Transgenic Lines Proven Unstable
By Dr Mae-Wan Ho

There is plenty of evidence that transgenic lines are unstable, which is why ISIS has long recommended that appropriate molecular methods must be used to document the stability of the GM insert before any transgenic line is released into the environment. The characterization of the insert must be ‘event-specific’, which not only gives the structure of the insert, but also the host genome sequences flanking the insert, proving that the insert remains stable in successive generations. This recommendation has been incorporated into the current European Directive (2001/18 /EC) on deliberate release of GMOs.

But to this day, pro-GM scientists advising the UK and other governments have refused to acknowledge the evidence on transgenic instability, and worse. In its latest reply to ISIS, the UK Advisory Committee on Releases to the Environment (ACRE) has gone as far as to say that event-specific molecular characterization is not necessary, thus going against the European Directive (see ISIS’ final response to ACRE: Let the people decide ).

ISIS has reiterated 5 experiments which should be done to address the ‘areas of uncertainty’, one of which calls for full event-specific molecular characterization of all transgenic lines to establish uniformity and genetic stability of the transgenic DNA insert(s), and “comparison with the original data supplied by the biotech company to gain approval for field trials or for commercial release.”

I am pleased to report that some effort has recently been made to do such experiments by French scientists from the Laboratory of Methods for Detecting GMOs in Versaille, and the Laboratory of Biometry and Artificial Intelligence, Domaine de Vilvert in Jouy-en-Josas. And they have presented their results in a poster at a conference in June 2003 [1].

The scientists recognized that, as labeling laws and thresholds are established for foods containing GMOs in Europe, Japan, Australia, New Zealand and elsewhere, “reliable GMO identification and quantification methods are needed to comply with the regulations.” And “in order for these tests to be specific, the sequence and detailed characterization of the GMO inserts and their edges are required.”

Five different commercially approved GMOs in Europe were analyzed: three from Monsanto, one from Bayer and one from Syngenta. All inserts were rearranged from their intended gene order. Moreover, all five inserts showed further rearrangements from the original structure submitted by the companies. In other words, either the companies were mistaken about the original structure, or more likely, further rearrangements had occurred after the crops had been commercially grown. The details are given in Box 1.

Box 1

Scrambling and further scrambling of GM inserts

T25 maize LibertyLink (Bayer)

Modified for tolerance to herbicide glufosinate. Company data showed insert includes a truncated ampicillin resistance bla gene in the plasmid vector pUC18, a CaMV 35S promoter (hereafter referred to as P35S) driving a synthetic pat gene (glufosinate tolerance) terminated by CaMV 35S terminator (hereafter referred to as T-35S). On analysis, the insert was found to have undergone further rearrangement, so that a second, truncated and rearranged P35S has been joined to the 5′ (left, or head) end of the insert, while additional pUC18 sequences were found at the 3′ (right, or tail) end.

Edges flanking the insert show homologies (similarities) with Huck retrotransposons (a class of mobile genetic elements) in the maize genome.

Mon 810 maize YieldGard (Monsanto)

Modified for resistance to lepidopteran insects (butterflies & moths). Company data showed insert has a P35S driving a CrylAb synthetic gene with terminator T-nos. Analysis revealed however, that T-nos and part of the 3′ (tail) end of the CrylAb gene have been deleted. T-nos has been detected elsewhere in the genome, indicating that it has moved from its original position.

The 5′ (head) end of the insertion site shows homology to the long terminal repeats (LTR) of the maize alpha Zein gene cluster, but no homology to the maize genome was detected at the 3′ site, indicating that there has been scrambling of the maize genome at the insertion site.

GTS 40-3-2 soybean (Monsanto)

Modified for tolerance to herbicide glyphosate (Roundup Ready). Company data showed insert with P35S driving a composite gene containing the N-terminal chloroplast transit peptide (CPT4) joined to modified epsps gene with T-nos terminator.Analysis revealed that a 254bp piece of DNA homologous to the epsps gene and 534bp of unknown DNA have been joined to the 3’end of the insert.

It was not possible to identify the insertion site at all, indicating substantial genome scrambling or deletion at the insertion site.

Bt 176 maize (Syngenta)

Modified for tolerance to herbicide glufosinate, male sterility and insect resistance. The structures of two inserts, originating from two GM constructs, were provided by the company. Only the simpler construct was analyzed. Company data showed insert contains P35S driving the bar gene (glufosinate tolerance) terminated by T35S, followed by the ampicillin resistance (bla) gene plus bacterial promoter, and plasmid origin of replication, ori. Analysis revealed several fragments, all containing CaMV 35S promoter, one with P35S joined to T35S, a second with P35S joined to an unknown sequence, and a third with P35S joined to the bar gene with the T35S deleted.

There were at least three insertion sites.

GA 21 maize (Monsanto)

Modified for tolerance to herbicide glyphosate (Roundup Ready). Company data indicated insert contains multiple copies of the cassette with the rice actin gene promoter (P-ract) driving the composite gene containing the N-terminal chloroplast transit peptide (CPT4) joined to modified epsps gene and T-nos. There were three complete cassettes flanked by a cassette with P-ract partially deleted at the 5′ end, and one cassette with 3′ deletion of epsps plus a lone P-ract at the 3’end. Analysis found partial deletion of P-ract and deletion of T-nos in two different cassettes.

The insertion site at the 3’end is flanked by sequences of pol polyprotein gene belonging to a PREM2-retrotransposon.

The results revealed that,

All GMO inserts had rearranged from the structure provided by the company.
Many of the breakpoints for rearrangement involve the CaMV 35S promoter, as can be predicted from its known recombination hotspot.
Scrambling of the genome at the site of insertion occurred in at least two out of five inserts.
GMO inserts appear to show a preference for mobile genetic elements (retrotransposons), with Long Terminal Repeats containing strong promoters, which would result in “altered spatial and temporal expression patterns of genes” nearby. In addition, it increases the chances that the inserts will move with the retrotransposons, resulting in further genome scrambling and horizontal gene transfer.
With considerable irony, whether intended or not is unclear, the authors conclude: “Studying GMO’s structure is necessary to develop reliable quantification and detection tests complying with the different regulations, but it also leads [one] to ask fundamental questions about genome fluidity. Many of the mechanisms involved in recombinant DNA integration are similar to those underlying genome evolution. Therefore, characterized GMO inserts are a very good model to study the molecular system involved in DNA rearrangements in general.”

Collonier C, Berthier G, Boyer F, Duplan M-N, Fernandez S, Kebdani N, Kobilinsky A, Romanuk M, Bertheau Y. Characterization of commercial GMO inserts: a source of useful material to study genome fluidity. Poster courtesy of Pr. Gilles-Eric Seralini, Président du Conseil Scientifique du CRII-GEN, www.crii-gen.org
This article can be found on the I-SIS website at http://www.i-sis.org.uk/TLPU.php