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Results for “DNA Transposable Elements”

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PubMed1996

The simian virus 40 packaging signal ses is composed of redundant DNA elements which are partly interchangeable.

Using the experimental system of simian virus 40 (SV40) pseudovirions we have previously shown that SV40 requires a specific DNA element for packaging, ses, which was mapped to the SV40 regulatory region. ses was previously found to play a role in facilitating the nucleosomal rearrangement required for chromatin condensation and viral packaging. Here, the fine structure of ses was investigated by genetic studies. Analyses of ses+ revertants indicated that in order to function, ses must be present in close proximity to the origin of replication (ori), supporting a role in the regulation of the viral life cycle. Fine dissection of ses was performed using a series of plasmids carrying mutations and deletions in this region. The results suggest that multiple DNA elements participate in the SV40 packaging process, including the GC-boxes and elements derived from the enhancer. The elements are redundant, and they can function in various combinations. Packaging efficiency correlated with the number of GC-boxes, known to bind Sp1. In addition, AP-2 binding elements appeared to more important than others. These findings were supported by experiments which showed that packaging was significantly enhanced by adding AP-2 binding sites to plasmids with large deletions and lacking those sites. The results imply that binding of Sp1 and/or AP-2 may participate in the packaging process.

Animals↗

PubMed1981

Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats.

We describe three related DNA alterations associated with transposon Tn10: precise excision of Tn10, nearly precise excision of Tn10 and precise excision of the nearly precise excision remnant. DNA sequence analysis shows that each of these alterations results in excision of all or part of the Tn10 element, and each involves specific repeat sequences at or near the ends of the element. Furthermore, all three events are structurally analogous: in each case, excision occurs between two short direct-repeat sequences, with resulting deletion of all intervening material plus one copy of the direct repeat; and in all three cases, the direct repeats involved occur at either end of an inverted repeat. Analysis of mutant Tn10 elements and characterization of bacterial host mutations suggest that all three types of excision events occur by pathways that are fundamentally distinct from the pathway(s) for Tn10-promoted transposition and other DNA rearrangements (deletions and inversions) actively promoted by the element. In addition, precise excision and nearly precise excision appear to occur by very closely related or identical pathways; and several lines of evidence suggest that the 1400 bp inverted repeats at the ends of Tn10 may play a structural role in both of these events. The third excision event appears to occur by yet another pathway.

Base Sequence↗

PubMed1988

A novel vector allowing the expression of genes in a wide range of gram-negative bacteria.

The construction and use of a novel vector allowing the expression of genes in a wide range of Gram-negative bacteria is described. The vector utilizes the regulatory region from IS50. The 70-bp promoter region was isolated from one of the terminal inverted repeats of Tn5 by creating EcoRI and Sa/I or PstI restriction sites by in vitro mutagenesis. This 70-bp region was shown to direct the expression of cat and lacZ genes in different bacterial genera including Alcaligenes, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas stutzeri, Pseudomonas fluorescens, and Serratia marcescens. Different strains containing the cat gene behind the regulatory elements of IS50 were able to tolerate high concentrations (300 micrograms/ml) of chloramphenicol in the medium. The 70-bp promoter region was cloned into a broad-host-range plasmid behind multiple cloning sites to create pAV10, which has unique restriction sites for BamHI, KpnI, SstI, and XbaI. Genes cloned into pAV10 can be expressed in a variety of Gram-negative bacteria.

DNA Transposable Elements↗

PubMed2004

ISSag1 in streptococcal strains of human and animal origin.

The chromosomal region of Streptococcus agalactiae harboring the C5a peptidase and the lmb genes displays the structure of a composite transposon. Its presence in a streptococcal strain is associated with the origin of this strain from a human host. In S. agalactiae it is flanked by two copies of the insertion element ISSag2, and the nucleotide sequence for a third IS element (ISSag1) can be found in this region. Based on amino acid sequence similarity of the deduced transposase ISSag1 belongs to the IS3 family. It is 1251 bp long and flanked by 37 bp imperfect inverted repeats. Horizontal gene transfer among different bacterial species is facilitated by mobile genetic elements. To investigate if ISSag1 homologues are also present in other streptococcal species, various species of pyogenic streptococci from animal and human origin were analyzed by Southern blot hybridization and PCR. Among the different streptococcal species, multiple copies of an ISSag1 homologue could only be detected in S. dysgalactiae subsp. dysgalactiae strains of animal origin. All of the S. agalactiae strains harbored only a single copy, that was always found in strains with the scpB-lmb composite transposon. A single copy of an ISSag1 homologue could also be detected in some of the S. pyogenes and S. dysgalactiae subsp. equisimilis strains. Nucleotide sequencing of the IS element in S. dysgalactiae subsp. dysgalactiae strains revealed several different variants. One of the variants showed the features of a regular IS3 element. The other two variants that were observed displayed a 500-bp deletion and a mosaic structure composed of ISSag1 and ISSag2 homologues. This mosaic structure suggests that recombination and horizontal gene transfer events in S. dysgalactiae strains of bovine origin could have played a role in the assembly of the scpB-lmb composite transposon structure.

