WESSEX
Campaign to ban genetically engineered food

Political Compliance V Sound Science - Biotech Debacle Set to Unfold Further?
( This posting looks at some important findings by the John Innes Centre on biosafety and other issues in relation to GM crops, and examines their significance in the context of the current political climate influencing the relationship between industry and bio-science.  It is available on-line at www.btinternet.com/~nlpwessex/Documents/compliance.htm )

2 February 2000

Proponents of GM crops are often fond of pretending that the technology used is sound because the methods deployed are so 'precise' - or so they would like everyone to think. This contention is a huge deception which is blazingly exposed when the trouble is taken to look beyond the hyperbole of genetic engineering 'breakthrough' press conferences [1], and into the realities of the basic scientific research.  

For those prepared to spend time rifling academic libraries a disturbing catalogue of scientific deficiencies begins to emerge.  The biotech 'spin' conferences are of course held on the assumption that no one - especially any deadline driven journalist - is likely to have the time and energy to do this.  

In this context the scientific paper below ('Transgene expression and stability in Brassica') is an interesting illustration of what transpires when this assumption does not work out in practice.  This paper is significant not for what it contains - critics of the technology are already aware of these concerns - but because of the 'pedigree' of the authors by whom it is produced.  Catalogued in its latest annual report the paper is written by some of the most senior scientists in the plant biotechnology world from the John Innes Centre (JIC) - a UK research institute funded by both government and industry-backed sponsors.  

The language and content of this paper clearly expose the reality that almost nothing is known about the basic science of genetic engineering when it comes to the creation of transgenic plants. The text is sprinkled with words and expressions indicating ignorance and doubt such as 'may', 'uncertain', 'probably', 'might', 'believed', 'it is thought', 'not known' etc.  

Put simply the fundamental science lying at the basis of one of the most radical technologies mankind has ever attempted to deploy has not been done.

Having clearly elucidated major areas of scientific ignorance the paper then spells out the consequences of this knowledge gap with the following anodyne but telling conclusion: "A knowledge of transgene stability, expression and inheritance is fundamental for the successful and safe use of transgenes in large scale agricultural production."  The astonishing significance of this statement is that these are precisely the areas of knowledge that the paper itself acutely demonstrates as being ubiquitously missing.  

Particular points of interest arising out of the extracts of the JIC paper detailed below include: ..............................................................................................................................................................................

1) the authors make it clear that, whilst focusing primarily on brassica crops such as oilseed rape, most of the issues raised in the paper also apply to other types of transgenic crops.  

2) the paper acknowledges that variation in transgene expression can, amongst other factors, be influenced by transgene position (on the chromosome).  This is important because, contrary to frequent claims by biotechnologists over the precision of their methods, genetic engineers have little or no control over where artificially inserted transgenes are placed in the host genome.  

3) whilst an individual transgene may behave in a particular way in a plant on its own this may change when another transgene is introduced. Such introduction can occur unintentionally through pollination and gene flow with sexually compatible species (although the authors somewhat unconvincingly claim that when such gene combinations are intentionally introduced through plant breeding such interactions are 'likely' to be picked up during the approvals process).  

4) the statement in the paper that "We believe there is no evidence to date to suggest that transgenes are more vulnerable to environmental instability than resident genes" seems to be contradicted by the authors' own reference to suggested evidence of 'promoter methylation' in work carried out by Meyer, et al.  (It is also interesting that research since reported in New Scientist November 1999 found that in high temperature environmental conditions Monsanto's herbicide resistant soya beans were prone to excessive stem splitting, although the precise cause of this has yet to be established. Additional severe systemic problems causing 'fruit abortion' and partial plant transpiration failure with some varieties of Monsanto's herbicide resistant cotton have also since been reported in the field by US extension specialists, although little is known about the causes.)  

5) the paper highlights problems associated with gene silencing events arising from the use of the CaMV 35 promoter, stating: "Intensive research at present is directed towards understanding this silencing mechanism and its significance....This is important....for assessing the significance of this effect for biosafety."  Clearly this promoter, which is used in nearly all transgenic plants (not just brassicas), has been introduced without a proper understanding of its functioning and behaviour.  Since the publication of this paper the JIC has also demonstrated that the CaMV promoter has a recombination 'hotspot'. This provides the possibility that new viruses may be created through its use in transgenic plants. The promoter is taken from a plant virus which is very similar to Hepatitis B and related to HIV. (For more information on this see: http://www.btinternet.com/~nlpwessex/Documents/camv.htm ).  

