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PubMed1982

Transposon-mediated site-specific recombination: identification of three binding sites for resolvase at the res sites of gamma delta and Tn3.

The tnpR gene product, resolvase, of the transposable element gamma delta mediates site-specific recombination between two copies of gamma delta directly repeated on the same replicon, and it negatively regulates transcription of the tnpA (transposase) gene and its own gene. The recombinational site, res, and the regulatory region both are located in the tnpA-tnpR intercistronic region. In studying the interaction of purified resolvase with DNA fragments derived from gamma delta and the related transposon, Tn3, that span this region, we have demonstrated that three sites specifically bind resolvase. Site I overlaps the recombinational crossover point and both transcriptional promoters. Sites II and III cover most of the DNA between the crossover point and the translational start of the tnpR gene. These are the only binding sites we have detected in a region of about 400 base pairs centered on the crossover point. Studies of cointegrates that contain only part of the region that binds resolvase indicate that site I is not sufficient for efficient site-specific recombination and suggest that all three sites are probably required.

Binding Sites↗
PubMed2005

The Genomes of Oryza sativa: a history of duplications.

We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000-40,000. Only 2%-3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.

Base Sequence↗
PubMed1979

Identification and characterization of a self-regulated repressor of translocation of the Tn3 element.

Gene fusions that bring expression of the lacZ gene under control of transcriptional and trnaslational signals within the transposable element Tn3 have been used to study regulation of Tn3-specified proteins. A gene encoding a 21,355-Mr peptide that represses translocation of Tn3 and acts at the level oquenced; amber, missense, and cis-dominant (operator-constitutive) point mutations in this gene have been isolated and characterized.

Bacterial Proteins↗
PubMed2005

I element distribution in mitotic heterochromatin of Drosophila melanogaster reactive strains: identification of a specific site which is correlated with the reactivity levels.

The I factor is a Drosophila melanogaster LINE-like element that efficiently transposes in the genetic system of I-R hybrid dysgenesis. It has been suggested that some of the I-related sequences located in the heterochromatin of D. melanogaster are involved in the regulation of I factor activity. In this work we have performed fluorescent in situ hybridization (FISH) mapping of I element sequences in mitotic heterochromatin of nine differentially reactive D. melanogaster strains. The results of our analysis showed that a single hybridization site mapping to region h28 of the distal heterochromatin of the X chromosome is present in three strains with low or intermediate levels of reactivity, while it is undetectable in six highly reactive strains. Together, these observations suggest a negative correlation between I sequences located at h28 and the level of reactivity. To this regard, it is intriguing that flamenco and COM, two loci that regulate the activity of D. melanogaster endogenous retroviruses also map to the distal heterochromatin of the X chromosome. Our data represent the first experimental evidence in favour of a silencing effect exerted by naturally occurring I element sequences located in pericentromeric heterochromatin.

Animals↗
PubMed2003

Vege and Mar: two novel hAT MITE families from Drosophila willistoni.

Two novel families of miniature inverted repeat transposable elements (MITEs), Vege and Mar, are described from Drosophila willistoni. Based on their structures, both element families are hypothesized to belong to the hAT superfamily of transposable elements. Both elements have perfect, inverted terminal repeats and 8-bp target site duplications and were found to have inserted within fixed copies of nonautonomous P elements. Vege is present in all studied D. willistoni populations and appears to have a relatively low copy number. Mar was identified in only a single D. willistoni population, and its copy number is presently unknown. Although MITEs occupy relatively large proportions of the genomes of a broad range of organisms, this may be their first unambiguous identification in any species of the genus Drosophila.

Animals↗
PubMed1993

Prospects of applying a combination of DNA transposition and site-specific recombination in plants: a strategy for gene identification and cloning.

The concept of gene identification and cloning using insertional mutagenesis is well established. Many genes have been isolated using T-DNA transformation or transposable elements. Maize transposable elements have been introduced into heterologous plant species for tagging experiments. The behaviour of these elements in heterologous hosts shows many similarities with transposon behaviour in Zea mays. Site-specific recombination systems from lower organisms have also been shown to function efficiently in plant cells. Combining transposon and site-specific recombination systems in plants would create the possibility to induce chromosomal deletions. This 'transposition-deletion' system could allow the screening of large segments of the genome for interesting genes and may also permit the cloning of the DNA corresponding to the deleted material by the same site-specific recombination reaction in vitro. This methodology may provide a unique means to construct libraries of large DNA clones derived from defined parts of the genome, the phenotypic contribution of which is displayed by the mutant carrying the deletion.

Chromosome Deletion↗
PubMed1987

Identification of base pairs in the outside end of insertion sequence IS50 that are needed for IS50 and Tn5 transposition.

