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Mobile DNA

, 7:6

First Online: 31 March 2016Received: 07 January 2016Accepted: 15 March 2016

Abstract

BackgroundThe revolutionary concept of -jumping genes- was conceived by McClintock in the late 1940s while studying the Activator-Dissociation Ac-Ds system in maize. Transposable elements TEs represent the most abundant component of many eukaryotic genomes. Mobile elements are a driving force of eukaryotic genome evolution. McClintock’s Ac, the autonomous element of the Ac-Ds system, together with hobo from Drosophila and Tam3 from snapdragon define an ancient and diverse DNA transposon superfamily named hAT. Other members of the hAT superfamily include the insect element Hermes and Tol2 from medaka. In recent years, genetic tools derived from the ‘cut’ and ‘paste’ Tol2 DNA transposon have been widely used for genomic manipulation in zebrafish, mammals and in cells in vitro.

ResultsWe report the purification of a functional recombinant Tol2 protein from E.coli. We demonstrate here that following microinjection using a zebrafish embryo test system, purified Tol2 transposase protein readily catalyzes gene transfer in both somatic and germline tissues in vivo. We show that purified Tol2 transposase can promote both in vitro cutting and pasting in a defined system lacking other cellular factors. Notably, our analysis of Tol2 transposition in vitro reveals that the target site preference observed for Tol2 in complex host genomes is maintained using a simpler target plasmid test system, indicating that the primary sequence might encode intrinsic cues for transposon integration.

ConclusionsThis active Tol2 protein is an important new tool for diverse applications including gene discovery and molecular medicine, as well as for the biochemical analysis of transposition and regulation of hAT transposon-genome interactions. The measurable but comparatively modest insertion site selection bias noted for Tol2 is largely determined by the primary sequence encoded in the target sequence as assessed through studying Tol2 protein-mediated transposition in a cell-free system.

KeywordsTol2 transposase hAT superfamily Recombinant transposase protein Zebrafish Transposition site preference AbbreviationsAc-DsActivator-Dissociation

TEstransposable elements

TIRsterminal inverted repeats

GBTgene-breaking transposon

dpfdays post-fertilization

Kankanamycin resistance

Electronic supplementary materialThe online version of this article doi:10.1186-s13100-016-0062-z contains supplementary material, which is available to authorized users.

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Autor: Jun Ni - Kirk J. Wangensteen - David Nelsen - Darius Balciunas - Kimberly J. Skuster - Mark D. Urban - Stephen C. Ekker

Fuente: https://link.springer.com/



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