6) additional concerns regarding large scale environmental releases of GM plants are explored in further JIC papers (see: http://www.btinternet.com/~nlpwessex/Documents/gmrisk.htm ). Detection of some of these potential effects are described as 'much more difficult, if not impossible'. ..............................................................................................................................................................................  

Given the blatant absence of an adequate understanding of transgenics at the molecular level as elucidated in this paper, it is absolutely clear that large scale releases of GM crops at this stage in the development of our knowledge of genetic modification constitute little short of gross professional negligence.

So extreme is this situation that it is difficult to avoid raising questions regarding the involvement of political compliance in the process. This is not the first time the possibility of such a linkage would have been identified.  

As was pointed out by the London Times 25 January : "The secrets of Germany's corruption crisis are almost certainly buried in Helmut Kohl's early years as a politician when he first struck up a mutually rewarding relationship with the chemicals and pharmaceutical industry."  

That industry has since evolved into what is now know as the 'biotechnology' or 'life sciences' sector, comprising a coalition of research and business interests largely responsible for the deployment of GM technology in food, manufacturing and medicine.  One German industrialist is reported in the same Times article boasting about his relationship with Kohl: "Even if I ring him at three in the morning, he has to jump."  

In this context it is interesting that the part industry-funded John Innes Centre is also a principal adviser to the British Government on GM crop risk assessment.  Why then does it appear that the UK Government is ignoring fundamental risk-related questions that work at the JIC itself has identified?  

Why is the technology being moved out into the environment at this early prototype stage when it should be being confined to the laboratory until such basic questions are resolved? This is far from being the only such paper from the JIC which raises such fundamental questions about the behaviour of transgenes in genetically modified plants.  Why is the British Government not demanding that the fundamental molecular science be completed prior to deployment of the technology in the open environment? Rarely, if ever, has so little been known about the functioning of a technology as radical and invasive as this prior to its societal and environmental introduction.   

As the UK Government prepares to allow a sea of large scale GM brassica trials to spread its shores across our 'green and pleasant land' ponder tonight - as you prepare to sleep peacefully in your bed - the concluding words of this both troublesome and troubling scientific paper: "For those concerned with the development of transgenic brassicas, the take home message from this paper is 'watch out for homology'."   This phenomenon is pointedly described by the JIC as a factor of "crucial importance".

How long must we wait before 'homology' is transformed into 'apology'?  Europe may have waited over 30 years for Helmut Kohl to make this type of progression. Francois Mitterand never got round to it.   Ironically it is oilseed rape developed by a subsidiary (AgrEvo) of a newly formed Franco-German transnational company, Aventis [2], which comprises the bulk of the brassica GM trials currently taking place in the UK. 

Aventis has been formed from the merger of two German and French chemical-pharmaceutical companies, Hoechst and Rhone-Poulenc. Aventis now becomes one of the world's largest 'life sciences' corporations, and has the additional distinction of being the only company involved in the UK Government's GM crop farmscale trials. (Trials for Aventis's GM oilseed rape in the UK are for resistance to its glufosinate ammonium based 'Liberty' herbicide. Bizarrely this is a chemical which, for biosafety reasons, does not have clearance from the Pesticides Safety Directorate for commercial use involving the type of herbicide application scenarios being tested on winter oilseed rape varieties in the trials.)  

Aventis has only within the last few days begun its corporate launch publicity campaign to UK farmers with multi-page colour advertisements in the agricultural press. The merger gives Hoechst's shareholders a 53% interest in the company.  Hoechst's previous track record of responsible deployment of technology is far from exemplary. According to the 'Ecologist' an American subsidiary of Hoechst admitted that between 1976 and 1994 it had kept secret 200 studies showing adverse health impacts from its chemicals.