Short DNA sequences at ends of transposable elements are needed as sites for transposition. Previous deletion mapping showed that, in Tn5 and its component IS50 elements, these essential sites are about 19 base pairs long. To determine which positions are important in transposition, we made one or more sequence changes at each position in the IS50 outside (O) end and assayed the effects of these changes on transposition. Our results indicate that the specific base pairs at 18 of the 19 positions are important in transposition. A 9-base-pair segment in the O end corresponds to a binding site for the Escherichia coli DnaA protein. Comparisons of effects of mutations at different positions in this site, and also measurements of Tn5 transposition in dnaA- cells, indicate that DnaA protein participates in O-end-mediated transposition.

Bacterial Proteins↗
PubMed2002

What's buzzing? Mosquito genomics and transgenic mosquitoes.

Genome projects and associated technologies are now being established for mosquito species that are vectors of human disease. The recent announcement of an award by the National Institute of Allergy and Infectious Diseases (NIAID) to Celera Genomics to sequence the Anopheles gambiae genome will further accelerate the completion of the sequencing of this genome. Completion of the An. gambiae sequence will mean that the genomes of all three organisms involved in the transmission of falciparum malaria--the mosquito, the parasite, and the human--will have been sequenced. This will greatly facilitate the identification of genes and pathways involved in the transmission of malaria. The recent genetic transformation of An. gambiae with the piggyBac transposable element and the transformation of another important malarial vector, Anopheles stephensi using the Minos element, now provide researchers with powerful tools with which to genetically manipulate these medically important vector species. Here we review the recent progress made in the extension of contemporary tools of modern genetics and genomics into these medically important insects.

Aedes↗
PubMed1998

Convergent transcription units and their promoters at both ends of pot2, an inverted repeat transposon from the rice blast fungus.

The rice blast fungus Pot2 is an inverted repeat transposon which shares structural features with the Tc1-mariner superfamily. In a search for the transposition regulatory mechanism of presently quiescent Pot2, we found truncated transcripts for the sense and antisense strands of the transposase (TPase) gene. Reverse transcription-PCR analysis identified the convergent overlapping transcription units that contain the entire coding region of the TPase gene. DNA fragments carrying presumed TATA box motifs at both ends of Pot2 proved to be functional as promoters in transient expression assays. Using these promoters on transformation vectors, stable transformants of the fungus were obtained at a frequency comparable to that with the established system. Furthermore, the antisense promoter transcribed a marker gene from one of the original sites on the chromosome. The above results indicate that the antisense RNA is transcribed by the antisense promoter at the 3' end of the element, which has implications for a possible regulatory mechanism. This is the first example of the identification of an antisense promoter within a class II transposon from a eukaryote.

Ascomycota↗
PubMed2005

Taf1: a class II transposon of Aspergillus fumigatus.

Aspergillus fumigatus is an important medical pathogen that lacks a known sexual cycle. Transposons may provide an important mechanism for the generation of genetic diversity in this organism. Here, we describe Taf1, the first class II transposon to be identified in A. fumigatus. Taf1, a member of the mariner superfamily and pogo family of transposons, is distinguished by the presence of extremely long (89 bp) inverted repeats that flank the transposase coding sequence. Taf1 is present in different locations and copy number among clinical strains of A. fumigatus and is transcribed. Analysis of multiple insertion sequences within a single strain suggests that Taf1 elements undergo inactivation by a repeat induced polymorphism-like mechanism. Taf1 insertion patterns were extremely stable despite multiple stressors including heat shock, serial passage, and infection in mice. Thus Taf1 may be useful for strain identification and molecular typing.

Amino Acid Sequence↗
PubMed2005

The map-based sequence of the rice genome.

Rice, one of the world's most important food plants, has important syntenic relationships with the other cereal species and is a model plant for the grasses. Here we present a map-based, finished quality sequence that covers 95% of the 389 Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 non-transposable-element-related protein-coding genes were identified, of which 71% had a putative homologue in Arabidopsis. In a reciprocal analysis, 90% of the Arabidopsis proteins had a putative homologue in the predicted rice proteome. Twenty-nine per cent of the 37,544 predicted genes appear in clustered gene families. The number and classes of transposable elements found in the rice genome are consistent with the expansion of syntenic regions in the maize and sorghum genomes. We find evidence for widespread and recurrent gene transfer from the organelles to the nuclear chromosomes. The map-based sequence has proven useful for the identification of genes underlying agronomic traits. The additional single-nucleotide polymorphisms and simple sequence repeats identified in our study should accelerate improvements in rice production.

Cell Nucleus↗
PubMed2004

Computational analysis of transposable element sequences.

This chapter provides a simple guide for the computational analysis of transposable element (TE) sequences. Web links are provided for a number of sequence analysis applications, and their potential use in the analysis of TE sequences is briefly described. The level of detail provided is intended to be sufficient for a naive user to begin to analyze TE sequences in silico. The emphasis is placed on the identification, retrieval and manipulation of TE sequences. Information is also provided on the evolutionary study of TE sequences including the use phylogenetics programs.

