Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants

Abstract

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of one inverted repeat. A phenotypic assay based on restored hygromycin resistance demonstrates that a dTam3 element harbouring a bacterial plasmid can be trans-activated with a low frequency. Molecular analysis confirms that the dTam3 element has been excised from the HPTII marker gene. Reintegration of the dTam3 element into the tobacco genome is detected only in one out of six hygromycin-resistant plants analysed. PCR analysis of empty donor sites shows that excision of the dTam3 element in tobacco results in rearrangements (deletions and additions), that have been shown to be characteristic of Tam3 excision in the original host Antirrhinum majus. This trans-activation assay allowed us to establish that, in contrast to what has been detected in Antirrhinum majus, a periodical temperature shift down to 15 °C does not enhance dTam3 transposition in regenerating tobacco calli.

Phylogenetic and Functional Characterization of the hAT Transposon Superfamily

“…Previous analyses of hAT transposon phylogeny focused only on transposons within what we now define as the Ac branch of the hAT element superfamily (Kempken and Windhofer 2001;Rubin et al 2001). The members of this branch known to be active are Ac, Tam3, hobo, Hermes, Herves, and Tol2 with all also being active in new host species (Martin et al 1989;Haring et al 1991;O'brochta et al 1994;Sarkar et al 1997a;Kawakamai and Noda 2004;Arensburger et al 2005;Evertts et al 2007) Our data show that members of the Buster family, which include the recently discovered SPIN transposons, are in fact capable of transposition upon introduction to new host species. Our data also illustrate the strong conservation of several domesticated Buster genes within several mammalian species including primates, dogs, horses, and cattle, strongly suggesting an essential, but as yet, unknown function for these genes.…”

The use of transgenic plants to understand transposition mechanisms and to develop transposon tagging strategies

“…Recently a two-element system for Tam3 has been established in transgenic tobacco plants [28]. Since no naturally occurring two-element system has been described for Tam3, an artificial system has been constructed consisting of an immobilized Tam3 element as the activator and a non-autonomous element in which a 1.3 kb internal fragment of Tam3 has been replaced by a bacterial plasmid.…”

Novel DNA structures resulting from dTam3 excision in tobacco

“…From D N A gel blot analyses it could be deduced that only in plant 4202 the dTam3 element had re-integrated (data not shown and [ 12]). …”

“…On the contrary, a Tam3 two-element system appears to be unable to perform the transposition process with the same efficiency and accuracy as the autonomous Tam3 element in both tobacco and Antirrhinum [20,21]. A non-autonomous Tam3 element (dTam3), which is activated in trans by a 'wings-clipped' Tam3 element (Tam3 ATIR), produces rearrangements in the HPT gene it excises from, while re-integration of the dTam3 element was detected in only one out of six plants analysed [ 12]. In this paper we present the analysis of the empty donor sites produced by aberrant dTam3 excision.…”

“…Analysis of this dTam3 element and its flanking sequences indicates deletion of the ends of the transposable element and integration by a process different from transposoninduced integration. [10,12].…”