Transpositionally active episomal hAT elements

<p>Abstract</p> <p>Background</p> <p><it>hAT </it>elements and V(D)J recombination may have evolved from a common ancestral transposable element system. Extrachromosomal, circular forms of transposable elements (referred to here as episomal forms) have been reported yet their biological significance remains unknown. V(D)J signal joints, which resemble episomal transposable elements, have been considered non-recombinogenic products of V(D)J recombination and a safe way to dispose of excised chromosomal sequences. V(D)J signal joints can, however, participate in recombination reactions and the purpose of this study was to determine if <it>hobo </it>and <it>Hermes </it>episomal elements are also recombinogenic.</p> <p>Results</p> <p>Up to 50% of <it>hobo/Hermes </it>episomes contained two intact, inverted-terminal repeats and 86% of these contained from 1-1000 bp of intercalary DNA. Episomal <it>hobo/Hermes </it>elements were recovered from <it>Musca domestica </it>(a natural host of <it>Hermes</it>), <it>Drosophila melanogaster </it>(a natural host of <it>hobo</it>) and transgenic <it>Drosophila melanogaster </it>and <it>Aedes aegypti </it>(with autonomous <it>Hermes </it>elements). Episomal <it>Hermes </it>elements were recovered from unfertilized eggs of <it>M. domestica </it>and <it>D. melanogaster </it>demonstrating their potential for extrachromosomal, maternal transmission. Reintegration of episomal <it>Hermes </it>elements was observed <it>in vitro </it>and <it>in vivo </it>and the presence of <it>Hermes </it>episomes resulted in lower rates of canonical <it>Hermes </it>transposition <it>in vivo</it>.</p> <p>Conclusion</p> <p>Episomal <it>hobo</it>/<it>Hermes </it>elements are common products of element excision and can be maternally transmitted. Episomal forms of <it>Hermes </it>are capable of integration and also of influencing the transposition of canonical elements suggesting biological roles for these extrachromosomal elements in element transmission and regulation.</p

DNA binding activities of the Herves transposase from the mosquito Anopheles gambiae

Determining the mechanisms by which transposable elements move within a genome increases our understanding of how they can shape genome evolution. Class 2 transposable elements transpose via a 'cut-and-paste' mechanism mediated by a transposase that binds to sites at or near the ends of the transposon. Herves is a member of the hAT superfamily of class 2 transposons and was isolated from Anopheles gambiae, a medically important mosquito species that is the major vector of malaria in sub-Saharan Africa. Herves is transpositionally active and intact copies of it are found in field populations of A gambiae. In this study we report the binding activities of the Herves transposase to the sequences at the ends of the Herves transposon and compare these to other sequences recognized by hAT transposases isolated from other organisms. We identified the specific DNA-binding sites of the Herves transposase. Active Herves transposase was purified using an Escherichia coli expression system and bound in a site-specific manner to the subterminal and terminal sequences of the left and right ends of the element, respectively, and also interacted with the right but not the left terminal inverted repeat. We identified a common subterminal DNA-binding motif (CG/AATTCAT) that is critical and sufficient for Herves transposase binding. The Herves transposase binds specifically to a short motif located at both ends of the transposon but shows differential binding with respect to the left and right terminal inverted repeats. Despite similarities in the overall structures of hAT transposases, the regions to which they bind in their respective transposons differ in sequence ensuring the specificity of these enzymes to their respective transposon. The asymmetry with which the Herves terminal inverted repeats are bound by the transposase may indicate that these differ in their interactions with the enzyme.https://doi.org/10.1186/1759-8753-2-

Targeting the X Chromosome during Spermatogenesis Induces Y Chromosome Transmission Ratio Distortion and Early Dominant Embryo Lethality in Anopheles gambiae

We have exploited the high selectivity of the homing endonuclease I-PpoI for the X-linked Anopheles gambiae 28S ribosomal genes to selectively target X chromosome carrying spermatozoa. Our data demonstrated that in heterozygous males, the expression of I-PpoI in the testes induced a strong bias toward Y chromosome–carrying spermatozoa. Notably, these male mosquitoes also induced complete early dominant embryo lethality in crosses with wild-type females. Morphological and molecular data indicated that all spermatozoa, irrespectively of the inheritance of the transgene, carried a substantial amount of I-PpoI protein that could attack the maternally inherited chromosome X of the embryo. Besides the obvious implications for implementing vector control measures, our data demonstrated the feasibility of generating synthetic sex distorters and revealed the intriguing possibility of manipulating maternally inherited genes using wild-type sperm cells carrying engineered endonucleases

Transposable Element Dynamics of the hAT Element Herves in the Human Malaria Vector Anopheles gambiae s.s.

