A chromosomal paracentric inversion associated with T‐DNA integration in Arabidopsis

Laufs, Patrick; Autran, Daphné; Traas, Jan

doi:10.1046/j.1365-313x.1999.00436.x

Cited by 63 publications

(37 citation statements)

References 34 publications

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“…Thus, the variable mutation frequency most likely was caused by position effects resulting in variable expression of the HS::QQR transgene and͞or variable accessibility of the QEQ target. The lack of mutations in line Q5 could possibly have resulted from rearrangement of the T-DNA insertion, which commonly occurs (38)(39)(40)(41). We do not know what limits the mutation frequency in these experiments.…”

Section: Discussionmentioning

confidence: 90%

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Lloyd

Plaisier

Carroll³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

375

247

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Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate targeted modifications in plant genes are not currently available. In this study we investigated the efficacy of a targeted-mutagenesis approach based on zinc-finger nucleases (ZFNs). In this procedure, ZFNs are used to generate double-strand breaks at specific genomic sites, and subsequent repair produces mutations at the break site. To determine whether ZFNs can cleave and induce mutations at specific sites within higher plant genomes, we introduced a construct carrying both a ZFN gene, driven by a heat-shock promoter, and its target into the Arabidopsis genome. Induction of ZFN expression by heat shock during seedling development resulted in mutations at the ZFN recognition sequence at frequencies as high as 0.2 mutations per target. Of 106 ZFN-induced mutations characterized, 83 (78%) were simple deletions of 1-52 bp (median of 4 bp), 14 (13%) were simple insertions of 1-4 bp, and 9 (8%) were deletions accompanied by insertions. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFNinduced mutations. These data indicate that ZFNs can form the basis of a highly efficient method for targeted mutagenesis of plant genes.gene targeting ͉ nonhomologous end joining A major focus of plant biotechnology is genetic modification and improvement of crop plants. With this aim, large-scale genome and͞or EST sequencing projects are underway for many important plant species including rice, maize, wheat, soybean, and tomato (www.ncbi.nlm.nih.gov͞genomes͞PLANTS͞ PlantList.html). The enormous amount of genome-sequence information becoming available has intensified the need for methods that can use this sequence information to generate targeted modifications in plant genes. Targeted-mutagenesis methods could be used experimentally to investigate plant gene function or for genetic modification of important crop plants. Such methods are especially important for species, including most crops, that lack readily available mutant collections (1, 2). Furthermore, targeted mutagenesis could facilitate development of genetically modified crops lacking transgenic DNA, including genes conferring resistance to antibiotics. To date, efficient and widely usable methods for targeted modification of higher plant genomes are not available.The most widely used targeted-mutagenesis strategy is gene targeting (GT) by homologous recombination (3-6). Efficient GT procedures have been available for Ͼ20 years in yeast (7) and mouse (8). In these systems, DNA fragments are introduced into cultured cells, and repair by homologous recombination causes the introduced DNA to be incorporated into the homologous locus. Typically, GT events ...

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Section: Discussionmentioning

confidence: 90%

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Lloyd

Plaisier

Carroll³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

375

247

View full text Add to dashboard Cite

show abstract

“…The "knockworst" category includes those T-DNA insertion events that lead to large-scale chromosomal rearrangements. Such T-DNA-induced rearrangements have been documented in Arabidopsis (Nacry et al, 1998;Laufs et al, 1999).…”

Section: Proposed Nomenclaturementioning

confidence: 95%

T-DNA as an Insertional Mutagen in Arabidopsis

1999

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“…Without sequencing the actual breakpoints (~10-55 kb away from the insertions) we are currently not able to identify the molecular event responsible for that inversion and to decide on one of the models proposed for the generation of inversions during insertion of two transgenes in cis configuration (Laufs et al, 1999).…”

Section: Fish Analyses Of the Transgenic Line El702c With Flankingmentioning

confidence: 99%

Tandem repetitive transgenes and fluorescent chromatin tags alter local interphase chromosome arrangement in Arabidopsis thaliana

Pečinka

Kato

Meister

et al. 2005

Journal of Cell Science

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Fluorescent protein chromatin tagging as achieved by the lac operator/lac repressor system is useful to trace distinct chromatin domains in living eukaryotic nuclei. To interpret the data correctly, it is important to recognize influences of the tagging system on nuclear architecture of the host cells. Within an Arabidopsis line that carries lac operator/lac repressor/GFP transgenes, the transgene loci frequently associate with each other and with heterochromatic chromocenters. Accumulation of tagged fusion protein further enhances the association frequency. Independent experiments with a transgenic plant carrying another multi-copy transgene also revealed, independent of its transcriptional state, unusually high frequencies of association with each other and with heterochromatin. From these results we conclude that the lac operator/lac repressor chromatin tagging system may alter the spatial chromatin organization in the host nuclei (in particular when more than one insertion locus is present) and also that loci of homologous transgenic repeats associate more often with each other and with endogenous heterochromatin than normal euchromatic regions.

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A chromosomal paracentric inversion associated with T‐DNA integration in Arabidopsis

Cited by 63 publications

References 34 publications

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

T-DNA as an Insertional Mutagen in Arabidopsis

Tandem repetitive transgenes and fluorescent chromatin tags alter local interphase chromosome arrangement in Arabidopsis thaliana

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