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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Control of gene expression and silencing in transgenic plants

Background and objectives (1)

The introduction of novel traits in plants is an extremely powerful tool in agricultural research and development. Engineering such traits can be classified into two groups. Either the introduced gene encodes a protein which itself brings a new structural or regulatory property to the plant, or the transgene serves as a tool to regulate the expression of an endogenous gene or gene family. For both strategies, it is essential that the engineered trait behaves as expected, over several generations and in different genetic backgrounds. A process known as gene silencing leads to alterations in gene expression. Extensive selection programmes performed by several biotechnology companies and in research laboratories, have shown that only a minor fraction of the transgenic lines obtained, meet the plant breeders' requirements.

The aim of this project was to develop 'know how' for strategies that maximise the chances of obtaining stable activity of transgenes, by understanding the molecular mechanisms (transcriptional and post-transcriptional) that regulate gene silencing in transgenic plants.

(1) This project was a direct follow-on of the Training and Mobility of Researchers network “Gene silencing in transgenic plants: Molecular analysis of underlying mechanisms and approaches towards stabilisation of transgene activity” (Contract Number: CHRX-CT94-0530; Contractual Period: 01.11.1994 - 31.10.1997).

image image Transgenic plant.



Approach and methodology

To identify the structural and context-specific requirements for the stable expression of transgenes, we wished to define the influence of transcription, DNA sequence, DNA methylation, sequence homology and secondary structures on gene silencing. Likewise, the role of the genetic background and the spatial arrangement of homologous sequences in the chromosomes and in the nucleus was also examined. Secondly, to understand and exploit gene silencing mechanisms, we studied the importance of RNA transcription, the production of aberrant transcripts and antisense RNA and the spatial arrangement of homologous sequences and transcripts. Finally, environmental and physiological conditions that favour stable transgene activity were defined by conducting a comparative study of factors favouring stable expression or gene silencing.
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Main findings and outcome

As a result of the work of the silencing network, we now understand that transcriptional silencing is due to chromatin remodelling processes which package the transgene DNA into a chromatin structure blocking the access of enzymes required for its transcription. Certain structural features, especially inverted repeats and other repetitive elements, appear to be recognised as specific targets for transcriptional silencing.

Post-transcriptional gene silencing (PTGS) is due to specific degradation of transgene RNA and also targets any other RNA with homology to the transgene, but does not affect transcription. PTGS therefore blocks the normal processing pathways of the transgene, and of endogenous genes which share homology with the transgene. A certain threshold level of transgene RNA is needed to induce PTGS and our studies have shown that specific structural features, such as stem loops, strongly enhance the susceptibility of a transgene to PTGS. This suggests that PTGS requires the production of double-stranded RNA. These results will be most valuable for the design of transgenes with highly enhanced PTGS features.

This project includes two research laboratories working on gene silencing mechanisms in filamentous fungus. Studies in Neurospora crassa enabled cloning of the first genes that have been identified as encoding proteins involved in PTGS. Genes with a similar function are probably responsible for PTGS in plants, making the Neurospora crassa genes highly valuable for future collaborative work seeking to prevent or enhance PTGS via modification of the activity of these crucial genes. Other partners in this project have identified genes involved in transcriptional gene silencing as well as specific target sequences for transcriptional silencing, allowing us to make predictions and recommendations to the scientific community about the likelihood of transcriptional silencing for individual transgenes.

Other results from the project have demonstrated the importance of considering genetic background (choice of cultivar) as a factor influencing the level of silencing of an endogenous gene. Furthermore, it was shown that RNA signals can trigger promoter methylation and transcriptional silencing and that viral sequences can be integrated into plant DNA and contribute to plant genome evolution. From our investigations, it was clear that gene silencing mechanisms are not specific for transgenes but that they represent ancient tools which have evolved to protect plants against mobile elements, such as transposons or retroviruses, and for protection against viruses.
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Conclusions

This research should assist breeders and farmers to define culture conditions and propagation techniques that improve the control of gene silencing. The collaboration of the network partners has not only opened a route towards improved control and exploitation of gene silencing mechanisms, it has also provided new insight into fundamental regulatory mechanisms used by plants, filamentous fungi and, quite probably, most other higher organisms.
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Major publications

Cogoni C. and Macino G., “Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase”.
Nature,
399, 1999, pp. 166-169.

Cogoni C. and Macino G., “Posttranscriptional Gene Silencing in Neurospora by a RecQ DNA Helicase”.
Science,
286, 1999, pp. 2342-2344.

Kooter J.M., Matzke M.A. and Meyer P., “Listening to the silent genes: Transgene silencing research identifies new mechanisms of gene regulation and pathogen control”.
Trends Plant. Sci.,
4, 1999, pp. 340-347.

Mette M.F., van der Winden J., Matzke M.A. and Matzke A.J.M., “Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans”.
EMBO J., 18, 1999, pp. 241-248.

Metzlaff M., O'Dell M., Cluster P.D. and Flavell R.B., “RNA-Mediated RNA degradation and Chalcone Synthase A silencing in Petunia”.
Cell,
88, 1997, pp. 845-854.
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imageResearch project
 

Contract number
BIO4-CT96-0253

Period
November 1996 - January 2000

Coordinator
P. Meyer
University of Leeds (UK)

Project website address
http://www.biology.leeds.
ac.uk/centres/liba/eu_web/
index.html

 
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Partners


M. Cornelissen
Plant Genetic Systems
Gent (BE)

A. Depicker
Universiteit Gent (BE)

R. Flavell, R. Hellens
John Innes Centre
Norwich (UK)

D. Grierson
University of Nottingham (UK)

J. Kooter
Vrije Universiteit
Amsterdam (NL)

G. Macino
Università "La Sapienza"
Roma (IT)

M. Matzke
Austrian Academy of Sciences
Salzburg (AT)

F. Meins,
O. Mittelsten Scheid

Friedrich Miescher Institute
Basel (CH)

W. Schuch
ZENECA Plant Sciences
Bracknell (UK)

H. Vaucheret
INRA
Versailles (FR)

R. Visser
Wageningen Agricultural University (NL)

J-L. Rossignol
Institut Jacques Monod
Paris (FR)

 
 
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