A comparative study of Tam3 and Ac transposition in transgenic tobacco and petunia 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. Ac excises from the HPTII gene with comparable frequencies (30~o) in both plant species. When the /~-glucuronidase (GUS) gene was used to detect transposition of Tam3, a significantly lower excision frequency (13~o) was found in both plant species. It could be shown that deletion of parts of the transposable elements Tam3 and Ac, removing either one of the terminal inverted repeats (TIR) or part of the presumptive transposase coding region, abolished the excision from the marker genes. This demonstrates that excision of the transposable element Tam3 in heterologous plant species, as documented for the autonomous element Ac, also depends on both properties. Southern blot hybridization shows the expected excision pattern and the reintegration of Tam3 and Ac elements into the genome of tobacco plants.

Novel DNA structures resulting from dTam3 excision in tobacco

“…This can be deduced from the sequence of the left end of the excision site because the Tam3 end fits the deletion that had been introduced by Bal31 deletion [ 10]. This is a surprising observation as we were unable to detect excision (hygromycin resistance) in control experiments with both pTT21808, pTT21806 (Tam3ATIR) and pTT21840 (dTam3).…”

“…1). As neither of these elements can excise autonomously [ 10] these results indicate that the non-autonomous dTam3 element is activated in trans by the immobilized element Tam3 ATIR. To confirm the excision of the dTam3 element from the HPT gene a DNA These sequence data reveal that none of the sequences produced upon dTam3 excision matches the empty donor site for autonomous Tam3 excision in Antirrhinum or tobacco [20,21].…”

“…DNA gel blots were performed as described [ 10]. The polymerase chain reaction [28] was carried out to amplify the empty donor site fragments from hygromycin-resistant plants using a CaMV 35S primer (GGCTGCAGTCTCCACTGACG-TAAGGGATGACG), a reverse HPT primer (AGCTGAAAGCA CCGAGAATTCTTCGC) and the following thermal cycle: denaturation 94°C 30 s, annealing 55 °C 30 s, extension 72 °C 1 min, performed 30 times.…”

“…An immobilized transposase donor Tam3 element and an internaUy deleted non-autonomous Tam3 element are both incapable of excision, as has been determined with a phenotypic assay [ 10]. When a plant containing either of the constructs is transformed with the second component of the trans-activation system hygromycin, resistant plants can be obtained at low frequencies (5 ~o of the transformed leaf-discs yields a resistant shoot).…”

“…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].…”