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

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 Anopheles stephensi Promoter-Trap: Augmenting Genome Annotation and Functional Genomics

The piggyBac transposon was modified to generate gene trap constructs, which were then incorporated into the genome of the Asian malaria vector, Anopheles stephensi and remobilized through genetic crosses using a piggyBac transposase expressing line. A total of 620 remobilization events were documented, and 73 were further characterized at the DNA level to identify patterns in insertion site preferences, remobilization frequencies, and remobilization patterns. Overall, the use of the tetameric AmCyan reporter as the fusion peptide displayed a preference for insertion into the 5′-end of transcripts. Notably 183 – 44882 bp upstream of the An. stephensi v1.0 ab initio gene models, which demonstrated that the promoter regions for the genes of An. stephensi are further upstream of the 5′-proximal regions of the genes in the ab inito models than may be otherwise predicted. RNA-Seq transcript coverage supported the insertion of the splice acceptor gene trap element into 5′-UTR introns for nearly half of all insertions identified. The use of a gene trap element that prefers insertion into the 5′-end of genes supports the use of this technology for the random generation of knock-out mutants, as well as the experimental confirmation of 5′-UTR introns in An. stephensi