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Results for “DNA Transposable Elements”

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PubMed1989

Intramolecular transposition by Tn10.

Transposon Tn10 promotes the formation of a circular product containing only transposon sequences. We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair. The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends. The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins. We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions.

Base Sequence↗

PubMed1983

The analysis of some new Drosophila repetitive DNA sequences isolated and cloned from two-dimensional agarose gels.

Drosophila melanogaster DNA (Dm) was sequentially cleaved by BamHI and EcoRI and separated by two-dimensional gel electrophoresis. Six different prominent bands, which are derived primarily from the cleavage of long sequences that are repeated 20-100 times per genome, were recovered from the gel and cloned in pBR322. Hybridization and restriction analysis of the cloned Dm segments showed that three of these bands are mainly derived from the ribosomal and histone gene repeating units. Segments cloned from the other three bands are not homologous to any known repeating elements that we have tested. They represent long repetitive sequences of moderate multiplicity that appear not to have been hitherto described. These segments have been restriction-mapped and hybridized to cDNA prepared from poly(A)RNA from adult flies. While two minority segments did hybridize to this probe, the majority failed to hybridize. The arrangement of genomic sequences homologous to each plasmid was tested by restriction analysis and Southern hybridization. The results indicate that the repetitive element is largely conserved intact although occupying numerous different positions in the genome. The DNAs from four different strains of D. melanogaster and two of D. simulans produced restriction patterns having some segment lengths in common and some showing clear differences, a fact that indicates that these sequences can move about to occupy different genomic locations in different strains.

Animals↗

PubMed1994

Distribution of the catabolic transposon Tn5271 in a groundwater bioremediation system.

The distribution of Tn5271-related DNA sequences in samples of groundwater and a groundwater bioremediation system at the Hyde Park (Niagara Falls, N.Y.) chemical landfill site was investigated. PCR amplification of target sequences within the cha genes of Tn5271 revealed similar sequences in the groundwater community and in samples from the sequencing batch reactors treating that groundwater. Cell dilution combined with PCR amplification indicated that cha sequences were carried in about 1 of 10 culturable bacteria from the treatment system. Characterization of isolates involved in chlorobenzoate and toluene biodegradation in the treatment system indicated that two phenotypic clusters, Alcaligenes faecalis type 2 and CDC group IVC-2, contained all of the Tn5271 probe-positive isolates from the community. These two groups differed phenotypically from recipient groups isolated following horizontal transfer of pBRC60 (Tn5271) in pristine freshwater microcosms. A genetic rearrangement in Tn5271 attributable to the intramolecular transposition of the flanking element IS1071R was detected in an isolate from the treatment system. Comparison of the structure of the intramolecular transposition derivative from groundwater isolate OCC13(pBRC13) with a laboratory-derived intramolecular transposition derivative of pBRC60 revealed similarities. The rearrangement was shown to increase the stability of the plasmid under starvation conditions.

Alcaligenes↗

PubMed1982

Changes in gene order and gene expression.

Studies on changes in gene position in germ line and somatic cell chromosomes during evolution and differentiation have led biologists to abandon the static view of chromosomes as invariant linear arrays of hereditary information. Prokaryotic cells contain several classes of DNA insertion elements which move from place to place in the genome and mediate chromosome rearrangements. Similar elements exist in a wide variety of eukaryotic organisms (yeasts, insects, plants, and vertebrates). In addition, both reversible and irreversible changes of chromosome primary structure provide developmental controls on gene activity in bacteria, bacteriophages, yeasts, trypanosomes, and mammalian lymphocytes. At least five recombination mechanisms are known to catalyze chromosome changes: 1) general homologous, 2) site-specific reciprocal, 3) illegitimate, 4) DNA splicing, and 5) replicative. Various combinations of these mechanisms can explain many different chromosome rearrangements and changes in gene dosage. Changes in gene position can alter gene expression in many ways, some of which we understand (such as insertional mutation and inversion of coding and regulatory sequences) and some of which are still unexplained. The activities of DNA insertion elements and somatic rearrangement systems are subjects to controls at several levels by specific regulatory systems, natural selection, and connection to cell lineage. Despite the recent increase in knowledge about the biological importance of changes in gene order on chromosomes, there are far more questions than answers, particularly about the mechanisms that coordinate recombination events and cell division in higher organisms.

