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PubMed2003

Evaluation of repetitive element sequence-based PCR as a molecular typing method for Clostridium difficile.

Repetitive element sequence-based PCR (rep-PCR) is a typing method that enables the generation of DNA fingerprinting that discriminates bacterial strains. In this study, we evaluated the applicability of rep-PCR in typing Clostridium difficile clinical isolates. The results obtained by rep-PCR were compared with those obtained by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping. A high correspondence between pattern differentiations produced by rep-PCR and PFGE was observed, whereas PCR ribotyping showed a lower level of discriminatory power.

Bacterial Typing Techniques↗
PubMed2006

Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor.

BACKGROUND: Repbase is a reference database of eukaryotic repetitive DNA, which includes prototypic sequences of repeats and basic information described in annotations. Updating and maintenance of the database requires specialized tools, which we have created and made available for use with Repbase, and which may be useful as a template for other curated databases. RESULTS: We describe the software tools RepbaseSubmitter and Censor, which are designed to facilitate updating and screening the content of Repbase. RepbaseSubmitter is a java-based interface for formatting and annotating Repbase entries. It eliminates many common formatting errors, and automates actions such as calculation of sequence lengths and composition, thus facilitating curation of Repbase sequences. In addition, it has several features for predicting protein coding regions in sequences; searching and including Pubmed references in Repbase entries; and searching the NCBI taxonomy database for correct inclusion of species information and taxonomic position. Censor is a tool to rapidly identify repetitive elements by comparison to known repeats. It uses WU-BLAST for speed and sensitivity, and can conduct DNA-DNA, DNA-protein, or translated DNA-translated DNA searches of genomic sequence. Defragmented output includes a map of repeats present in the query sequence, with the options to report masked query sequence(s), repeat sequences found in the query, and alignments. CONCLUSION: Censor and RepbaseSubmitter are available as both web-based services and downloadable versions. They can be found at http://www.girinst.org/repbase/submission.html (RepbaseSubmitter) and http://www.girinst.org/censor/index.php (Censor).

Animals↗
PubMed2001

Use of long-range repetitive element polymorphism-PCR to differentiate Bacillus anthracis strains.

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains.

Bacillus anthracis↗
PubMed1999

The characterization of DINE-1, a short, interspersed repetitive element present on chromosome and in the centric heterochromatin of Drosophila melanogaster.

The banded portion of chromosome 4 (the "dot" chromosome) in Drosophila melanogaster displays some properties of beta-heterochromatin, which is normally found within the centric domain of the chromosomes. The nature and distribution of repetitive elements on chromosome 4 could play a role in the establishment of this unusual chromatin configuration. We describe here one such element: a short, interspersed repetitive sequence named DINE-1. Determination of a consensus sequence for the element reveals that there are two conserved regions (A and B) separated by a highly variable spacer. The conserved sequences are approximately 400 bp long but degenerate at both ends, opening the possibility that a yet-to-be-discovered mother element may be present in the genome. DINE-1 bears few of the properties of the mammalian short interspersed elements (SINEs) to which it bears a superficial resemblance in size. It does not appear to be the product of reverse transcription and lacks any polymerase III promoter consensus. The elements are not flanked by target site duplications and their termini lack direct or inverted repeats, suggesting that they themselves are not transposable. Our analysis of cosmid clones from chromosome 4, and elsewhere in the genome, revealed that the euchromatic locations of DINE-1 are almost exclusively confined to chromosome 4. In situ hybridization of a DINE-1 probe to polytene chromosomes confirmed the preferential distribution along 4, in addition to its presence in the centric heterochromatin. This unusual genomic distribution of bias toward chromosome 4 is also seen in the sibling species, D. simulans, whose dot chromosomes exhibit poorly resolved polytene bands and lack crossing over during meiosis like those of D. melanogaster. However, the dot chromosome of D. virilis, which exhibits a well-defined banded structure on polytene chromosomes and can cross over, has only a single, discrete site of DINE-1 element hybridization. The presence of DINE-1 within these regions suggests a role in the heterochromatic nature of chromosome 4 in D. melanogaster and supports the contention that repeats accumulate in regions of diminished crossing over.

Animals↗
PubMed2000

Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli.