How many studies in the genetic engineering sector are going to be kept out of sight from the public and governmental decision makers? The popular former Secretary of State for Northern Ireland, Mo Mowlam, was recently appointed Cabinet Office Minister in charge of the British Government's biotechnology policy 'presentation' unit. But to what extent is she is aware of the literature on GM crop risks coming out of the JIC?  Certainly if her speech at the Bioindustry Association dinner in January is anything to go by there is little in her current approach which encourages political reticence on the subject: "I would urge everyone here tonight to speak up more loudly in the informed public debate.....Rest assured, the government is ready to support and enhance the competitiveness of the biotechnology industry. We believe you are a real success in the UK...." 

Significantly she acknowledged that benefits from GM crops and foodstuffs were "more difficult" to establish - but what "best example" of genetic engineering success did Mo Mowlam chose to draw attention to at the Bioindustry dinner in order to demonstrate her knowledge of the genetic engineering debate? Yes indeed, GM insulin for diabetics - the most widely used GM product in medicine.  Unfortunately, unbeknown to most of the general public this genetically engineered product has caused severe health problems for many diabetic sufferers including at least 50 suspected deaths to date [3], - as it happens a figure similar to the number of deaths so far from BSE related human illness.  

Where is Mo Mowlam getting her biotechnology briefings from? As will be seen from the acknowledgements at the end of the JIC paper below both MAFF and the Department of Environment, Transport and the Regions are aware of this work. Does this situation therefore represent political ignorance or political compliance?    

The reason people in the biotechnology industry do not speak up more loudly on these subjects as Mo Mowlam has called for, is because they are precisely the people who know what really lies under the carpet.  The last thing they want is the carpet being lifted.  Often under the guise of 'commercial confidentiality' secrecy has been fundamental to the introduction of this technology precisely because its core elements are not capable of standing up to intense scientific and public scrutiny.  

In a pan-European and global political context what are the implications of all this in a situation where the authority and autonomy of previously sovereign national governments have increasingly come under threat from the financial and political influence of multi-national corporations? The implications certainly go well beyond the technical issues of genetic engineering itself.  

A follow up article in the Times on 28 January examines further the Kohl-Mitterand corruption allegations. It refers to other alleged dealings with industry involving former French foreign minister Roland Dumas diverting 'easy money' from business to private and political recipients. The paper quotes Dumas's former mistress Christine Deviers-Joncour as saying: "These secret operations are...intimately linked to the functioning of our instititions." Mme Deviers-Joncour admits to lying when previously denying her role in acting on behalf of Elf-Aquitane (the French oil company linked to the Kohl-Mitterand corruption allegations) in an apparently successful attempt to influence Dumas over his role in an £1.6 billion Taiwanese arms deal. She claims that on the instructions of politicians Elf had been used to "sprinkle money on French political parties of both Right and Left and even foreign parties."  

Could it be then that Mowlam's new found enthusiasm for biotechnology is in fact more than the result of inadequate briefing? Interestingly the front page of the 'Independent on Sunday' 30 January reports that Mowlam had complained to friends before Christmas about her new job as British Cabinet Enforcer, where she is allowed to champion everything from drugs to rural affairs. She told them that she was "sick of going out each day and being told to tell lies."  The Independent quotes a political insider as claiming that Downing St "is convinced she is going to speak out against government policy at any moment", and it reports that the security guards traditionally provided to protect former Northern Ireland ministers had been removed within the previous few days.  

Meanwhile global political support for GMOs is waning fast. Given that the biosafety protocol agreed in Montreal at the end of January allows countries to ban imports of GM foods on the basis of the precautionary principle - even in the absence of scientific evidence - it would seem that the scientific literature that has emerged out of the JIC may well be sufficient to bring a halt to international trade in GMOs altogether. We are no longer in a situation where there is no evidence. The evidence is there and it is plain. What government can justify the importation or domestic deployment of GM crops in these circumstances?  

We may now be entering a period where a global ban on the environmental release of GM crops gains a toe-hold on the political agenda. After all the global ban on British beef was eventually adopted and applied with great vigour especially by countries like the US, Germany and France, despite years of previous risk-denial by eminent members of the scientific and political communities. With GMOs we are now well beyond the risk-denial stage if the Government supported work carried out at the JIC is to mean anything at all.  