Transposable elements are being considered as genetic drive agents for introducing phenotype-altering genes into populations of vectors of human disease. The dynamics of endogenous elements will assist in predicting the behavior of introduced elements. Transposable element display was used to estimate the site-occupancy frequency distribution of Herves in six populations of Anopheles gambiae s.s. The site-occupancy distribution data suggest that the element has been recently active within the sampled populations. All 218 individuals sampled contained at least one copy of Herves with a mean of 3.6 elements per diploid genome. No significant differences in copy number were observed among populations. Nucleotide polymorphism within the element was high (π = 0.0079 in noncoding sequences and 0.0046 in coding sequences) relative to that observed in some of the more well-studied elements in Drosophila melanogaster. In total, 33 distinct forms of Herves were found on the basis of the sequence of the first 528 bp of the transposase open reading frame. Only two forms were found in all six study populations. Although Herves elements in An. gambiae are quite diverse, 85% of the individuals examined had evidence of complete forms of the element. Evidence was found for the lateral transfer of Herves from an unknown source into the An. gambiae lineage prior to the diversification of the An. gambiae species complex. The characteristics of Herves in An. gambiae are somewhat unlike those of P elements in D. melanogaster

Intrinsic Characteristics of Neighboring DNA Modulate Transposable Element Activity in Drosophila melanogaster

Identifying factors influencing transposable element activity is essential for understanding how these elements impact genomes and their evolution as well as for fully exploiting them as functional genomics tools and gene-therapy vectors. Using a genetics-based approach, the influence of genomic position on piggyBac mobility in Drosophila melanogaster was assessed while controlling for element structure, genetic background, and transposase concentration. The mobility of piggyBac elements varied over more than two orders of magnitude solely as a result of their locations within the genome. The influence of genomic position on element activities was independent of factors resulting in position-dependent transgene expression (“position effects”). Elements could be relocated to new genomic locations without altering their activity if ≥500 bp of genomic DNA originally flanking the element was also relocated. Local intrinsic factors within the neighboring DNA that determined the activity of piggyBac elements were portable not only within the genome but also when elements were moved to plasmids. The predicted bendability of the first 50 bp flanking the 5′ and 3′ termini of piggyBac elements could account for 60% of the variance in position-dependent activity observed among elements. These results are significant because positional influences on transposable element activities will impact patterns of accumulation of elements within genomes. Manipulating and controlling the local sequence context of piggyBac elements could be a powerful, novel way of optimizing gene vector activity

An Active Transposable Element, Herves, From the African Malaria Mosquito Anopheles gambiae

Transposable elements have proven to be invaluable tools for genetically manipulating a wide variety of plants, animals, and microbes. Some have suggested that they could be used to spread desirable genes, such as refractoriness to Plasmodium infection, through target populations of Anopheles gambiae, thereby disabling the mosquito's ability to transmit malaria. To achieve this, a transposon must remain mobile and intact after the initial introduction into the genome. Endogenous, active class II transposable elements from An. gambiae have not been exploited as gene vectors/drivers because none have been isolated. We report the discovery of an active class II transposable element, Herves, from the mosquito An. gambiae. Herves is a member of a distinct subfamily of hAT elements that includes the hopper-we element from Bactrocera dorsalis and B. cucurbitae. Herves was transpositionally active in mobility assays performed in Drosophila melanogaster S2 cells and developing embryos and was used as a germ-line transformation vector in D. melanogaster. Herves displays an altered target-site preference from the distantly related hAT elements, Hermes and hobo. Herves is also present in An. arabiensis and An. merus with copy numbers similar to that found in An. gambiae. Preliminary data from an East African population are consistent with the element being transpositionally active in mosquitoes