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PubMed1991

Tec2, a second transposon-like element demonstrating developmentally programmed excision in Euplotes crassus.

The analysis of a repetitive DNA interruption of the micronuclear precursor to a 0.85-kb macronuclear gene in the hypotrich Euplotes crassus has led to the identification of a second transposon-like element named Tec2. Two copies of this element, one inserted into the other, compose the interruption. The Tec2 element resembles the previously characterized Tec1 element in overall size, copy number, length, and extreme terminal sequence of its inverted repeats and in the apparent use of a 5'-TA-3' target site. In addition, extrachromosomal circular forms of Tec2 appear in DNA isolated from cells undergoing macronuclear development at the same time and with the same conformation as extrachromosomal circular forms of Tec1. These similarities suggest that the Tec1 and Tec2 elements may be under the same type of regulation during macronuclear development.

Animals↗
PubMed2002

Genomic comparisons among Escherichia coli strains B, K-12, and O157:H7 using IS elements as molecular markers.

BACKGROUND: Insertion Sequence (IS) elements are mobile genetic elements widely distributed among bacteria. Their activities cause mutations, promoting genetic diversity and sometimes adaptation. Previous studies have examined their copy number and distribution in Escherichia coli K-12 and natural isolates. Here, we map most of the IS elements in E. coli B and compare their locations with the published genomes of K-12 and O157:H7. RESULTS: The genomic locations of IS elements reveal numerous differences between B, K-12, and O157:H7. IS elements occur in hok-sok loci (homologous to plasmid stabilization systems) in both B and K-12, whereas these same loci lack IS elements in O157:H7. IS elements in B and K-12 are often found in locations corresponding to O157:H7-specific sequences, which suggests IS involvement in chromosomal rearrangements including the incorporation of foreign DNA. Some sequences specific to B are identified, as reported previously for O157:H7. The extent of nucleotide sequence divergence between B and K-12 is < 2% for most sequences adjacent to IS elements. By contrast, B and K-12 share only a few IS locations besides those in hok-sok loci. Several phenotypic features of B are explained by IS elements, including differential porin expression from K-12. CONCLUSIONS: These data reveal a high level of IS activity since E. coli B, K-12, and O157:H7 diverged from a common ancestor, including IS association with deletions and incorporation of horizontally acquired genes as well as transpositions. These findings indicate the important role of IS elements in genome plasticity and divergence.

Chromosome Mapping↗
PubMed1992

Ty4, a new retrotransposon from Saccharomyces cerevisiae, flanked by tau-elements.

We have isolated and sequenced a genomic clone from Saccharomyces cerevisiae that shows structural features of a novel retrotransposon, designated Ty4. The element is 6.2 kilobases in length, and its genetic organization of the deduced functional domains is similar to Ty1 and Ty2 and thus different from Ty3. In contrast to hitherto known Ty elements from yeast, Ty4 is flanked by long terminal tau-element repeats instead of delta or sigma sequences. Ty4 contains two overlapping open reading frames. The first open reading frame, TYA4, is 1230 base pairs long and encodes a protein with a motif found in the nucleic acid-binding gag-protein of retroviruses. The second 4395-base pair open reading frame, TYB4, encodes a polyprotein that has domains with significant homology to retroviral protease, integrase, reverse transcriptase, and RNase H, structurally arranged in that order. The deduced amino acid sequence shows the greatest similarity with Ty2 and Ty1. The overall identity of the deduced functional protein domains is 28% with Ty2, 25% with Ty1, 19% with copia from Drosophila, and 18% with Ty3. Examination of genomic DNA from several laboratory strains indicates that Ty4 is present in two to four copies. Ty4 mRNA is of low abundance as compared to other Ty retrotransposons. At the 3' end of Ty4, two "solo" delta-elements, a full length and an overlapping, truncated one, are associated.

Amino Acid Sequence↗
PubMed1984

Insertion element IS5 contains a third gene.