Adhesins, Bacterial↗

PubMed2006

Insertion sequence ISEcp1-like element connected with a novel aph(2'') allele [aph(2'')-Ie] conferring high-level gentamicin resistance and a novel streptomycin adenylyltransferase gene in Enterococcus.

Enterococcus casseliflavus HZ95 is an enterococcus with high-level resistance to aminoglycosides. Nine genes responsible for high-level aminoglycoside resistance, including aac(6')-Ie-aph(2'')-Ia, aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id, aph(3')-IIIa, aac(6')-Ii, ant(3')-Ia, ant(4')-Ia and ant(6')-Ia, were not detected in HZ95. An 8 kb fragment from unconjugative plasmids of HZ95 was cloned, and expressed gentamicin resistance in Escherichia coli DH5alpha. The genetic structures ( approximately 8 kb DNA fragment) containing these aminoglycoside-modifying enzyme genes in Ent. casseliflavus HZ95 were determined. The deduced amino acid sequence of the novel aph(2'') allele, aph(2'')-Ie, had 93.7 % amino acid identity with APH(2'')-Id. The aph(2'')-Ie gene was bracketed upstream by an insertion sequence (IS)Ecp1-like element and downstream by a streptomycin adenylyltransferase gene (str). The streptomycin adenylyltransferase encoded by the str gene had 80.3 % amino acid identity with the protein encoded by aadE. The plasmid of approximately 16 kb could hybridize with a PCR-generated aph(2'')-Ie intragenic probe. The ISEcp1-like element had 91 % identity with ISEcp1. ISEcp1, which commonly acts as a key factor in the dissemination of CTX-M-type beta-lactamase genes in Gram-negative bacteria, has not been reported in Enterococcus.

Alleles↗

PubMed1998

Prevalence and distribution of the hmw and hia genes and the HMW and Hia adhesins among genetically diverse strains of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. In previous work we identified high-molecular-weight adhesins referred to as HMW1 and HMW2, expressed by nontypeable strain 12, and determined that most strains of nontypeable H. influenzae express one or two antigenically related proteins. More recently, we determined that some strains lack HMW1- and HMW2-like proteins and instead express an adhesin called Hia. In the present study, we determined the prevalence and distribution of the hmw and hia genes in a collection of 59 nontypeable strains previously characterized in terms of genetic relatedness. Based on Southern analysis, 47 strains contained sequences homologous to the hmw1 and hmw2 genes and nine strains contained homologs to hia. No strain harbored both hmw and hia, and three strains harbored neither. Although the hmw and hia genes failed to define distinct genetic divisions, the hmw-deficient strains formed small clusters or lineages within the larger population structure. Additional analysis established that the IS1016 insertion element was uniformly absent from strains containing hmw sequences but was present in two-thirds of the hmw-deficient strains. As IS1016 is associated with the capsule locus (cap) in most encapsulated strains of H. influenzae, we speculate that hmw-deficient nontypeable strains evolved more recently from an encapsulated ancestor.

Adhesins, Bacterial↗

PubMed1987

Effect of irradiation and mutagenic chemicals on the generation of ADH2-constitutive mutants in yeast. Significance for the inducibility of Ty transposition.

Mutations caused by the insertion of a Ty element resulting in an antimycin-A-resistant phenotype in an adh1- strain of Saccharomyces cerevisiae were used as an assay for the quantitative detection of Ty transposition. Antimycin-A-resistant mutants were found to be inducible by ethyl methanesulfonate (EMS) as well as by gamma- and UV irradiation. DNA analysis of gamma-induced mutants showed an increase of the fraction of Ty insertions in the ADH2 locus with increasing dose.

Alcohol Dehydrogenase↗

PubMed2006

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses.

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we speculate that Mavericks represent an evolutionary missing link between seemingly disparate invasive DNA elements that include bacteriophages, adenoviruses and eukaryotic linear plasmids.