At this point perhaps it is appropriate to finish with the insertion of a 'phonetic modification' into the concluding words of this particular piece of JIC GM risk analysis: "For those who are concerned about the development of transgenic technology, the take home message from this paper has now mututated to 'watch out for apology'."  

NATURAL LAW PARTY WESSEX
nlpwessex@bigfoot.com
www.btinternet.com/~nlpwessex  

(more information available at our GM homepage: www. btinternet.com/~nlpwessex/Documents/gmocarto.htm )  

Footnotes:

[1] For a couple of blatant examples of bio-industry press conference hyperbole where deficiencies in GM 'breakthough' products are conveniently kept in the closet see:
www.netlink.de/gen/Zeitung/1999/990715.htm
www. btinternet.com/~nlpwessex/Documents/gmindust.htm

[2] For more information on Aventis (AgrEvo) see http://www.corporatewatch.org/publications/GEBriefings/aventis1.html

[3] See http://www.netlink.de/gen/Zeitung/2000/000414a.html

BBC News June 2001 - French elite hit by sleaze claims
BBC News Feb 200 - German sleaze - The story so far

Dale P.J., Al-Kaff N., Bavage A., Irwin J., Senior I. (1998) Transgene expression and stability in Brassica. ACTA Horticulturae 459, 167-171.  

John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK  

Keywords: Biosafety assessment, Brassica napus, Oilseed rape, Gene silencing,

Transgene expression, Transformation.

Abstract

Transgenes are likely to be used widely for the development of new plant varieties for agriculture. An understanding of the causes of transgene stability and expression is important for various reasons: 1) to determine the utility of transgenes in agriculture; 2) for assessing their potential impacts on human health and the environment and; 3) to achieve a better basic understanding of the ways in which genes act and interact. Many companies and laboratories have experienced poor transgene expression, transgene instability and silencing. Transgene expression is influenced by various factors including: the nature of the construct; transformation events; background genotype; interactions with other transgenes; environmental conditions; and, in certain specific cases, by pathogen infection. Genetic homology between the transgene construct and other segments of nucleic acids within the cell can have a fundamental effect on the way transgenes work. It is, therefore, important to understand the underlying genetic mechanisms of these interactions.

1. Introduction  

In a recent survey of at least thirty companies developing transgenic plants for use in agriculture, all companies observed some transgene instability (Finnegan, et al., 1994). Many research laboratories have also reported difficulties in obtaining sufficiently high expression of certain transgenes or in stabilising transgene expression of an introduced gene. In a recent study in our laboratory, one hundred Brassica napus transgenic lines were produced and half of them displayed unstable or unusual transgene behaviour (Jones, et al., 1995). An understanding of the factors influencing transgene expression and stability are important for three reasons:(l) for assessing the utility of particular transgenes in agriculture; (2) for biosafety assessment; and (3) to gain a better basic understanding of the ways in which genes act and interact. The objective in this paper is
to consider some of the factors which may influence the way in which transgenes work. If we imagine a transgene on a Brassica chromosome, what variables might influence its level of expression and stability?  

Many of the variables considered in this paper are believed to interact with one another. For example, transgene copy number, transgene position or homology with other nucleic acid sequences in the genome, may cause the transgenes to display irregular patterns of expression and stability. This is probably the main reason why an increase in the number of copies of a transgene, or the effect of DNA homology within and between transgenes, do not have a predictable effect on plant phenotype.  

Although we are considering transgene expression and stability with particular reference to Brassica species, relevant data from other species will be included to illustrate principles.  

2. Transgene construct  

The way in which the introduced genetic construct is designed, clearly, influences the expression of the transgenes it contains. A detailed discussion on construct design is outside the scope of this paper. There is a wide range of different promoters that can be used and each have different strengths of expression and tissue specificity. Any areas of internal homology or of inverted DNA sequence repeats may potentially increase the likelihood of instability and unusual transgene expression. The influence of DNA homology will be returned to later.  