Animals↗

PubMedDate not supplied

[Phage-plasmid interactions in bacteria].

A review is devoted to consideration of the basic types of interaction of moderate and virulent bacteriophages and extrachromosomal elements in bacterial cells. Various experimental data confirm diversity of such interactions as well as resistance of bacteria to productive phage infection. Role of plasmids in such processes as abortion infection, lysogenic state, reparation of phage DNA as well as the effect of plasmids on the basic stages of phage development in the cells are analyzed. Possible mechanisms for limiting development of phages as a result of plasmids' presence in bacterial cells are considered.

Bacteria↗

PubMed1987

Interactions between the transposable element IS21 on R68.45 and TN7 in Pseudomonas aeruginosa PAO.

Tn7 transposes from the chromosome of Pseudomonas aeruginosa into the plasmid R68.45 with tandem IS21, at up to 400 times the frequency that it transposes into R68, which has only one copy of IS21. While R68::TN7 derivatives are stable, R68.45::Tn7 isolates undergo frequent deletions. Instability of R68.45 occurs whether Tn7 is inserted into the plasmid (cis configuration) or into the bacterial chromosome (trans configuration). The deletions of R68.45 start at the junction between the tandem IS21 copies and proceed clockwise, ending in the region of oriT. It appears that Tn7 and IS21 can mutually stimulate transposition of each other.

Chromosome Deletion↗

PubMed1986

DNA inversion in bacteriophage Mu: characterization of the inversion site.

Gin-mediated site-specific recombination promotes inversion of the G segment of phage Mu. The crossover takes place between two 34 bp-long inverted repeat sequences flanking the G segment. We have characterized the inversion site, the target for the site-specific recombination mechanism. An artificial invertible segment was constructed which consists of parts of the invertible segments of Mu and phage P1, which in this respect are largely homologous. Upon inversion of this hybrid segment the crossover site could be located, by DNA sequencing, in the ACCT sequence of the centre of symmetry in the inverted repeat in Mu. The hybrid Mu-P1 segment inverts at a lower frequency than its parental invertible segments probably because of the mismatches between the inverted repeats of Mu and P1. This suggests that base pairing between the inverted repeats is an intermediate step in recombination. Plasmids with subcloned G segments lacking the adjacent beta region of Mu or the corresponding region in P7, a relative of P1, are deficient in inversion. By analysis through site-specific mutagenesis of Mu DNA, an enhancer element with multiple recognition sites was identified which is necessary for efficient inversion. This component of the inversion site was located in a 170 bp segment within the Mu beta region, 30 bp to the right of the inverted repeat sequence, but can be separated from the crossover site by a 1200 bp insertion without losing its effect.

Bacteriophage mu↗

PubMed1999

Ac as a tool for the functional genomics of rice.

To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations. All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions. In nine of the 12 independent transpositions analyzed, their germinal transmission was detected. Partial sequencing of 99 Ac-flanking sequences revealed that 21 clones exhibited significant similarities with protein-coding genes in databases and four of them matched rice cDNA sequences. These results indicate preferential Ac transposition into protein-coding rice genes. To examine the feasibility of PCR-based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three-dimensional matrix. Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny. Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome.

Amino Acid Sequence↗

PubMed1991

Retroviral integrase domains: DNA binding and the recognition of LTR sequences.