Repetitive-element PCR (rep-PCR) fingerprinting is a promising molecular typing tool for Escherichia coli, including for discriminating between pathogenic and nonpathogenic clones, but is plagued by irreproducibility. Using the ERIC2 and BOXA1R primers and 15 E. coli strains from the ECOR reference collection (three from each phylogenetic group, as defined by multilocus enzyme electrophoresis [MLEE], including virulence-associated group B2), we rigorously assessed the effect of extremely elevated annealing temperatures on rep-PCR's reproducibility, discriminating power, and ability to reveal MLEE-defined phylogenetic relationships. Modified cycling conditions significantly improved assay reproducibility and discriminating power, allowing fingerprints from different cyclers to be analyzed together with minimal loss of resolution. The correspondence of rep-PCR with MLEE with respect to tree structure and regression analysis of distances was substantially better with modified than with standard cycling conditions. Nonetheless, rep-PCR was only a fair surrogate for MLEE, and when fingerprints from different days were compared, it failed to distinguish between different clones within all-important phylogenetic group B2. These findings indicate that although the performance and phylogenetic fidelity of rep-PCR fingerprinting can be improved substantially with modified assay conditions, even when so improved rep-PCR cannot fully substitute for MLEE as a phylogenetic typing method for pathogenic E. coli.

DNA Fingerprinting↗
PubMed1987

A new repetitive element of the CR1 family downstream of the chicken vitellogenin gene.

We have analyzed a repetitive DNA sequence found in the 3'-flanking region of the chicken vitellogenin gene. By its sequence, the repetitive DNA has been identified as a hitherto unreported member of the chicken CR1 family of repetitive elements. The CR1 sequence displays the structural characteristics of a long terminal repeat located at the 3' end of an avian retrovirus. The CR1 element lies 2.2 kb downstream of the vitellogenin gene and 'points' away from the gene rather than toward it. In this respect, this element differs from other CR1 repeats. The CR1 element is embedded in a region showing changes in chromatin structure implying a potential role for this sequence in determining the structural state of the local chromatin.

Alpharetrovirus↗
PubMed1989

A long interspersed repetitive element--the I factor of Drosophila teissieri--is able to transpose in different Drosophila species.

Long interspersed repetitive elements (LINEs) are transposable elements present in many species. In mammals they are difficult to study because most of them are defective and their transposition frequency is low. The I factor of Drosophila melanogaster is a LINE element that is particularly interesting because its transposition occurs at high frequency during I-R hybrid dysgenesis. This phenomenon occurs when males from the class of inducer strains are crossed with females from the class of reactive strains. Inducer strains contain several complete 5.4-kilobase I factors at various sites on the chromosomal arms. Reactive strains are devoid of complete I factors. Many results indicate that active I factors have invaded the D. melanogaster genome recently. To study the evolutionary history of I elements, we have cloned and sequenced a potentially active I factor from Drosophila teissieri. It is flanked by a target-site duplication and terminates at the 3' end by tandem repeats of the sequence TAA. When introduced into the germ line of a reactive strain of D. melanogaster by P element-mediated transformation, it is able to transpose and induces hybrid dysgenesis. This strengthens the hypothesis of a recent reinvasion of the D. melanogaster genome by active I factors giving rise to the inducer strains. They could have originated by horizontal transfer from another species. Such events also could occur for other LINE elements and might explain the spread of new variants in mammalian genomes. Moreover, the results give a further insight into I factor functional organization.

Amino Acid Sequence↗
PubMed2006

Mycobacterium avium restriction fragment length polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains.

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 AIDS inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Acquired Immunodeficiency Syndrome↗
PubMed1982

A short interspersed repetitive element found near some mouse structural genes.

We have isolated and characterized a family of interspersed repetitive elements which make up about 1% of the mouse genome. The elements represent a group of homologous but non-identical units about 400 bp in length. Individual members of the family show considerable divergence from one another. The spacial relationships between members of the family and a number of other identified mouse sequences including structural genes have been determined; these elements are found on the 5' as well as 3' sides of various genes at distances ranging from less than 1 to 7.5 kilobases (Kb). The sequences are present in the DNA of all species of Mus. Related sequences are present in the rat genome at a repetition frequency similar to that in the mouse genome. A partial sequence of one member of the family is presented.

Animals↗
PubMed1987

Integration of a vector containing rodent repetitive elements in the rat genome.

We have previously shown that integration of a polyoma vector containing rodent repetitive elements into rat cellular DNA is non-random (Wallenburg et al. J. Virol. 50: 678-683). Junctions between the polyoma vector and the host DNA occur in the repetitive sequences of the vector about ten times more frequently than would be expected if sequences from the vector were used randomly for integration. In this paper we looked at the host sequences involved in these junctions. Our analysis did not reveal any repetitive or specific sequences and we presume therefore that the repetitive sequences of the vector acted as hot spots for illegitimate recombination. We also analysed the integration mechanism and found that: First, even though the polyoma vector was transfected in the presence of carrier DNA, integration did not involve the formation of a transgenome. Second, in at least one of the clones analysed, integration resulted in deletion of host DNA sequences. Third, the host DNA displaced at the integration site was considerably longer than the integrated segment.

