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PubMed2005

The Caenorhabditis briggsae genome contains active CbmaT1 and Tcb1 transposons.

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.

Amino Acid Sequence↗
PubMed2006

A hAT superfamily transposase recruited by the cereal grass genome.

Transposable elements are ubiquitous genomic parasites with an ancient history of coexistence with their hosts. A few cases have emerged recently where these genetic elements have been recruited for normal function in the host organism. We have identified an expressed hobo/Ac/Tam (hAT) family transposase-like gene in cereal grasses which appears to represent such a case. This gene, which we have called gary, is found in one or two copies in barley, two diverged copies in rice and two very similar copies in hexaploid wheat. No gary homologues are found in Arabidopsis. In all three cereal species, an apparently complete 2.5 kb transposase-like open reading frame is present and nucleotide substitution data show evidence for positive selection, yet the predicted gary protein is probably not an active transposase, as judged by the absence of key amino acids required for transposase function. Gary is expressed in wheat and barley spikes and gary cDNA sequences are also found in rice, oat, rye, maize, sorghum and sugarcane. The short inverted terminal repeats, flanked by an eight-nucleotide host sequence duplication, which are characteristic of a hAT transposon are absent. Genetic mapping in barley shows that gary is located on the distal end of the long arm of chromosome 2H. Wheat homologues of gary map to the same approximate location on the wheat group 2 chromosomes by physical bin-mapping and the more closely related of the two rice garys maps to the syntenic location near the bottom of rice chromosome 4. These data suggest that gary has resided in a single genomic location for at least 60 Myr and has lost the ability to transpose, yet expresses a transposase-related protein that is being conserved under host selection. We propose that the gary transposase-like gene has been recruited by the cereal grasses for an unknown function.

Amino Acid Sequence↗
PubMed2006

Master: a novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.).

Members of a novel Master family of class II transposons were identified in the carrot genome. Two elements, 2.5 kb long DcMaster1 and 4.4 kb long DcMaster-a, are characterized by 22 bp imperfect terminal inverted repeats and by 3 bp target site duplications. GenBank search revealed that related elements are also present in Medicago truncatula, including a 5.1 kb element MtMaster-a. Both DcMaster-a and MtMaster-a contain open reading frames encoding for putative transposases with the complete DDE domain typical for plant class II transposable elements belonging to PIF/Harbinger superfamily, where the Master elements form a distinct group. Less than 10 copies of the DcMaster element containing the DDE domain are present in genomes of carrot and other Apiaceae, but more copies with internal deletions or insertions may occur. DcMaster elements were associated with putative coding regions in 8 of 14 identified insertion sites. PCR amplification of carrot genomic DNA using a primer complementary to TIRs of DcMaster gave products < 400 bp in size. We speculate that these may all represent a MITE-like family of transposable elements that we named Krak, present in the carrot genome in at least 3,600 copies.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori.

piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species.

Amino Acid Sequence↗
PubMed2000

Phylogenetic analysis of the functional domains of mariner-like element (MLE) transposases.

We have analyzed the sequences of mariner-like element (MLE) transposases, in order to obtain a clearer picture of their phylogenetic relationships. In particular, we have considered their two known structural domains, as well as the nucleic acid sequences of the MLE inverted terminal repeats (ITR). The most consistent tree was obtained using sequences of the catalytic domain of the transposase. The trees obtained with the amino acid sequences of the ITR-binding domain and the ITR sequences themselves were similar to that obtained with the catalytic domain. However, a major difference indicated that the cecropia sub-family is divided into two sub-groups. These new trees were used to examine the evolutionary divergence of mariner-like transposable elements, with particular reference to the possibility that recombination events or gene conversions created mosaic elements during the evolution of transposons.

Amino Acid Sequence↗
PubMed2001

Integrated structures of the linear plasmid SCP1 in two bidirectional donor strains of Streptomyces coelicolor A3(2).