We have previously shown that IS5 contains two genes encoded on opposite DNA strands within the same stretch of DNA. Here we present evidence that a third gene and its promoter are present on IS5. The newly discovered gene, ins5C , is contained within the longest gene of IS5, ins5A , but encoded by the complementary DNA strand. The three genes comprise a total of 519 codons present on the 1195-bp element. The arrangement of these genes represents a coding structure of unprecedented compactness.

Codon↗
PubMedDate not supplied

Creation of ARS activity in yeast through iteration of non-functional sequences.

Replication origins in Saccharomyces cerevisiae have been identified through the cloning of autonomous replication sequence (ARS) elements that allow the extrachromosomal maintenance of plasmid molecules. ARS activity requires a close match to an 11 bp consensus sequence and A + T-rich flanking DNA. ARS elements with a wide range of capacities for promoting plasmid maintenance have been described. We determined the ARS activity of plasmids with inserts consisting of repetitions of a 64 bp 100% A + T sequence that has sequence similarities to known ARS elements. An insert with approximately four repeats did not yield transformants, but inserts with either eight or eleven repeats did. The cooperative production of ARS activity did not require a contiguous arrangement since a plasmid containing two inserts of four repeats each, separated by about 1 kb, was functional. Our results show that a change from non-function to function can be accomplished by the cumulative action of individually inactive sequences. We conclude that the probability of replication initiation is too low with only four repeats to allow plasmid maintenance, but the overall probability is increased by further sequence iteration to provide origin activity. We suggest that chromosomes may contain stretches with dispersed, weak origin elements, each undetected by the conventional ARS assay, that in sum provide origin function.

DNA Replication↗
PubMed1984

Inverted repeats in the DNA of Streptomyces plasmids pMG110 and pMG120.

Two cryptic plasmids pMG110 (10.5 kb) and pMG120 (14.5 kb) isolated from Streptomyces luteolutescens were cleaved by restriction endonucleases Bg/II, KpnI, and SalGI. A physical map was constructed for pMG110. After denaturation and intrastrand reannealing, two types of snap-back structures were identified by electron microscopy, differing in the size of the loop (type 1, 1 kb; type 2, 1.6 kb), whereas the stem of both structures was about 190 bp long. Stem-loop structures of similar size were also observed in pMG120. In rare cases, both types of elements were present on the same DNA molecule. The analysis of Bg/II- and KpnI-generated fragments allowed the localization of the elements at two alternative positions on the physical map of pMG110.

Bacterial Proteins↗
PubMed1980

Specificity in the formation of delta tra F-prime plasmids.

Twenty-three independent delta tra F-prime plasmids from three different Escherichia coli K-12 sublines were isolated from Hfr strains whose points of origin coincided with the IS3 element alpha 3 beta 3 or alpha 4 beta 4 in the lac-purE region of the E. coli chromosome. Electrophoretic analysis of plasmid deoxyribonucleic acid digested with EcoRI and hybridization analysis of plasmid deoxyribonucleic acid digested with BglII revealed that at least 14 of these plasmids were formed by processes involving specific bacterial and F loci. Two of the specific bacterial loci involved in delta tra F-prime formation were located at approximately 3.3 and 11.7 min on the E. coli chromosomal map. Two of the delta tra F-prime plasmids contained bacterial deoxyribonucleic acid with circularization endpoints that mapped very near the termini of the IS2 element that is normally located between lac and proC.

Chromosome Deletion↗
PubMed1993

Genome organization of repetitive elements in the rodent, Peromyscus leucopus.