Animals↗
PubMed1993

Whole-cell repetitive element sequence-based polymerase chain reaction allows rapid assessment of clonal relationships of bacterial isolates.

Repetitive element sequence-based polymerase chain reaction (rep-PCR) enables the generation of DNA fingerprints which discriminate bacterial species and strains. We describe the application of whole-cell methods which allow specimens from broth cultures or colonies from agar plates to be utilized directly in the PCR mixture. The rep-PCR-generated DNA fingerprints obtained with whole-cell samples match results obtained with genomic DNA templates. Examples with different gram-negative bacteria (e.g., Citrobacter diversus, Escherichia coli, and Pseudomonas aeruginosa) and gram-positive bacteria (e.g., Staphylococcus aureus and Streptococcus pneumoniae) are demonstrated. Rapid specimen preparation methods enable rep-PCR-based fingerprinting to be completed in several hours and, therefore, allows the timely analysis of epidemiological relationships.

Bacteriological Techniques↗
PubMed2005

Chompy: an infestation of MITE-like repetitive elements in the crocodilian genome.

Interspersed repeats are a major component of most eukaryotic genomes and have an impact on genome size and stability, but the repetitive element landscape of crocodilian genomes has not yet been fully investigated. In this report, we provide the first detailed characterization of an interspersed repeat element in any crocodilian genome. Chompy is a putative miniature inverted-repeat transposable element (MITE) family initially recovered from the genome of Alligator mississippiensis (American alligator) but also present in the genomes of Crocodylus moreletii (Morelet's crocodile) and Gavialis gangeticus (Indian gharial). The element has all of the hallmarks of MITEs including terminal inverted repeats, possible target site duplications, and a tendency to form secondary structures. We estimate the copy number in the alligator genome to be approximately 46,000 copies. As a result of their size and unique properties, Chompy elements may provide a useful source of genomic variation for crocodilian comparative genomics.

Alligators and Crocodiles↗
PubMed1996

Characterization of the transcript of a new class of retroposon-type repetitive element cloned from the powdery mildew fungus, Erysiphe graminis.

The putative master transcript of a novel class of repetitive element has been cloned from the fungus erysiphe graminis f.sp. hordei. Sequence analysis of the cDNA revealed that the element, designated Eg-R1, is a member of the retroposon superfamily with properties in common with SINEs and LINEs (short or long interspersed elements). SINE-like properties include the transcript size (approximately 700 bp), and the lack of major open reading frames. In contrast, the fact that the transcript is polyadenylated and is most probably transcribed by RNA polymerase II, suggests a functional relationship to LINEs. Except for a short, but striking, sequence identity to a published SINE from the same fungus, no similar sequence was found in database searches. A constitutively high transcript level is found throughout the asexual life cycle of the fungus. Small differences in band patterns of Southern blots were observed between two isolates of E. graminis f.sp. hordei, while the band patterns in an isolate of the very close relative E. graminis f.sp. tritici in general appear dissimilar. This may imply that the element is currently active. Recent dispersal is confirmed by the observation that an approximately 550 bp internal hinfI fragment is conserved in the majority of the copies in all three isolates. Approximately 50 copies are present in E. graminis f.sp. hordei.

Ascomycota↗
PubMed1994

A short interspersed repetitive element provides a new 3' acceptor site for trans-splicing in certain ribosomal P2 beta protein genes of Trypanosoma cruzi.

Four Trypanosoma cruzi genomic DNA fragments carrying different TcP2 beta genes have been isolated and sequenced. Three of them had a single TcP2 beta gene, while the 3.8-kb-long DNA segment encoding the TcP2 beta-H1.8 locus showed two TcP2 beta genes arranged in tandem. These genes were physically connected by a 428-bp-long DNA sequence that was also located immediately 5' to the first gene and immediately 3' to the second. Comparison of the 4 TcP2 beta gene loci, suggested that the insertion of this repeated element originated the duplication of its target sequence, a poly(dT) stretch. Approximately 1200 copies of this short sequence, named short interspersed repetitive element (SIRE), were found scattered in the genome. Analysis of the 5' non-coding regions of different TcP2 beta mRNAs, and RNA-PCR experiments suggested that the insertion of a SIRE upstream of a TcP2 beta-H1.8 gene introduced a new 3' spliced leader (SL) acceptor site in the TcP2 beta-H1.8 pre-mRNAs, encoded within the SIRE. Consequently, in the mature H1.8 mRNA the SL sequence is followed by 38 bases directly transcribed from the SIRE. Structural and functional features of this repeated element reveal similarity to the short interspersed repetitive DNA sequences detected in the genomes of several microorganisms.

Animals↗
PubMed1993

Different organization of the tRNA-gene-associated repetitive element, DRE, in NC4-derived strains and in other wild-type Dictyostelium discoideum strains.