3. Transformation variables  

It is common to observe substantial variation in transgene expression between independently transformed plants.  The source of this variation is often attributed to transgene position or copy number effects. It is believed that if transgenes insert into
heterochromatic or highly methylated regions of the chromosome, this will reduce the expression of those transgenes. The presence of high copy numbers of transgenes does not always decrease levels of expression, but is likely to do so (Hobbs, et al., 1993). Structural rearrangements associated with the transformation process, including deletions, duplications and repeated areas of homology are likely to influence transgene stability and expression. Recent evidence suggests that repeat T-DNA structures are produced by co-integration of multiple T-DNAs into competent plant cells at the time of transformation (De Neve. et al., 1997). Exactly how this co-integration occurs is uncertain, at present, but reducing the number of transferred T-DNA complexes, could potentially reduce the formation of repeals.  

Transgene stability and expression can be increased by using scaffold attachment regions flanking the gene of interest. Higher levels of expression have been obtained using both yeast- and tobacco-derived SARs (Allen, et al., 1993; Allen, et al., 1996). However, the use of SARs did not avoid transgene expression variability, especially when multiple copies were present. This was rationalised by the suggestion that multi-copies can still interact and result in reduced expression and gene silencing (Allen, et al., 1996).  

The insertion of Ti plasmid DNA sequences from outside the T-DNA region of Agrobacterium is surprisingly common. In Petunia, 15% of transformants were observed to contain large fragments of non T-DNA binary vector plasmid (Cluster, et al., 1996). In tobacco 75% contained sequences from outside T-DNA and in 20% of these plants there was gene expression (gus) from these regions (Kononov, et al., 1997). In current studies in our laboratory, analyses show that about half (15/31) of transgenic lines of B. napus contain up to 350 bases of T-DNA flanking sequences (Bavage, et al., unpublished). One objective of our study is to determine whether these vector plasmid sequences affect stability of transgene expression and inheritance.  

4. Background genotype  

An effect of background genotype is well known from classical genetics (Harland,1936. Sanda and Amasino. 1996). When particular genes, e.g. those for disease resistance, are moved from one genetic background to another, they can vary in efficiency. Plant breeders are already moving transgenes into a range of different genetic backgrounds, and this practice will increase in the future as more transgenic lines become part of the genetic resources at plant breeders' disposal. Evidence from our laboratory, following the transfer of a construct into a range of different spring and winter lines of Brassica napus has shown that in some cases the transgenes behaved normally, and in other cases abnormally, with evidence of loss of particular transgenes (Irwin, et al., unpublished).  

In another study, a stable line containing a single insert of the bar herbicide tolerance gene in the hemizygous condition in Brassica napus was hybridized with a range of ecolypes of 5 species; B. napus, B. rapa, B. juncea, R. raphanistrum and S. arvensis. The observed segregation of the transgene to the Fl progeny varied from one species to another. The control (B. napus x B. napus) exhibited normal Mendelian segregation (1: 1). Transgene expression in the genetic backgrounds tested was found to be similar to that in B. napus.(Al-Kaff, et al., unpublished).  

The precise cause of the interaction between transgenes and the background genotype is not known. One mechanism which may play some part is cosuppression, in which there is interaction between a transgene and homologous DNA sequence in the background genome. Single copy transgenes do not elicit the phenomenon efficiently, whereas multi-copy transgenes, particularly in an inverted repeat structure, can down regulate or silence gene expression (Stam, et al., 1997). It is thought that the inverted repeat structure induces the production of abnormal RNA, which is subsequently recognised by the cell as aberrant. Any transcript which has homology to this aRNA is thought to be degraded and removed from the cell, resulting in no native or transgene expression. Where there is homology between transgenes and resident genes, therefore, there is likely to be some interaction (Senior. 1998).  

5. Interaction between transgenes  

Different transgene constructs will combine in plants by two routes:1) intentionally, by breeders as part of their Brassica breeding programmes; and 2) unintentionally, by cross pollination and gene flow to sexually compatible species. It is known from basic research studies, some of them in tobacco, that one gene construct can switch off expression of another. One of the most detailed studies has been by Matzke, et al., (1989), where they combined two gene constructs by performing sequential transformations on the same plant line. In this work, it was found that one transgene could switch off the expression of another transgene. When the transgenes were separated sexually, the previously silenced transgene was able to express. When the same two constructs were combined again sexually, the silenced phenotype was re-imposed (Matzke, et al.1993).  