To document the frequency and distribution of repetitive elements in Peromyscus leucopus, the white-footed mouse, a cosmid genomic library was examined. Two thousand thirteen randomly chosen recombinants, with an average insert size of 35 kb and representing 2.35% of the haploid genome of P. leucopus, were screened with probes representing microsatellites, tandem repeats, and transposable elements. Of the four dinucleotides, (GT)n was present in 87% of the clones, (CT)n was present in 59% of the clones, and (AT)n and (GC)n each was represented in our sample by a single clone (0.05%). (TCC)n was present in 8% of the clones. Of the tandem repeats, the 28S ribosomal probe and the (TTAGGG)n telomere probe were not represented in the library, whereas a heterochromatic fragment was present in 9% of the clones. A transposable element, mys, was estimated to occur in 4700 copies, whereas a long interspersed element (LINE) was estimated to occur in about 41,000 copies per haploid genome. LINE and mys occurred together in the same clones more frequently than expected on the basis of chance. Hybridizing the library to genomic DNA from P. leucopus, Reithrodontomys fulvescens, Mus musculus, and human produced general agreement between phylogenetic relatedness and intensity of hybridization. However, dinucleotide repeats appeared to account for a disproportionately high number of positive clones in the more distantly related taxa.

Animals↗
PubMed1997

Cloning and sequence analysis of a novel insertion element from plasmids harbored by the carbofuran-degrading bacterium, Sphingomonas sp. CFO6.

Sphingomonas sp. CFO6 (a member of the alpha group of Proteobacteria) was isolated from a Washington soil by enrichment on the insecticide carbofuran as a sole source of carbon and energy. This strain has been shown to harbor five plasmids, at least some of which are required for catabolism of carbofuran. Rearrangements, deletions, and loss of individual plasmids resulting in the loss of the carbofuran-degrading phenotype were observed following treatment with heat or introduction of Tn5. Several putative insertion sequence elements of different sizes were cloned from these plasmids by trapping in pUCD800, a positive selection vector for isolation of transposable elements. Three of the most common putative IS elements (designated IS1412, IS1487, and IS1488) in the clone library were of different sizes and cross-hybridize with each other. An element hybridizing with IS1412, IS1487, and IS1488 was mobilized during growth of CFO6 at 42 degrees C and inserted into one of CFO6's plasmids (pCFO4), corresponding to a deletion in the plasmid and a loss of catabolic function. IS1412 was completely sequenced and its sequence analyzed. IS1412 is 1656 bp in length and possesses terminal partially matched inverted repeats of unequal length (17 and 18 bp). In addition, IS1412 contains an open reading frame which encodes a putative transposase with significant homology to the putative transposases of IS1380 from Acetobacter pasteurianus, HRS1 from Bradyrhizobium japonicum, and IS1247 from Xanthobacter autotrophicus. These related IS elements form part of a family of common IS elements distributed among members of the alpha group of the Proteobacteria.

Amino Acid Sequence↗
PubMed1987

Chromosome- and plasmid-mediated gentamicin resistance in Staphylococcus aureus encoded by Tn4001.

DNA sequences corresponding to the 4.7-kb gentamicin, tobramycin and kanamycin resistance (GmrTmrKmr) transposon Tn4001 have been detected on a series of nine structurally-related plasmids that mediate this phenotype in Australian isolates of Staphylococcus aureus. Tn4001 sequences have also been demonstrated on the chromosomes of GmrTmrKmr isolates that do not possess these plasmids, and the exhibited diversity of chromosomal sites occupied by this element implies that Tn4001 has transposed to the chromosome on numerous occasions in vivo. These results suggest that the rapid emergence of nosocomial GmrTmrKmr S. aureus in the early 1980s may have been the result of the transposition of Tn4001 from a chromosomal site to a readily disseminated plasmid.

Chromosomes, Bacterial↗
PubMed2001

Elimination of active tad elements during the sexual phase of the Neurospora crassa life cycle.