Integration of retroviral DNA into the host chromosome requires a virus-encoded integrase (IN). IN recognizes, cuts and then joins specific viral DNA sequences (LTR ends) to essentially random sites in host DNA. We have used computer-assisted protein alignments and mutagenesis in an attempt to localize these functions within the avian retroviral IN protein. A comparison of the deduced amino acid sequences for 80 retroviral/retrotransposon IN proteins reveals strong conservation of an HHCC N-terminal 'Zn finger'-like domain, and a central D(35)E region which exhibits striking similarities with sequences deduced for bacterial IS elements. We demonstrate that the HHCC region is not required for DNA binding, but contributes to specific recognition of viral LTRs in the cutting and joining reactions. Deletions which extend into the D(35)E region destroy the ability of IN to bind DNA. Thus, we propose that the D(35)E region may specify a DNA-binding/cutting domain that is conserved throughout evolution in enzymes with similar functions.

Amino Acid Sequence↗

PubMed1991

Construction of an IS946-based composite transposon in Lactococcus lactis subsp. lactis.

An artificial composite transposon was constructed based on the lactococcal insertion sequence IS946. A 3.0-kb element composed of the pC194 cat gene (Cmr) flanked by inversely repeated copies of IS946 was assembled on pBluescript KS+. When subcloned into the shuttle vector pSA3 (Emr), two putative transposons were created on the recombinant plasmid pTRK128: the 3.0-kb Cmr element (Tn-CmA) and an inverse 11.5-kb Emr element (Tn-EmA). pTRK128 was electroporated into the recombination-deficient strain Lactococcus lactis MMS362, which contains the self-transmissible plasmid pRS01. An MMS362 Cmr Emr transformant was used to assay for transposition events via conjugal mobilization of pTRK128-encoded Cmr or Emr to L. lactis LM2345. Transfer of either marker alone occurred at frequencies of ca. 2 x 10(-4) per input donor. Approximately 19% of the Emr transconjugants were Cms, indicating loss of the cat gene marker. No Cmr Ems transconjugants were recovered (n = 550). Plasmid analysis showed that the Cms Emr isolates contained a single large plasmid that was determined to be a cointegrate between pRS01 and the Tn-EmA element. A 32P-labeled pSA3 probe hybridized specifically to pTRK128 sequences and revealed different junction fragments within each of the cointegrate plasmids. DNA sequence analysis of the Tn-EmA::pRS01 junctions from a representative cointegrate verified transposition by Tn-EmA. This represents the first example of a functional composite transposon in the genus Lactococcus and serves as an experimental tool and model for the genetic analyses of transposons in these organisms.

Base Sequence↗

PubMedDate not supplied

IS elements as constituents of bacterial genomes.

We provide here an overview of our present understanding of the distribution of different insertion sequences (ISs) within bacterial genomes (both chromosomes and plasmids). This is at present fragmentary and a significant effort is needed in the analysis of the increasing number of genomes whose sequence has been determined. We also consider some of the properties of ISs which are important in their role of assembling, reassorting, and transmitting groups of genes.

Bacteria↗

PubMed1989

Recombination mediates production of an extrachromosomal circular DNA containing a transposon-like human element, THE-1.

An abundant class of HeLa extrachromosomal circular DNA containing the transposon-like element, THE-1, is shown to arise via site specific recombination. The chromosomal locus from which these circles are derived, however, is single-copy. Northern blot analysis detects homology to two polyadenylated RNAs in HeLa cells. The possible presence of an origin of replication and its role in generating these small polydisperse circles is discussed.

Base Sequence↗

PubMed1995

Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination.

Chromosomal rearrangements are useful genetic and breeding tools but are often difficult to detect and characterize. To more easily identify and define chromosome deletions and inversions, we have used the bacteriophage P1 Cre-lox site-specific recombination system to generate these events in plants. This involves three steps: (i) the introduction of two lox sites into one locus in a plant genome, including one site within a modified Ds transposon; (ii) Ac transposase-mediated transposition of the Ds-lox element to a new locus on the same chromosome; (iii) Cre-mediated site-specific recombination between the two lox sites that bracket a chromosome segment. We report the production of a deletion and three inversion events in tobacco. The utility of chromosomal segments bracketed by lox sites for targeted manipulation and cloning is discussed.

