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Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events

Lieve Herman (UGent) , Anni Jacobs (UGent) , Marc Van Montagu (UGent) and Anna Depicker (UGent)
(1990) MOLECULAR & GENERAL GENETICS. 224(2). p.248-256
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Abstract
During Agrobacterium tumefaciens infection, the T-DNA flanked by 24 by imperfect direct repeats is transferred and stably integrated into the plant chromosome at random positions. Here we measured the frequency with which a promoterless reporter gene is activated after insertion into the Nicotiana tabacum SR1 genome. When adjacent to the right or left T-DNA border sequences, at least 35% of the transformants express the marker gene, suggesting preferential T-DNA insertion (>70%) in transcriptionally active regions of the plant genome. When the promoterless neomycin phosphotransferase II (nptII) gene is located internally in the T-DNA, the activation frequency drops to 1% since gene activation requires T-DNA truncation. These truncation events in the nptII upstream region occur independently of the nature of the upstream sequence and of the T-DNA length. Deletion of the right border region prevents the detection of activated marker genes. Therefore, T-DNA truncation probably occurs after synthesis of a normal T-DNA intermediate during the transfer and/or integration process. In the absence of border regions, expression of the nptII selectable marker directed by the nopaline synthase promoter was detected in 1 out of 105 regenerated calli, suggesting the possibility that any DNA sequence from the Ti plasmid can be transformed into the plant genome, albeit at a low frequency.
Keywords
Agrobacterium tumefaciens, T-DNA borders, Intergeneric gene transfer, Pseudoborder, Insertional activation, Aberrant T-DNAs

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Chicago
Herman, Lieve, Anni Jacobs, Marc Van Montagu, and Anna Depicker. 1990. “Plant Chromosome/marker Gene Fusion Assay for Study of Normal and Truncated T-DNA Integration Events.” Molecular & General Genetics 224 (2): 248–256.
APA
Herman, Lieve, Jacobs, A., Van Montagu, M., & Depicker, A. (1990). Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events. MOLECULAR & GENERAL GENETICS, 224(2), 248–256.
Vancouver
1.
Herman L, Jacobs A, Van Montagu M, Depicker A. Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events. MOLECULAR & GENERAL GENETICS. 1990;224(2):248–56.
MLA
Herman, Lieve, Anni Jacobs, Marc Van Montagu, et al. “Plant Chromosome/marker Gene Fusion Assay for Study of Normal and Truncated T-DNA Integration Events.” MOLECULAR & GENERAL GENETICS 224.2 (1990): 248–256. Print.
@article{8501192,
  abstract     = {During Agrobacterium tumefaciens infection, the T-DNA flanked by 24 by imperfect direct repeats is transferred and stably integrated into the plant chromosome at random positions. Here we measured the frequency with which a promoterless reporter gene is activated after insertion into the Nicotiana tabacum SR1 genome. When adjacent to the right or left T-DNA border sequences, at least 35% of the transformants express the marker gene, suggesting preferential T-DNA insertion (>70%) in transcriptionally active regions of the plant genome. When the promoterless neomycin phosphotransferase II (nptII) gene is located internally in the T-DNA, the activation frequency drops to 1% since gene activation requires T-DNA truncation. These truncation events in the nptII upstream region occur independently of the nature of the upstream sequence and of the T-DNA length. Deletion of the right border region prevents the detection of activated marker genes. Therefore, T-DNA truncation probably occurs after synthesis of a normal T-DNA intermediate during the transfer and/or integration process. In the absence of border regions, expression of the nptII selectable marker directed by the nopaline synthase promoter was detected in 1 out of 105 regenerated calli, suggesting the possibility that any DNA sequence from the Ti plasmid can be transformed into the plant genome, albeit at a low frequency.},
  author       = {Herman, Lieve and Jacobs, Anni and Van Montagu, Marc and Depicker, Anna},
  issn         = {0026-8925},
  journal      = {MOLECULAR & GENERAL GENETICS},
  keywords     = {Agrobacterium tumefaciens,T-DNA borders,Intergeneric gene transfer,Pseudoborder,Insertional activation,Aberrant T-DNAs},
  language     = {eng},
  number       = {2},
  pages        = {248--256},
  title        = {Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events},
  url          = {http://dx.doi.org/10.1007/BF00271558},
  volume       = {224},
  year         = {1990},
}

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