The retrotransposon DRE (Dictyostelium repetitive element) was discovered in the course of an extensive study concerning the genomic organization of tRNA genes in the NC4-derived strains AX2 and AX3 of the cellular slime mold Dictyostelium discoideum. As a striking feature, DRE was found exclusively in a constant orientation and at a constant distance upstream from different tRNA genes. About 150-200 DRE with intact 5'-terminal-repeat structures are present in NC4-derived strains. These strains were termed high-copy DRE strains (HCD strains) as opposed to low-copy DRE strains (LCD strains) such as the wild-type D. discoideum isolates DD61, WS380B, OHIO and V12. LCD strains contain only 3-15 DRE with intact 5'-terminal-repeat-structures. However, in addition to these few intact elements, many 5'-truncated DRE elements are present in LCD strains. In HCD strains, most DRE show typical structural characteristics of retrotransposons containing terminal repeats at both ends, which seems to be one prerequisite for active transposition. In LCD strains, however, most DRE elements are 5'-truncated, which is a common feature of eukaryotic LINE elements. Despite their truncated 5'-ends, DRE in LCD strains retain unique integration specificities, i.e. they are always found position-specifically and orientation-specifically integrated in front of tRNA genes, flanked by a 12-16-bp target-site duplication.

Animals↗
PubMed1994

Characterization of a repetitive element detected by NheI in the genomes of Salmo species.

The genomes of the two species in the genus Salmo (Atlantic salmon, S. salar; brown trout, S. trutta) contain a 380-bp repetitive element that is flanked by the recognition sequence of the restriction enzyme NheI. These elements, which comprise approximately 1.2% of the salmon genome, do not exist in long tandem arrays as is typical of satellite DNA. A comparison of the sequences of 16 salmon and 7 trout elements revealed that members of this family of repetitive DNA are closely related to one another (over 95% identity). Subfamily structure exists and there is evidence that members of the same subfamilies are found in both Salmo species. A search of the GenBank database indicated that sequences homologous to the NheI repeat are located within a 1424-bp segment inserted immediately downstream of the 5' end of a Tc1 transposon-like sequence isolated from Atlantic salmon (A.R. Radice, B. Bugaj, D.H. Fitch, and S.W. Emmons, unpublished data; GenBank accession No. L12206).

Animals↗
PubMed1997

Fingerprinting of Ehrlichia species by repetitive element polymerase chain reaction.

To facilitate identification of ehrlichial pathogens, we developed a new technique based on fingerprints resulting from repetitive element polymerase chain reaction (rep-PCR). This technique uses consensus tRNA primers to generate amplification products that reflect distance polymorphisms between adjacent tRNA genes. Species-specific fingerprint patterns were obtained for seven Ehrlichia spp., as well as the unnamed causative agent of human granulocytotropic ehrlichiosis. Bands ranged in size from approximately 50 to 1,000 base pairs. Banding patterns varied depending on dilution of template DNA, with lower dilutions giving more complex banding patterns. These preliminary data indicate that repetitive-sequence-based PCR appears to be a useful technique for identifying ehrlichial organisms to the species, and perhaps the strain level. Compared with other conventional molecular-biologic methods, rep-PCR offers the advantages of ease of performance and rapid availability of results.

Bacterial Typing Techniques↗
PubMed1993

Molecular characterization of a short interspersed repetitive element from tobacco that exhibits sequence homology to specific tRNAs.

We have characterized a family of tRNA-derived short interspersed repetitive elements (SINEs) in the tobacco genome. Members of this family of SINEs, designated TS, have a composite structure and include a region structurally similar to a rabbit tRNA(Lys), a tRNA-unrelated region, and a TTG repeat of variable length at the 3' end. Southern blot hybridization, together with a search of the GenBank data base, showed that various plants belonging to the families Solanaceae and Convolvulaceae contain sequences homologous to the TS family in the introns and flanking regions of many genes, whereas Arabidopsis in the family Cruciferae and several species of monocoytledonous plants do not. The TS family is widely involved in structural and genetic variations in the genomes of many plants that belong to the order Tubiflorae. All of nine sequences identified in a data base search are truncated at their 5' regions and lack the tRNA-related region of the TS family. We characterized the entire sequence of the members of the TS family and found that this family can be categorized as a member of a group of SINEs with a tRNA(Lys)-like structure, as can several animal SINEs. The TS family can be divided into two major subfamilies by analysis of diagnostic positions, and one of the subfamilies is clearly younger than the other. Amplification of many copies of the full sequence of the younger subfamily occurred during the recent evolution of the tobacco lineage. We also discuss mechanisms that could be involved in the generation of SINEs in animals and also in plants.

Base Sequence↗