Amino Acid Sequence↗

PubMed1997

Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells.

Members of the Tc1/mariner superfamily of transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to construct a synthetic transposon, Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations. Sleeping Beauty transposase binds to the inverted repeats of salmonid transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells. Sleeping Beauty is an active DNA-transposon system from vertebrates for genetic transformation and insertional mutagenesis.

Amino Acid Sequence↗

PubMed1996

[Endogenous retroviral sequences as a factor in the pathogenesis of systemic lupus erythematosus].

Endogenous retroviral sequences (ERV) are integrated parts of the human genome. They make up at least 1% of the total genomic DNA. This pool of genetic material might help explain the long discussed role of retroviruses in autoimmune disease. Their proviral features suggest two possible models leading to autoimmune disease: the mobile insertion into a or near a somatic gene, changing its function, and the expression of proteins by ERV, which then might act as autoantigens or superantigens. These mechanisms are supported by prior studies of systemic lupus erythematosus (SLE). In MRL-lpr/lpr mice with SLE-like disease the insertion of a mobile retroviral element, the early transposon (ETn), into the second intron of the fas gene leads to reduced apoptosis, accumulation of lymphocytes and earlier mortality. Investigations of murine and human SLE demonstrate autoantibodies against self-proteins, which crossreact with retroviral proteins. Future investigations may further establish the interrelation between the activation of endogenous retroviral sequences and SLE with its multifactorial genetic determinants.

Animals↗

PubMed1994

Identification and nucleotide sequence analysis of a transfer-related region in the streptococcal conjugative transposon Tn5252.

To obtain a functional map of Tn5252, a 47.5-kb streptococcal conjugative transposon, a series of defined deletion and insertion mutations were introduced within the transposon. Interruptions at several regions were found to affect the conjugal transposition functions of the element in filter-mating experiments. The nucleotide sequence of the left terminus of Tn5252 showed two open reading frames, ORF1 and ORF2, adjoining the att site. The organization of this region and the structure of the predicted integrase encoded by ORF1 were found to be similar to those of other site-specific recombination systems.

Amino Acid Sequence↗

PubMed2000

Isolation of Tn916-like conjugal elements from swine lot effluent.

Isolates of Enterococcus faecalis obtained from a swine farrowing house outflow were examined for genetic elements similar to Tn916. Of the enterococci isolated, 71% were resistant to tetracycline. Among the tetracycline-resistant enterococci isolated from the outflow samples, approximately 34% were able to transfer the tetracycline resistance phenotype to Bacillus thuringiensis in cross-genus matings. The frequencies of transfer for 10 random isolates were comparable to those for transfer of Tn916 from E. faecalis to B. thuringiensis. In addition, these elements were shown to mobilize plasmid pC194 between Bacillus species, as did Tn916. Southern blot and polymerase chain reaction (PCR) analysis showed these elements share extensive structural homology with Tn916. The selected conjugal elements were capable of transfer to a Bacillus recipient in a soil environment. When the swine waste was introduced into the soil, the tetracycline resistant fecal enterococci levels rose from essentially undetectable levels to approximately 4 x 10(4) and remained at this level for 4 weeks. After six months, including one winter, levels had decreased to 5 x 10(3).

Animals↗

PubMed2002

Sequence rearrangement in the AT-rich minisatellite of the novel rice transposable element Basho.

In the process of characterizing a rice wx deletion mutant, an AT-rich minisatellite sequence that consisted of units of approximately 80 bp was detected about 2.3 kb downstream of the wx gene. This AT-rich minisatellite was a multiple-copy element (1 x 10(3) to 2 x 10(3) copies per haploid genome) and interspersed in the rice genome. By BLAST homology search it was indicated that not only the tandem repeat but also both flanking sequences were conserved among copies. According to the characteristics of the termini (5'-CHH ... CTAG-3') and a target site preference for T, this AT-rich minisatellite accompanying the flanking sequences was classified into a novel transposon, Basho. The results of direct amplification of Basho showed that relatively large variation in size existed in the Basho family. We estimate the variation to be generated by not only alteration of the number of units in the minisatellite but also by duplications of larger blocks including the conserved flanking sequences caused by single-strand mispairing (SSM) at noncontiguous repeats. Because the AT-rich minisatellite contained in Basho possessed several motifs of the matrix attachment region (MAR) in its repeat unit, the functional role as MAR in the rice genome was discussed.