The linear plasmid SCP1 is integrated into the central region of the chromosome of Streptomyces coelicolor A3(2). The integrated structures of SCP1 in two bidirectional donor strains, 2612 and A634, were analyzed by cloning and sequencing of the junctions between the SCP1 DNA and the chromosomal DNA. In the NF (normal fertility) strain 2612, SCP1 is integrated in a right-handed direction into ORF-X at the left end of the IS cluster in AseI fragment E. An almost intact left end of SCP1 is retained, while the right terminal inverted repeat (TIR-R) of SCP1 and a 33-kb chromosomal DNA segment including the IS cluster are deleted. In the NF-like strain A634, SCPI is also integrated into AseI fragment E in a left-handed direction. The left junction is composed of IS466 with complete deletion of TIR-R of SCP1, and the right junction is located at the left end of IS468A* with half of TIR-L being deleted. During the integration event, a 5.4-kb chromosomal DNA segment including IS468A, IS468B, IS469 and IS466A was duplicated so that this sequence is now present on both sides of SCP1. Since 2612 and A634 exhibit a similar bidirectional gradient of gene transfer, it is surprising that their chromosomal structures are so different.

Base Sequence↗
PubMed2001

The ITR binding domain of the Mariner Mos-1 transposase.

Mariner-like elements are widespread eukaryotic transposons, but Mos-1 is the only natural element that is known to be active. Little is known about the biochemistry of mariner transposition. The first step in the process is the binding of the transposase to the 5' and 3' inverted terminal repeats (ITRs) of the element. Using the 3' ITR of the element, we have determined the binding properties of a recombinant Mos-1 transposase produced in bacteria, and we have used deletion derivatives to localize the minimal ITR binding domain between amino acids 1 and 141. Its features and structure indicate that it differs from the ITR binding domain of the transposase encoded by Tc1-related elements.

Amino Acid Motifs↗
PubMed1999

Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni.

The Lepidopteran transposable element piggyBac is being recognized as a useful vector for genetic engineering in a variety of insect species. This transposon can mediate transformation in the Dipteran species Ceratitis capitata, and can potentially serve as a versatile vector for transformation of a wide variety of insect species. Using a plasmid-based interplasmid transposition assay, we have demonstrated that this transposon, of the short inverted terminal repeat type, is capable of transposition in embryos of three different insect species, Drosophila melanogaster, the yellow fever mosquito Aedes aegypti, and its host of origin, Trichoplusia ni. This assay can confirm the potential utility of piggyBac as a gene transfer tool in a given insect species, and provides an experimental model for assessing molecular mechanisms of transposon movement.

Aedes↗
PubMed1997

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea.

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.

Amino Acid Sequence↗
PubMed1997

Isolation of the transposable element hupfer from the entomopathogenic fungus Beauveria bassiana by insertion mutagenesis of the nitrate reductase structural gene.

A transposable element has been isolated from the entomopathogenic fungus Beauveria bassiana by trapping it in the nitrate reductase structural gene, which has been cloned from this species. The element had inserted in the first exon of the nia gene and appeared to have duplicated the sequence TA at the site of insertion. It was 3336 bp long with 30-bp imperfect, inverted, terminal repeats. The element, called hupfer, contained an open reading frame encoding a 321-amino acid protein similar to the IS630- or mariner-Tcl-like transposases, and a residual sequence of about 2 kb which was not significantly similar to any published sequence. There are fewer than five copies of this transposable element present per genome in the fungus.

Amino Acid Sequence↗
PubMed1998

Tfo1: an Ac-like transposon from the plant pathogenic fungus Fusarium oxysporum.

A transposable element from a plant pathogenic fungus, Fusarium oxrysporum, was isolated and characterized. Four clones carrying moderately repetitive DNA were selected from a genomic library of the strain MAFF305118 of F. oxysporum f. sp. lagenariae, which causes wilt of bottle gourd. One the four clones was found to include a transposable element, which we have named Tfo1. This element is 2763 bp in size and appears to contain a long ORF that can encode a polypeptide of 777 amino acids. The amino acid sequence shows significant similarity to transposases from the hAT family of transposons, such as the maize transposon Activator (Ac). The element has 15-bp terminal inverted repeats and causes an 8-bp target site duplication upon insertion, as expected for an hAT-family transposon. Northern analysis detected a transcript, which hybridized to the putative transposase-encoding region of Tfo1. The size of this transcript (about 2.3 kb) corresponds to that of the ORF. A Southern analysis using pulsed-field gel electrophoresis showed that multiple chromosomal bands carry Tfo1 elements. PCR amplification of the Tfo1 elements with a 15-base inverted repeat primer produced a single DNA fragment of about 2.8 kb in all bottle gourd-infecting strains used. The element was found in multiple copies in the genome of all these strains and also in strains from other formae speciales tested. The sequence similarity of the Tfo1 element to other transposons, together with its transcriptional expression and genomic distribution, strongly suggests that Tfo1 is a member of the hAT transposon family.

