Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement.

  title={Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement.},
  author={Sergei Svitashev and Wojciech P. Pawlowski and Irina Makarevitch and David W. Plank and David a. Somers},
  journal={The Plant journal : for cell and molecular biology},
  volume={32 4},
To more fully characterize the internal structure of transgene loci and to gain further understanding of mechanisms of transgene locus formation, we sequenced more than 160 kb of complex transgene loci in two unrelated transgenic oat (Avena sativa L.) lines transformed using microprojectile bombardment. The transgene locus sequences from both lines exhibited extreme scrambling of non-contiguous transgene and genomic fragments recombined via illegitimate recombination. A perfect direct repeat of… 

Figures and Tables from this paper

Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation

This is the first report revealing near border T-DNA truncation and vector backbone integration in wheat transgenic lines produced by Agrobacterium-mediated transformation and reports on the presence of plasmid backbone DNA sequence in transgene loci detected using primer pairs outside the left and right T- DNA borders and within the plasmids selectable marker (NptI) gene.

Complex structures of transgene rearrangement implicate novel mechanisms of RNA-directed DNA methylation and convergent transcription

An RNAi construct for silencing FtsZ gene functioning on plastid division in higher plants was transformed into tobacco genome and revealed that the construct had rearranged, resulting in convergent transcription of nptII gene by 35S promoter and nos promoter with both transcripts expressing at low level.

Dosage-Dependent Gene Expression from Direct Repeat Locus in Rice Developed by Site-Specific Gene Integration

D dosage-dependent transgene expression can be obtained by integrating full-length copies, and site-specific gene integration approach can serve as an efficient tool for generating precise multi-copy integrations.

Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize[OPEN]

The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles, presumably by homology-dependent repair (HDR).

Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco.

Transfer of organelle DNA into the nuclear genome has been significant in eukaryotic evolution, because it appears to be the origin of many nuclear genes. Most studies on organelle DNA transfer have

In situ methods to localize transgenes and transcripts in interphase nuclei: a tool for transgenic plant research

In situ methods to visualize transgenes and their transcripts during interphase from different tissues and plant species are developed, which reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transGene activity.

Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment.

It is proposed that gene transfer using multiple gene cassettes offers an efficient and rapid method to obtain the single-copy transgenic wheat.

Efficient Targeted Gene Insertion in Maize using Agrobacterium-Mediated Delivery

An efficient, heritable, selectable marker-free site-specific gene insertion in maize using Agrobacterium-mediated delivery is reported, enabling the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacteria-mediated transformation.

Evaluation of the recombination efficiencies of FLP proteins

It is recommended to recommend the use of FLPo in plant genetic engineering after the relative recombination efficiencies of FLPwt, FLPe and FLPo for marker gene excision from the transgene locus in rice were evaluated and revealed that FLPo is relatively more efficient than FLPe.

Effect of gene order in DNA constructs on gene expression upon integration into plant genome

No significant effect of the gene order on gene expression was found in the transformed rice lines containing precise site-specific integrations and stable gene expression in plant cells could be obtained with altered gene orders.



Association of transgene integration sites with chromosome rearrangements in hexaploid oat

The results suggest that particle bombardment-mediated transgene integration may result from and cause chromosomal breakage and rearrangements in oat lines produced by microprojectile bombardment.

Genomic interspersions determine the size and complexity of transgene loci in transgenic plants produced by microprojectile bombardment.

It is proposed that copies of transgene along with other extrachromosomal DNA fragments are used as patches to repair double-strand breaks (DSBs) in the plant genome resulting in the formation of transGene loci.

Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA.

  • W. PawlowskiD. Somers
  • Biology
    Proceedings of the National Academy of Sciences of the United States of America
  • 1998
It is proposed that transgene integration at multiple clustered DNA replication forks could account for the observed interspersion of transgenic DNA with host genomic DNA within transgenic loci.

Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots.

The data indicate that transformation through direct DNA transfer generally results in a single transgenic locus as a result of this two-phase integration mechanism, and transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenes will be easier to characterize genetically.

High-resolution structural analysis of biolistic transgene integration into the genome of wheat

In the transgenic plants, the type of promotor used, rather than the chromosomal site of transgene integration, was most critical for transgenes expression, and the integration of the transgenes was not associated with detectable chromosomal rearrangements.

Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns

The efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors, and robust GUS activity in hygromycin-resistant plants was observed, confirming co-expression of the selectable and nonselectable markers.

The structures of integration sites in transgenic rice.

Since only three junctions of transgenic rice were implicated in the illegitimate recombination and extensive rearrangement of the rice genome, rice protoplasts may be active in this process.

Organizational Complexity of a Rice Transgene Locus Susceptible to Methylation‐Based Silencing

Observations of a rice (Oryza sativa L.) transgene locus introduced using biolistic techniques strongly support the concept that intrusive DNA is recognized by host surveillance systems and that transgenes loci with anomalous structural organization are subjected to inactivation by processes such as methylation.

Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters in trans.

To investigate the origins of trans-silencing ability and susceptibility, the structures, flanking DNA sequences and chromosomal locations of a nopaline synthase promoter silencing locus, H2, and a sensitive target locu, K81, are analyzed.