key: cord-014674-ey29970v
authors: nan
title: Dreizehnter Bericht nach Inkrafttreten des Gentechnikgesetzes (GenTG) für den Zeitraum vom 1.1.2002 bis 31.12.2002 : Die Arbeit der Zentralen Kommission für die Biologische Sicherheit (ZKBS) im Jahr 2002
date: 2003
journal: Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz
DOI: 10.1007/s00103-003-0614-5
sha: 
doc_id: 14674
cord_uid: ey29970v

nan

Die Zentrale Kommission für die Biologische Sicherheit (ZKBS) prüft und bewertet sicherheitsrelevante Fragen nach den Vorschriften des Gentechnikgesetzes (GenTG), gibt hierzu Empfehlungen und berät die Bundesregierung und die Länder in sicherheitsrelevanten Fragen der Gentechnik. Da das GenTG hauptsächlich aus der nationalen Umsetzung der EU-Gentechnikrichtlinien hervorgegangen ist, sind die Entwicklungen im Bereich der internationalen und der nationalen Gentechnik-Regelungen für die ZKBS von besonderem Interesse.

Aus dem Bereich der internationalen Regelungen zur Gentechnik ist für das Berichtsjahr 2002 hervorzuheben, dass das "Intergovernmental Committee for the Cartagena Protocol" (ICCP) eingerichtet wurde,das die Vorbereitungen zur Ratifizierung und sachgerechten Umsetzung des "Biosafety Protocols" begleitet. Mit In Vorbereitung auf das "Dritte Gesetz zur Änderung des Gentechnikgesetzes"das vorrangig die Richtlinie 2001/18/ EU auf nationaler Ebene umsetzt, befassten sich 2 Arbeitskreise aus ZKBS-Mitgliedern mit darüber hinausgehenden, fachlich schwierigen Aspekten (u. a. "Sicherheitseinstufung", § 7 GenTSV).Bei diesem "Dritten Gesetz zur Änderung des Gentechnikgesetzes"wirdvoraussichtlichauch die Implementierung des Cartagena-Protokolls berücksichtigt werden (s. oben).

Vom Dezember 2001 bis zum September 2002 fand auf Initiative des Bundesministeriums für Verbraucherschutz, Ernährung und Landwirtschaft (BMVEL) ein "Diskurs zur Grünen Gentechnik" statt,an dem Repräsentanten gesellschaftlicher Gruppen und betroffener Verbände beteiligt waren. Während der 10 Monate wurden verschiedene Aspekte der Nutzung der Gentechnik in Landwirtschaft und Ernährung erörtert 1 . Am Ende jedes Teildiskurses wurde ein Resü-mee gezogen. Ein gemeinsam getragener Ergebnisbericht, in dem auch Minderheitspositionen dargestellt wurden, wurde auf einer Schlussveranstaltung im September 2002 vorgestellt. Die ZKBS nahm zur Kenntnis, dass dieser "Diskurs zur Grünen Gentechnik" keine Fortsetzung der sog."Kanzlerinitiative" war,da die für die Bewertung der biologischen Sicherheit wesentlichen Aspekte, die in der "Kanzlerinitiative" eine vorrangige Rolle spielen sollten (Gewinnung praktischer Erfahrungen aus bereits abgeschlossenen oder laufenden Anbauprojekten mit GVOs), schon in der Konzeption des Diskurses ausgeschlossen wurden.Die ZKBS konnte keine Sachargumente für die Durchführung dieses "Diskurs zur Grünen Gentechnik" erkennen.