Tad is an active LINE-like retrotransposon isolated from the Adiopodoumé strain of Neurospora crassa. Extensive analysis of other Neurospora strains has revealed no other strain with active Tad, but all strains tested have multiple copies of defective Tad elements. We have examined the ability of Tad to survive during the sexual cycle of Neurospora and find that active Tad is rapidly eliminated. The characteristics of this elimination suggest that the repeat-induced point mutation (RIP) mechanism was responsible. By the use of transformation to switch the mating type of the Adiopodoumé strain we concluded that this strain is not defective in the RIP process. Analysis of defective Tad elements isolated from a variety of strains indicates that the major difference between these elements and active Tad is due to the presence of a large number of G-C to A-T transition mutations. This would be expected if the changes were due primarily to the RIP process. Mapping of a selection of defective Tad elements reveals that they are present on all of the chromosomes; however, many of the elements are not widely shared among strains. This suggests that repeated introduction and elimination of Tad elements has occurred. Mechanisms that might be responsible for this repeated introduction are discussed.

Blotting, Southern↗
PubMed2002

Palindromic repetitive elements in the mitochondrial genome of Volvox.

Group I introns were found in the cob and cox I genes of Volvox carteri. These introns contain tandem arrays of short palindromic sequences that are related to each other. Inspection of other regions in the mtDNA revealed that similar palindromic repetitive sequences are dispersed in the non-protein coding regions of the mitochondrial genome. Analysis of the group I intron in the cob gene of another member of Volvocaceae, Volvox aureus, has shown that its sequence is highly homologous to its counterpart in V. carteri with the exception of a cluster of palindromic sequences not found in V. carteri. This indicates that the palindromic clusters were inserted into the introns after divergence of the two species, presumably due to frequent insertions of the palindromic elements during evolution of the Volvocaceae. Possible involvement of the palindromic repetitive elements in the molecular evolution of functional RNAs is discussed.

Base Sequence↗
PubMed2002

The ICESt1 element of Streptococcus thermophilus belongs to a large family of integrative and conjugative elements that exchange modules and change their specificity of integration.

The 34,734-bp element ICESt1 from Streptococcus thermophilus CNRZ368 is site-specifically integrated into the 3(') end of the gene fda. ICESt1 encodes integrative functions and putative transfer functions. Six proteins of the putative conjugative system of ICESt1 are related to those encoded by the conjugative transposon Tn916 from Enterococcus faecalis. A comparison of these proteins with those encoded by the complete or partial genome sequences of various low G+C bacteria including Bacillus subtilis, Clostridium difficile, E. faecalis, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus mutans revealed the presence of numerous putative site-specific integrative conjugative elements and/or conjugative transposons within these genomes. Sequence comparisons revealed that these elements possess a modular structure and that exchanges of unrelated or distantly related modules and genes have occurred between these elements, and also plasmids and prophages. These exchanges have probably led to modifications in the site specificity of integration of these elements. Therefore, a distinction between low specificity integrative conjugative elements (i.e., conjugative transposons) and site-specific integrative conjugative elements does not appear to be relevant. We propose to call all the conjugative elements that excise by site-specific recombination and integrate by recombination between a specific site of a circular intermediate and another site, "Integrative and Conjugative Elements" (ICEs), irrespective of the integration specificity.

Base Composition↗
PubMed1997

The Deinococcus radiodurans uvr A gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence element from deinobacteria.

Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC(S)) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS4 of Escherichia coli (Ec) and to a resolvase found in ISXc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS2621.

Adenosine Triphosphatases↗
PubMed2002

Identification and sequence of an unstable DNA element in the entomopathogenic bacteria Photorhabdus temperata strain K122.

AIMS: A search was conducted for a difference in genome composition between phenotypic variants of the insect pathogenic bacteria, Photorhabdus temperata. METHODS: An unstable 300 bp fragment of DNA was identified by amplified fragment length polymorphism (AFLP) analysis, which was not, however, associated with phenotypic variation. RESULTS: During prolonged culturing of the bacteria, one copy of the repeated fragment was deleted and a restriction site linked to one of the copies was lost or gained. The sequence did not show substantial identity to any in the database, but a 16-bp region was identical to part of the marR gene of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The work has implications for the understanding of genetic instability in this and other pathogenic species of bacteria. In addition, the complete unstable element may be useful as a genetic tool in Photorhabdus spp.

Animals↗