Base Sequence↗

PubMed1997

The complete genome sequence of Escherichia coli K-12.

The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer.

Bacterial Proteins↗

PubMedDate not supplied

[Detection of intermediates of RNA reverse transcription for mobile dispersed MDG 1 and MDG 3 genes in Drosophila cells].

Poly(A)+RNA-DNA hybrids complementary to mobile dispersed genetic elements MDG 1 and MDG 3 have been isolated from Drosophila melanogaster culture cells. DNA and RNA in these complexes are represented by perfect hybrids. Three types of single-stranded DNA are detected in these hybrids: full-length DNA molecules of MDG containing one and two long terminal repeats (minus strand) and the long terminal repeat itself (plus strand). The results are consistent with the model of reverse transcriptional replication of MDG similar to that of retroviruses.

Animals↗

PubMed1992

The structure of an integrated copy of the giant linear plasmid SCP1 in the chromosome of Streptomyces coelicolor 2612.

In NF strain 2612 of Streptomyces coelicolor, a giant linear plasmid SCP1 is integrated into the chromosome at the 9 o'clock position. To characterize the integrated structure of SCP1, cloning and sequence analysis of the two junctions between the SCP1 DNA and the chromosomal DNA was carried out. The left junction was revealed to retain an almost intact left terminus of SCP1. On the other hand, the right junction was composed of IS466, deleting completely the right terminal inverted repeat of SCP1. This junction might have been formed by recombination of two IS466 elements, one present at the end of the right terminal inverted repeat of SCP1 and one on the chromosome. Based on these results, we have proposed a model for the integration of SCP1 into the chromosome. The unique conjugal transfer of NF strains and the origin of the chromosomal antibiotic biosynthetic genes in Streptomyces species are also discussed in relation to this model.

Base Sequence↗

PubMed1992

A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.

A new procedure is described to recombine plasmid-borne lacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flanking lacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncated lacZ and aadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carrying lacZ and aadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy in Pseudomonas putida the regulatory elements controlling expression of the XylS-activated Pm promoter of the TOL catabolic plasmid pWWO. We show here that the Pm promoter undergoes a XylS-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.

Blotting, Southern↗

PubMed1990

Molecular biology of Crohn's disease mycobacteria.

A Glasgow surgeon, T.K. Dalziel, published a detailed description of chronic enteritis in humans in 1913. He proposed that the disease was caused by the same organisms as those responsible for chronic enteritis, Johne's disease, in animals described a few years earlier (1895). Dalziel's dilemma was that he could see acid-fast bacilli in the diseased animal tissues but not in the diseased human tissues. Little real progress in the medical understanding of the causes of chronic enteritis in humans occurred over the next half a century or more. From 1978, a decade of research in many laboratories using improved methods for the culture of environmental mycobacteria showed that these could be grown in bacillary form from about one in five cases of Crohn's disease, from the same proportion of cases of ulcerative colitis, and from about one in ten control tissues. Spheroplasts were grown from two in five cases of Crohn's disease, one in five cases of ulcerative colitis, and rarely from control tissues. The nature of these agents was often uncertain. We describe work which began in 1985 and led rapidly to the identification of IS900, a DNA repetitive element in an uncharacterized Crohn's disease mycobacterial isolate. With other isolates, these were then shown by DNA fingerprinting to be indistinguishable from Mycobacterium paratuberculosis, Johne's bacillus. Similar techniques also demonstrated the wood-pigeon strain of M. avium in some Crohn's disease cultures. This bacillus can also cause chronic enteritis in calves. IS900 is the first of a family of unusual DNA insertion sequences which extend widely throughout environmental mycobacteria. Use of assays based on PCR amplification of highly specific DNA sequences from these insertional elements, and recombinant and synthetic peptides from their predicted proteins, will revolutionize the detection and characterization of these agents. These methods, applied to animal, human and environmental samples, will indicate new ways for the prevention and treatment of chronic enteritis, as well as other disorders associated with infections by environmental mycobacteria.

Actinomycetales↗