Animals↗
PubMed1987

Construction of hybrid Tn501/Tn21 transposases in vivo: identification of a region of transposase conferring specificity of recognition of the 38-bp terminal inverted repeats.

In order to study the transposase enzymes of Class II prokaryotic transposable elements, we have constructed genes encoding hybrid transposase proteins. This was done by recombination in vivo between the tnpA genes of transposons Tn501 and Tn21. These hybrid genes can complement in trans a transposition-defective mutant of Tn501. The structures of the products of this complementation indicate whether the specificity of the hybrid transposase in recognising the 38 bp terminal inverted repeats is that of Tn501 or that of Tn21. The determinant of this specificity is in the N-terminal region of the transposase protein, between amino acids 28 and 216. The predicted amino acid sequences so far determined of transposases from the Class II family reveal an area of homology in this region.

Amino Acid Sequence↗
PubMed2001

Mobility of the piggyBac transposon in embryos of the vectors of Dengue fever (Aedes albopictus) and La Crosse encephalitis (Ae. triseriatus).

The re-emergence of arboviral diseases such as Dengue Fever and La Crosse encephalitis is primarily due to the failure of insect vector control strategies. The development of a procedure capable of producing stable germ-line transformants in the insect vectors of these diseases would bridge the gap between gene expression systems being developed to curb vector transmission and the identification of important genes and regulatory sequences and their reintroduction back into the insect genome in the form of vector control strategies. The transposable element piggyBac is capable of transposition in a variety of insect species, and could serve as a versatile insect transformation vector. Using plasmid-based excision and transposition assays, we report that this short-ITR transposon undergoes precise, transposase-dependent excision and transposition in embryos of Aedes albopictus and Aedes triseriatus, the vectors of Dengue fever and LaCrosse encephalitis, respectively. These assays allow us easily and rapidly to confirm and assess the potential utility of piggyBac as a gene transfer tool in a given species. piggyBac is an exceptionally mobile and versatile genetic transformation vector, comparable to other transposons currently in use for the transformation of insects. The mobility of the piggyBac element seen in both Ae. albopictus and Ae. triseriatus is further evidence that it can be employed as a germ-line vector in important insect disease vectors.

Aedes↗
PubMed2004

Further evidence of microcolinearity between barley and rice genomes at two orthologous regions.

Two genetic markers, BCD135 and RZ567 were used to select clones from genomic BAC libraries of barley and rice for sequencing and subsequent sequence comparisons. A set of two orthologous BACs each from barley and rice was selected by hybridization with BCD135 and RZ567 cDNA probes. A total of 556-kb stretch including two barley BACs (773K135 and 745C13) and two orthologous rice BACs (24K23 and 49D11) was completely sequenced. Comparative sequence analysis between orthologous BACs from the two species revealed presence of two conserved genes at BCD135 region and only one gene at the RZ567 regions. The two conserved genes were in the same order and orientation in both the species however, separated by significantly larger distance in barley. The larger distance between two barley genes was mainly due to presence of different retrotransposable elements and their derivatives (78.9% of the intergenic region) that expanded the barley BCD135 region at the rate of 9.1X. An additional gene of unknown function was also inserted along with several retrotransposable elements between two conserved genes at barley BCD135 region. More genome expansion rate (10X) around barley RZ567 locus was estimated by extremely high proportion (> 70%) of retrotransposons. Among different retrotransposons, the Sabrina elements rather than BARE were more prevalent in both the regions. Contrary to it, the BCD135 region of rice was composed of only 17.1% retrotransposable elements and no significant retrotransposons except 14 miniature inverted transposable elements (MITEs) were observed in its RZ567 region. The sequence comparison between orthologous regions of rice and barley genomes was useful for gene identification and determination of individual gene structure indicating the possibility of effective utilization of rice genome sequences in understanding the large genome of barley. (The sequence data described in this paper have been submitted to the GenBank data library under the accession no. AF474072 (773K14), AF474071 (745C13), AF480497 (24K23) and AF480496 (49D11)).

Chromosomes, Artificial, Bacterial↗
PubMed1981

The nucleotide sequence and protein-coding capability of the transposable element IS5.

The nucleotide sequence of IS5, a bacterial insertion sequence, has been determined. It is 1195 bp long and contains an inverted terminal repetition of 16 bp with one mismatch. One open reading frame, spanning nearly the entire length of the element, could encode a polypeptide of 338 amino acids. Upon insertion into a DNA segment, IS5 causes a duplication of 4 bp. Based on seven examples, this site of insertion appears to be nonrandom, and the consensus target site sequence is C . T/A . A . G/A (or C/T . T . A/T . G on the opposite strand). The nucleotide sequences of IS5 insertions into the B and cim genes of bacteriophage Mu have allowed tentative identification of the protein-coding frames of B and cim.

Bacteriophage mu↗