AT Rich Sequence↗

PubMed1999

A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites.

To construct large-insert libraries for the sequencing, mapping, and functional studies of complex genomes, we have constructed a new modular bacterial artificial chromosome (BAC) vector, pBACe3.6 (GenBank Accession No. U80929). This vector contains multiple cloning sites located within the sacB gene, allowing positive selection for recombinant clones on sucrose-containing medium. A recognition site for the PI-SceI nuclease has also been included, which permits linearization of recombinant DNA irrespective of the characteristics of the insert sequences. An attTn7 sequence present in pBACe3.6 permits retrofitting of BAC clones by Tn7-mediated insertion of desirable sequence elements into the vector portion. The ability to retrofit BAC clones will be useful for functional analysis of genes carried on the cloned inserts. The pBACe3.6 vector has been used for the construction of many genomic libraries currently serving as resources for large-scale mapping and sequencing.

Binding Sites↗

PubMed1996

An insertion element prevents phycobilisome synthesis in N2-fixing Synechocystis sp. strain BO 8402.

The unicellular diazotrophic cyanobacterium Synechocystis sp. strain BO 8402, isolated from Lake Constance, contains a novel insertion sequence, IS8402, in the apcA gene encoding a pigmented protein of phycobilisomes. IS8402 comprises 1,322 bp, flanked by two inverted repeats of 15 bp. Upon insertion in the target DNA, direct duplications of 8 nucleotides were generated. One open reading frame, potentially coding for a protein of 399 amino acids, was found. The deduced amino acid sequence shows homology to putative transposases of the IS4 family. Precise excision of the insertion element resulted in a spontaneous revertant, Synechocystis sp. strain BO 9201, that had regained the ability to form hemidiscoidal phycobilisomes. Apart from the unique insertion of IS8402 into apcA in strain BO 8402 both strains contain at least 12 further homologous insertion elements at corresponding sites in the genomes. The unique insertion in strain BO 8402 prevents the expression of apcABC operon and hence abolishes the formation of intact phycobilisomes. This decreases the quantum efficiency of photosystem II and promotes anaerobic N2 fixation in a unicellular cyanobacterium with a highly oxygen-sensitive nitrogenase.

Amino Acid Sequence↗

PubMed2004

Composition and structure of the centromeric region of rice chromosome 8.

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)-related sequences; together, these account for approximately 60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.

Base Composition↗

PubMed1994

Tad1-1, an active LINE-like element of Neurospora crassa.

Tad is a LINE-like retrotransposon of Neurospora crassa. The element was originally detected and cloned using the am gene as a transposon trap in hybrid strains derived from a cross of Adiopodoume (a wild collected strain) and a laboratory strain devoid of Tad elements. We report the cloning and sequencing of an active Tad element, Tad1-1, which is capable of independent transposition. Transposition was demonstrated by screening for transfer of the element from a donor nucleus that contained the Tad1-1 element as the only active Tad, into a naive nucleus within a forced heterokaryon. We also report here the sequence analysis of Tad1-1, and its comparison with the sequence of another active element, Tad3-2. These elements are approximately 7 kb in length. They contain two long open reading frames (ORFs) encoded on the strand of the same polarity as the full-length transcript. ORF1 encodes a putative protein of 486 amino acids. Homology to the first ORF of other LINE elements is confined to three cysteine-rich motifs, located near the carboxy-terminus, that are thought to be involved in binding nucleic acids. The second ORF is 1156 amino acids in length and shows homology to the reverse transcriptase domains of various retroviruses and retrotransposons. Tad1-1 and Tad3-2 differ in only ten positions over their whole length.

Base Sequence↗

PubMed1998

Evidence for circular transposition derivatives from the fungal hAT-transposon Restless.

The structure and function of eukaryotic hAT-transposons has already been elucidated; however, their transposition mechanism is barely understood. We recently have discovered Restless, a fungal member of the hAT-transposon family, which shows transposition in its host Tolypocladium inflatum. Investigations of two strains from T. inflatum, carrying either about 15 copies or only a single copy of the Restless element, indicate the presence of circular transposition intermediates. Using PCR technology, amplicons were identified which carry the joined end of the Restless transposon fused at its inverted repeats. All of eight sequenced PCR fragments contained the intact transposon ends with a short insertion of 1-93 bp of genomic DNA. Remarkably, one of the discovered genomic sequences matches a previously described integration site. Our data are discussed with respect to the transposition mechanism and the horizontal transfer of hAT-transposons.

DNA Transposable Elements↗