Amino Acid Sequence↗
PubMed2001

Crypt1, an active Ac-like transposon from the chestnut blight fungus, Cryphonectria parasitica.

A moderately repetitive element was identified previously in the nuclear genome of the chestnut blight fungus, Cryphonectria parasitica, and has been used as a probe for population studies of the fungus. We report here that the repetitive element is a class II transposon of the hAT family of Activator (Ac)-like transposable elements. The element, named Crypt1, has a size of 3563 bp, including 21-bp terminal inverted repeats. A unique 8-bp direct repeat sequence flanking Crypt1 was identified in each of three clones examined. A single large ORF with the potential to encode a putative transposase of 946 amino acid residues was deduced from the sequence of Crypt1. Based on amino acid sequence alignments, Crypt1 is most closely related to other Ac-like transposons of filamentous ascomycetes. A single transcript of approximately 3.0 kb was identified by Northern hybridization experiments from Crypt1-containing isolates, suggesting that Crypt1 is an active element. An isolate containing a single, possibly defective, copy of Crypt1 was identified in C. parasitica isolates from China; no Crypt1 transcript was identified in this isolate. Transposition of Crypt1 was inferred from Southern and inverse PCR analyses of C. parasitica isolates maintained in the laboratory, but transposition appears to be a rare event.

Amino Acid Sequence↗
PubMed2003

Sequence mapping of the Californian MSW strain of Myxoma virus.

Partial sequence mapping of the MSW Californian strain of Myxoma virus was performed by cloning EcoRI and SalI restriction fragments of viral DNA and sequencing the ends of these. In this way, regions of 74 MSW open reading frames were sequenced and mapped onto the complete genome sequences of the related leporipoxviruses South American Myxoma virus and Rabbit fibroma virus to form a partial map of the MSW strain. In general, gene locations and sequences were conserved between the three viruses. However the Californian Myxoma virus was more closely related to South American myxoma virus than to Rabbit fibroma virus based on sequence comparisons and the presence of three genes that have been lost from the Rabbit fibroma virus genome. Compared to the other two viruses, the main difference found in the MSW genome was that the terminal inverted repeats were extended with the duplication of 5 complete open reading frames (M151R, M152R, M153R, M154L, M156R) and partial duplication of one open reading frame (M150R). This rearrangement was associated with the loss of the majority of the M009L open reading frame. Three known virulence genes, including the serine proteinase inhibitor (SERPIN) genes M151R and M152R and leukemia associated protein (LAP) gene M153R, and the potential virulence gene M156R are now present in two copies.

Animals↗
PubMed2006

Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains.

In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630-Tc1-mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning.

Amino Acid Sequence↗
PubMed2006

The N-terminus of Himar1 mariner transposase mediates multiple activities during transposition.

Mariner family transposons are perhaps the most widespread transposable elements of eukaryotes. While we are beginning to understand the precise mechanism of transposition of these elements, the structure of their transposases are still poorly understood. We undertook an extensive mutagenesis of the N-terminal third of the transposase of the Himar1 mariner transposon to begin the process of determining the structure and evolution of mariner transposases. N and C-terminal deletion analyses localized the DNA binding domain of Himar1 transposase to the first 115 amino acids. Alanine scanning of 23 selected sites within this region uncovered mutations that not only affected DNA binding but DNA cleavage as well. The behavior of other mutations strongly suggested that the N-terminus is also involved in multimerization of the transposase on a single inverted terminal repeat and in paired ends complex formation which brings together the two ends of the transposon. Finally, two hyperactive mutations at conserved sites suggest that mariner transposases are under a pattern of stabilizing selection in nature with regard to how efficiently they mediate transposition, resulting in a population of "average" transposons.

Alanine↗
PubMed2007

First evidence of methylation in the genome of Drosophila willistoni.

