized, suggesting the presence of functional Atmar-1 elements elsewhere in the genome. Phylogenetic analysis demonstrated that Atmar-1 elements belong to the irritans subfamily and Atmar-2 elements to the cecropia subfamily of mariner elements.

Amino Acid Sequence↗
PubMed2005

Isolation and characterization of a native composite transposon, Tn14751, carrying 17.4 kilobases of Corynebacterium glutamicum chromosomal DNA.

A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 x 10(2) transformants per mug of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

Bacterial Proteins↗