The interaction of different transgenes is of considerable significance for plant breeding, because transgenic lines will become an important part of the germplasm available for conventional breeding. Because of the intensive selection and evaluation that is necessary before a new plant variety can be released and used in agriculture, it is likely that any interactions of the kind described, will be identified before release into agriculture. Within the European Union regulatory system at present, when any two independent transgene inserts which have received previous commercialisation approval are combined together to form a new breeding line, they will need to be resubmitted for regulatory approval.  

There is a possibility of interaction between transgene constructs that become combined by sexual hybridization and gene flow under agricultural conditions. If more than one transgene construct becomes combined in weed species, there is the potential for genetic interaction. The most likely consequence is that, if transgene expression is affected at all, it will be to down regulate or silence their expression. In most cases, this will result in the plants involved reverting to a wild-type phenotype. However, the phenomenon of genetic interaction needs to be considered carefully during the environmental impact assessment phase.  

6. Environmental effects  

There are a number of cases where environmental stimuli affect transgene stability (Senior, 1998). The most comprehensive data to date is from the modification of flower colour in Petunia. Meyer, et al., (1992) performed a field experiment of 30,000 transgenic Petunia plants carrying the maize Al gene (which gave a salmon-red flower colour). Early in the season, flowers were predominantly salmon-red, but after a period of high light and temperature, approximately two-thirds of new flowers showed a reduction in A1 gene expression and pale red to white flowers were produced. Meyer, et al., (1992) showed this was correlated with increased promoter methylation. Analysis of progeny from the early season blossoms showed them to be resistant to inactivation. However, progeny of late flowers showed reduced Al gene expression. Meyer (1995aandb)rationalised this by suggesting an increased degree of methylation was imposed on transgenes as plants aged, and such methylation pattern imprinting was transferred to the progeny. These would be more susceptible to environmental stimuli because of the imprinted methylation pattern. Meyer (1995aandb) suggested the youngest possible material should be used for pollination, to avoid such imprinting problems in plant breeding.  

There is, of course, evidence of the instability of genes bred by conventional plant breeding methods. The variety of wheat Moulin failed to crop satisfactorily in some satisfactorily in some seasons (PBI. 1985). We believe there is no evidence to date to suggest that transgenes are more vulnerable to environmental instability than resident genes.  

7. Interaction with pathogens  

DNA sequences from pathogens (promoters, terminators and coding regions) have been used regularly in gene constructs for the transformation of plants. There is, however, no research to date which demonstrates the possible interaction between an infecting pathogen and homologous DNA sequences of that pathogen transformed stably into the plant. Recent research in our laboratory with Brassica napus plants containing the 35S promoter from the mosaic virus (CaMV) has shown that upon infection with the CaMV the driven transgene is silenced (Al-Kaff et al unpublished). Intensive research at present is directed towards understanding this silencing mechanism and its significance.  

As the 35S promoter is widely used to regulate transgenics in brassicas, it is important that we strive to obtain a clear understanding of the mechanisms of this silencing and its significance. This is important for two reasons, for assessing the use of the 35S promoter in agriculture and also for assessing the significance of this effect for biosafety. The presence of homology between the 35S promoter and the infecting virus is clearly important in determining this silencing phenomenon (Al-K-aff, et al., unipublished). However, we believe that the phenomenon may be part of a mechanism that Brassica napus has to resist attack from CaMV (Covey, et al., 1997). When Brassica napus is infected by CaMV, it is likely that viral genes and any transgenes regulated by the 35S promoter are simultaneously silenced. This work will be reported in detail elsewhere.  

8. Conclusions

A knowledge of transgene stability, expression and inheritance is fundamental for the successful and safe use of transgenes in large scale agricultural production. Many factors influence the ways in which transgenes express, but a factor of crucial importance is the effect of DNA sequences that are homologous to areas of transgene constructs. For those concerned with the development of transgenic brassicas the take home message from this paper is "watch out for homology".  

Acknowledgements

We thank BBSRC, MAFF and the Department of the Environment, Transport and the Regions, for their support. We thank Non Owen and Chris Jones for the initial transformation and characterisation of the B.napus transgenic lines. We also thank Eric Belfield, Maria Kreike, Neil McKenzie, Anthony Page, Rachel Pinder and Penny Sparrow for their contribution to the research, and Helen Ghirardello for preparation of the manuscript.  

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