Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

Cardon, Guillermo H.; Frey, Monika; Saedler, Heinz; Gierl, Alfons

doi:10.1007/bf00021428

Cited by 18 publications

(11 citation statements)

References 64 publications

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“…Differences in the ability of dicot plant species to correctly process the pre-mRNA of En has been noted in the past (Cardon et al, 1993) but not researched in any great detail. In this instance it was noted that Arabidopsis could splice correctly the En transcript at a level that was superior to that observed in tobacco and potato, and similar to that observed in maize.…”

Section: The Maize Ac Transposase Transcript Is Alternatively Splicedmentioning

confidence: 99%

Alternative processing of the maize Ac transcript in Arabidopsis

et al. 1997

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SummaryThe successful application of the maize transposable element system AciDs as a genome mutagen in heterologous plant species has recently proved the versatility and power of this technique in plant molecular biology. However, the frequency of Ac/Ds transposition is considerably lower in Arabidopsis thaliana than in most other dicot plant species that have been studied. Since previous research has established that transcripts derived from monocot genes can be alternatively processed in dicot plants, we have investigated both the efficiency of intron splicing and poiyadenylation of the maize Ac transposase pre-mRNA in Arabidopsis thaliana, Nicotiana tabacum, Nicotiana plumbaginifolia and Zea mays. In this paper, we demonstrate that intron 4 is alternatively spliced within Arabidopsis, using cryptic 5' and 3' splice sites within the intron sequence, leading to a heterogeneous population of full length transposase transcript. Furthermore, analysis of transposase transcript polyadenylation revealed that at least four alternative poly(A) sites were utilized between introns 2 and 3, resulting in truncated transposase transcripts. Finally, by Northern blotting, we established that the truncated transposase transcript was the most abundant form of transposase message in Arabidopsis. In contrast to these findings, the alternative splicing and premature polyadenylation of Ac message in Arabidopsis was unparalleled in the other species examined. We suggest that the poor frequency of transposition of Ac in Arabidopsis may be in part due to the low quantity of correctly processed transposase transcript available in this species.

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Section: The Maize Ac Transposase Transcript Is Alternatively Splicedmentioning

confidence: 99%

Alternative processing of the maize Ac transcript in Arabidopsis

et al. 1997

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“…The excision frequencies for these modified I/dSpm were nevertheless lower than for wild-type elements (Aarts 1996). Though the insertion of transcribed genes in the I/dSpm element has been shown to have a negative influence on the transposition rate in tobacco and Arabidopsis (Cardon et al 1993a;Tissier et al 1999), an additional negative effect due to a sub-optimal length of the left border cannot be ruled out. Based on these results, a longer fragment of 0.27 kb and a 0.64-kb fragment were used as left and right junctions of the I/dSpm element in the enhancer trap construct En -ET1 developed for rice transformation.…”

Section: Discussionmentioning

confidence: 96%

“…Further improvements, such as providing the TnpA and TnpD cDNAs for transposase expression as has been done successfully in tobacco (Cardon et al 1993a), and using longer I/dSpm transposon ends, could make En/Spm a practical alternative to Ac/Ds, the other maize system used for transposon mutagenesis in rice.…”

Section: Discussionmentioning

confidence: 97%

“…Previous reports on transposition of En/Spm in the heterologous systems Arabidopsis and tobacco made use of unmodified autonomous and non-autonomous elements (Pereira and Saedler 1989;Cardon et al 1993b;Aarts et al 1995;Wisman et al 1998;Speulman et al 1999). When the I/dSpm transposon was modified to carry selectable markers, left and right arms no smaller than 1015 bp and 786 bp, respectively, were used Cardon et al 1993a;Tissier et al 1999). In our laboratory, I/dSpm elements modified to function as gene traps in Arabidopsis revealed excision ability only in cases where a minimal left junction (I -Lj) of at least 0.19 kb was used, in combination with a 0.64-kb right junction (I -Rj; Aarts, 1996).…”

Section: Discussionmentioning

confidence: 97%

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Transcription and somatic transposition of the maize En / Spm transposon system in rice

Greco¹,

Ouwerkerk

Taal

et al. 2003

Mol Genet Genomics

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Transposition of the maize En/Spm system in rice was investigated using a two-component construct consisting of an immobilised transposase source driven by the CaMV 35S-promoter, and a modified I/dSpm transposon. Mobilization of I/dSpm in somatic sectors was demonstrated by sequencing of excision products and isolation of flanking genomic sequences in T0 and T1 progeny plants. Since the transposition efficiency appeared to be considerably lower than that observed in maize or in other heterologous systems like Arabidopsis, we examined En/Spm transcription and splicing in the transgenic rice plants. Northern analysis revealed the presence of transcripts encoding the active TnpA and TnpD transposases, with the latter predominating; this is the reverse of what is seen in maize and Arabidopsis. RT-PCR analysis confirmed the occurrence of correct splicing and the formation of the two other alternatively spliced transcripts (TnpB and TnpC), as previously described for maize. Two alternative splice donor sites at the end of exon 1 were identified in maize at positions 578 and 704. We observe that rice is similar to maize in that TnpA is preferentially spliced at position 578. We also show that in Arabidopsis splicing occurs preferentially at position 704, as in other dicots like tobacco. These observations indicate differences in the splicing of transcripts of the maize En/Spm element between dicot and monocot hosts. Nevertheless, the ratio in which the transcripts for the active transposases are produced seems to determine the efficiency of transposition, irrespective of the host considered. A limiting amount of TnpA might therefore be responsible for the lower transposition activity of En/Spm in rice. Alternatively, reduced mobility of the modified I/dSpm element used may have resulted from the absence of critical sequences necessary for transposition. The influence of endogenous, autonomous, En/Spm -related elements present in the rice genome on the transposition behaviour of the exogenous maize element is also considered.

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“…Esta colección fue generada mediante la inserción del transposón En-1/Spm (En-1, acc. M25427) en su forma activa (Cardon et al, 1993), lo que provoca mutaciones al azar en el genoma de Arabidopsis (Col-0).…”

Section: Mutantes Por Inserción De Elementos Transponiblesunclassified

Análisis funcional de los factores de trascripción Tower-of-Pisa1 y Tower-of-Pisa2 y su implicación en el desarrollo del gineceo de Arabidopsis.

González¹

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Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

Cited by 18 publications

References 64 publications

Alternative processing of the maize Ac transcript in Arabidopsis

Alternative processing of the maize Ac transcript in Arabidopsis

Transcription and somatic transposition of the maize En / Spm transposon system in rice

Análisis funcional de los factores de trascripción Tower-of-Pisa1 y Tower-of-Pisa2 y su implicación en el desarrollo del gineceo de Arabidopsis.

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