The Bg-rbg system of transposable elements consists of autonomous Bg elements which encode all the functions needed for its transposition, and non-autonomous members (rbg) which can only transpose if an autonomous element is present in the same cell. Both elements have been isolated and characterized at the molecular level. The Bg element, isolated at the waxy locus, is 4,869bp in length, carries terminal inverted repeats of 5bp and generates 8bp long target site duplications upon transposition. With respect to the autonomous element, the receptor element has undergone numerous deletions but it has retained the terminal inverted repeats of the Bg element as well as a 76bp long subterminal sequence. This subterminal region is supposed to be of importance for transposition, since it contains a number of 6bp long motifs, both in direct and inverted orientation. A similar motif has been found in the subterminal region of the Ac transposon of maize, and it has been demonstrated that the putative transposase of Ac binds to these sequences.
In order to study the mechanism of transposition of the Bg element, we have introduced the autonomous element in tobacco plants. For this purpose we have constructed two types of binary vectors both based on pBIN 19. The first vector contains the complete Bg element flanked by small fragments of the waxy gene. This construct was denominated pBINBg. The second binary vector contains a CaMV 35S promoter and a rolC gene from Agrobacterium rhizogenes. The transcription of the rolC gene in this vector has been interrupted by placing the complete Bg element plus flanking waxy sequence between the CaMV promoter and rolC. Bg was inserted in both orientations between promoter and rolC giving rise to the constructs pBINBg53, carrying the CaMV and Bg promoter in the same orientation, and pBINBg35 with the two promoters in opposite directions. Tobacco plants were transformed by the leaf-disk method and 84 transgenic plants were obtained. The excision of the Bg element from its original position was monitored by PCR analysis with two oligos complementary to the flanking waxy sequences. Only if Bg excises will the PCR analysis give rise to an amplification product since the oligos span a distance of almost 5kb in the presence of Bg. All plants tested showed amplification of specific products, indicating the excision of the Bg element. The PCR products were subcloned and sequenced. All fragments contained the expected waxy sequence with an empty donor site for the Bg element. Moreover, typical footprints were observed. A second series of experiments to demonstrate excision of Bg in tobacco was carried out with the use of the pBINBg53 and pBINBg35 plasmids. Plants transformed with these constructs are expected to express rolC upon excision of the Bg element. This would originate plants with pale green to yellow sectors. Sixteen plants transformed with pBINBg53 and 26 plants with pBINBg35 were analyzed. All showed characteristic sectors on the leaves. No differences were observed between the two types of constructs.
In order to investigate whether the excision of Bg was followed by reintegration of the element, Southern analyses were performed on plants transformed with the pBINBg53 and pBINBg35 constructs. Upon digestion with EcoRI or HindIII restriction enzymes and hybridization with a Bg probe, expected fragments were seen in most of the plants analyzed. A number of plants showed additional bands, indicating a reintegration of the Bg element. Some plants showed no hybridization with the Bg probe, indicating the loss of the element upon excision. This phenomenon was observed in plants transformed with both the pBINBg53 construct and the pBINBg35 construct. Striping of the blots and rehybridization with a rolC probe produced bands for all plants, confirming that all plants tested were actually transformed. The majority of plants integrated a single copy of the construct.
The available data indicate that the Bg element is capable of excising from and reintegrating into the tobacco genome. Furthermore, the addition of a strong promoter does not influence the rate of transposition of the Bg element in tobacco. Studies aimed at quantifying the amount of Bg transcript in plants containing the different constructs are in progress.
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