DNA methylation has been studied abundantly in vertebrates and recent evidence confirms that this phenomenon could be disseminated among some invertebrates groups, including Drosophila species. In this paper, we used the Methylation-Sensitive Restriction Endonuclease (MSRE) technique and Southern blot with specific probes, to detect methylation in the Drosophila willistoni species. We found differential cleavage patterns between males and females that cannot be explained by Mendelian inheritance, pointing to a DNA methylation phenomenon different from the Drosophila melanogaster one. The sequencing of some of these bands showed that these fragments were formed by different DNA elements, among which rDNA. We also characterized the D. willitoni dDnmt2 sequence, through a Mega Blast search against the D. willistoni Trace Archive Database using the D. melanogaster dDnmt2 nucleotide sequence as query. The complete analysis of D. willistoni dDnmt2 sequence showed that its promoter region is larger, its dDnmt2 nucleotide sequence is 33% divergent from the D. melanogaster one, Inverted Terminal Repeats (ITRs) are absent and only the B isoform of the enzyme is produced. In contrast, ORF2 is more conserved. Comparing the D. willistoni and D. melanogaster dDnmt2 protein sequences, we found higher conservation in motifs from the large domain, responsible for the catalysis of methyl transfer, and great variability in the region that carries out the recognition of specific DNA sequences (TRD). Globally, our results reveal that methylation of the D. willistoni genome could be involved in a singular process of species-specific dosage compensation and that the DNA methylation in the Drosophila genus can have diverse functions. This could be related to the evolutionary history of each species and also to the acquisition time of the dDnmt2 gene.

Amino Acid Sequence↗
PubMed2006

High-frequency Ds remobilization over multiple generations in barley facilitates gene tagging in large genome cereals.

Transposable elements have certain advantages over other approaches for identifying and determining gene function in large genome cereals. Different strategies have been used to exploit the maize Activator/dissociation (Ac/Ds) transposon system for functional genomics in heterologous species. Either large numbers of independent Ds insertion lines or transposants (TNPs) are generated and screened phenotypically, or smaller numbers of TNPs are produced, Ds locations mapped and remobilized for localized gene targeting. It is imperative to characterize key features of the system in order to utilize the latter strategy, which is more feasible in large genome cereals like barley and wheat. In barley, we generated greater than 100 single-copy Ds TNPs and determined remobilization frequencies of primary, secondary, and tertiary TNPs with intact terminal inverted repeats (TIRs); frequencies ranged from 11.8 to 17.1%. In 16% of TNPs that had damaged TIRs no transposition was detected among progeny of crosses using those TNPs as parental lines. In half of the greater than 100 TNP lines, the nature of flanking sequences and status of the 11 bp TIRs and 8-bp direct repeats were determined. BLAST searches using a gene prediction program revealed that 86% of TNP flanking sequences matched either known or putative genes, indicating preferential Ds insertion into genic regions, critical in large genome species. Observed remobilization frequencies of primary, secondary, tertiary, and quaternary TNPs, coupled with the tendency for localized Ds transposition, validates a saturation mutagenesis approach using Ds to tag and characterize genes linked to Ds in large genome cereals like barley and wheat.

Base Sequence↗
PubMed2006

Characterization of the genome structure of Bombyx mori densovirus (China isolate).

The genome of Bombyx mori densovirus (China isolate), termed as BmDNV-3, is composed of two kinds of different single-stranded linear DNA molecules (VD1 and VD2). In this study, the viral DNA molecules were purified and cloned into pUC119 vector, and the complete nucleotide sequence was determined. Sequence analysis showed that VD1 genome consisted of 6,543 nts including inverted terminal repeats (ITRs) of 224 nts, and VD2 genome consisted of 6,022 nts including ITRs of 524 nts. Comparison of the complete genome sequence between BmDNV-3 and BmDNV-2 (Yamanashi isolate) showed an identity of 98.4% in VD1 and 97.7% in VD2, with a total number of 228 bp substitutions, 11 bp deletions and 3 bp insertions found in BmDNV-3. A single nucleotide "A" deletion at nt 1589 in BmDNV-3 caused a frame shift mutation and brought about a premature stop codon, thus dividing VD2 of BmDNV-3 into two ORFs (named VD2 ORF1a and VD2 ORF1b) within that region, while there was only one ORF (named VD2 ORF1) in the corresponding region of BmDNV-2 (Yamanashi isolate). Comparative polymorphisms of ORFs and ITR regions of the two viral genomes showed that highly variable regions were mainly located in VD1 ORF3, VD1 ORF4, VD2 ORF2, and ITRs of BmDNV-3. Northern blots analysis revealed that VD1 had 1.1 kb and 1.5 kb transcripts from the left half of its plus strand, and one transcript about 3.3 kb from the right half of its minus strand. Sequencing of 3' and 5' RACE products showed that the 1.1 kb transcript started at nt 290 and ended at nt 1437, the 1.5 kb transcript started at nt 1423 and ended at nt 2931, and the 3.3 kb transcript started at nt 6287 and ended at nt 2922. These results help us to further understand the variation between different DNV genera and its possible causes, providing clues for studying the evolutionary history of densoviruses.

Amino Acid Sequence↗