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Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

Lisson R, Hellert J, Ringleb M, Machens F, Kraus J, Hehl R - Plant Mol. Biol. (2010)

Bottom Line: Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication.The fourth intron of the transposase was found to be partially misspliced.In addition, the second and third exon were found to harbour two and three novel introns, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institut für Genetik, Technische Universität Braunschweig, Germany.

ABSTRACT
The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain.

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Schematic representaion of Ac and Ds integration events in sugar beet. Three IPCR products, 35–43(Ds), 36–43(Ds), and 001R-20(Ac) were obtained with primer pairs number 5/6 and 11/12 (arrows). Genomic integration sites (WT) were amplified with primer pairs number 7/8, 13/14, and 15/16. The hatched boxes represent Ac or Ds terminal sequences and the white boxes genomic sugar beet DNA. Primer numbers correspond to primers in Table 1
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Fig2: Schematic representaion of Ac and Ds integration events in sugar beet. Three IPCR products, 35–43(Ds), 36–43(Ds), and 001R-20(Ac) were obtained with primer pairs number 5/6 and 11/12 (arrows). Genomic integration sites (WT) were amplified with primer pairs number 7/8, 13/14, and 15/16. The hatched boxes represent Ac or Ds terminal sequences and the white boxes genomic sugar beet DNA. Primer numbers correspond to primers in Table 1

Mentions: Excision of Ds in phosphinothricin resistant transgenic hairy roots was analysed by PCR. Genomic DNA was prepared according to the following protocol. A 4–5 cm long root piece was used for genomic DNA extraction with the ‘REDEXtract-N-AmpTM Plant PCR Kit’ from Sigma–Aldrich GmbH (Taufkirchen, Germany) according to the protocol from the manufacturer. The sequences of all primers used in the PCR and IPCR analyses are shown in Table 1. The positions of the primers are shown schematically in Figs. 1 and 2. For the amplification of the empty donor site of Ds, the primers number 1 and 2 were employed. PCR amplifications were performed in a final volume of 20 μl harbouring 2 μl template DNA (1–100 ng), 11.8 μl dH2O, 2 μl 10xExtension-buffer (ExTaqTMBuffer, Appligene Oncor, Illkirch, France), 2 μl dNTP-mix (2.5 mM each), 1 μl of each primer (20 μM), and 0.2 μl Taq polymerase (5 u/μl, Qbiogene—MP Biomedicals, Illkirch, France). The PCR program used was: 3 min 94°C, 35× (1 min 94°C, 1 min 55°C, 2 min 72°C), 10 min 72°C, subsequently 4°C. Results of PCR analyses were always analysed gel electrophoretically.Table 1


Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

Lisson R, Hellert J, Ringleb M, Machens F, Kraus J, Hehl R - Plant Mol. Biol. (2010)

Schematic representaion of Ac and Ds integration events in sugar beet. Three IPCR products, 35–43(Ds), 36–43(Ds), and 001R-20(Ac) were obtained with primer pairs number 5/6 and 11/12 (arrows). Genomic integration sites (WT) were amplified with primer pairs number 7/8, 13/14, and 15/16. The hatched boxes represent Ac or Ds terminal sequences and the white boxes genomic sugar beet DNA. Primer numbers correspond to primers in Table 1
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2921059&req=5

Fig2: Schematic representaion of Ac and Ds integration events in sugar beet. Three IPCR products, 35–43(Ds), 36–43(Ds), and 001R-20(Ac) were obtained with primer pairs number 5/6 and 11/12 (arrows). Genomic integration sites (WT) were amplified with primer pairs number 7/8, 13/14, and 15/16. The hatched boxes represent Ac or Ds terminal sequences and the white boxes genomic sugar beet DNA. Primer numbers correspond to primers in Table 1
Mentions: Excision of Ds in phosphinothricin resistant transgenic hairy roots was analysed by PCR. Genomic DNA was prepared according to the following protocol. A 4–5 cm long root piece was used for genomic DNA extraction with the ‘REDEXtract-N-AmpTM Plant PCR Kit’ from Sigma–Aldrich GmbH (Taufkirchen, Germany) according to the protocol from the manufacturer. The sequences of all primers used in the PCR and IPCR analyses are shown in Table 1. The positions of the primers are shown schematically in Figs. 1 and 2. For the amplification of the empty donor site of Ds, the primers number 1 and 2 were employed. PCR amplifications were performed in a final volume of 20 μl harbouring 2 μl template DNA (1–100 ng), 11.8 μl dH2O, 2 μl 10xExtension-buffer (ExTaqTMBuffer, Appligene Oncor, Illkirch, France), 2 μl dNTP-mix (2.5 mM each), 1 μl of each primer (20 μM), and 0.2 μl Taq polymerase (5 u/μl, Qbiogene—MP Biomedicals, Illkirch, France). The PCR program used was: 3 min 94°C, 35× (1 min 94°C, 1 min 55°C, 2 min 72°C), 10 min 72°C, subsequently 4°C. Results of PCR analyses were always analysed gel electrophoretically.Table 1

Bottom Line: Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication.The fourth intron of the transposase was found to be partially misspliced.In addition, the second and third exon were found to harbour two and three novel introns, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institut für Genetik, Technische Universität Braunschweig, Germany.

ABSTRACT
The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain.

Show MeSH
Related in: MedlinePlus