Insgesamt kann die ZKBS die Veränderungen im Bereich der internationalen und der nationalen Regelungen für die "Grüne Gentechnik" als auch ihre Entwicklung und Anwendung nicht unbedingt als positiv einstufen.Aus diesem Grund spricht die ZKBS für das Jahr 2003, in dem die politische Verantwortung für den Bereich Gentechnik vom Bundesministerium für Gesundheit und soziale Sicherung (BMGS) zum Bundesministerium für Verbraucherschutz, Ernährung und Landwirtschaft (BMVEL) überwechseln wird, erneut ihre Erwartung auf eine Wende aus, die -unter Wahrung sachgerechter, wissenschaftlich begründbarer Vorsorgemaßnahmen -auch "den rechtlichen Rahmen für die Erforschung, Entwicklung, Nutzung und Förderung der wissenschaftlichen, technischen und wirtschaftlichen Möglichkeiten der Gentechnik" (GenTG § 1, Abs. 2) schafft. Die Situation innerhalb der Europäischen Union für die Genehmigungsverfahren zum Inverkehrbringen von Produkten, die gentechnisch veränderte Organismen enthalten, stagniert nun schon im vierten Jahr unverändert seit 1998. Weder die z. T. seit einigen Jahren anhängigen Genehmigungsverfahren gemäß der Richtlinie 90/220/EWG noch solche nach der Novel-Foods-Verordnung wurden abgeschlossen (Tabelle 4 in [3] ).

Zur Erfüllung der Aufgaben der ZKBS bei der Prüfung sicherheitsrelevanter Fragen der Gentechnik werden die Mitglieder der Kommission aus unterschiedlichen Disziplinen berufen. Maßgeblich für die Zusammensetzung der ZKBS ist § 4 des Gentechnikgesetzes. Darin ist geregelt, dass sich die Kommission zusammensetzt aus ◗ 10 Sachverständigen, die über besondere und möglichst auch internationale Erfahrung in den Bereichen der Mikrobiologie, Zellbiologie, Virologie, Genetik, Hygiene, Ökologie und Sicherheitstechnik verfügen; von diesen müssen mindestens 6 auf dem Gebiet der Neukombination von Nukleinsäuren arbeiten; jeder der genannten Bereiche muss durch mindestens einen Sachverständigen, der Bereich der Ökologie muss durch mindestens 2 Sachverständige vertreten sein, 

In ihrer Publikation "Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico" [7] 

Anhang "Letter to the editor" der Zeitschrift 'Nature'

Ladies and Gentlemen, in their article Quist and Chapela (1) report on the detection of transgenic DNA constructs in native maize landraces grown in remote mountains in Oaxaca, Mexico. They raise therewith concerns about the unintended introgression of transgenic maize traits into landraces ('criollo') in the centre of their origin resulting in a danger for the natural diversity of this crop plant. By use of molecular methods including PCR,inverted PCR (iPCR) and sequencing of the amplified DNA,they obtained data from which they conclude (i) that the nucleotide sequence of the cauliflower mosaic virus (CMV) 35S promoter [p-35S; contained in various lines of genetically modified maize; 2] is present in the maize genomes of several 'criollo' samples, (ii) that in two instances these promoter sequences were flanked by adh1-sequences which are also neighbouring the p-35S in the transgenic construct of Novartis Bt11 maize, and (iii) that the transgenic p-35S sequences were "embedded within various genomic contexts" of the 'criollo' samples.

We have closely examined the experimental data and the analyses of the nucleotide sequences presented in the report.We find that aside from problematic details of the experimental design and some erratic presentations of the data the results of the study do not provide evidence for the introgression of recombinant DNA from transgenic crop plants into the genomes of 'criollo' maize. Our detailed analyses of the data including the nucleotide sequences (1) which the authors have deposited in the nucleotide sequence data base of GenBank clearly show that none of the authors conclusions are justified and therefore the far reaching interpretations on the endangered diversity of landraces is lacking any basis. Our position with respect to the presented data are detailed in the following.

1. In order to prove that the p-35S sequences that were amplified by PCR from the DNA samples prepared from the corn cobs were not derived from contaminating CMV it is necessary to show that the observed p-35S sequences are linked on one side or on both to maize DNA. To identify the sequences flanking the p-35S the authors applied iPCR. The template for iPCR were EcoRV restriction fragments of the maize DNA circularized by ligation. EcoRV cuts at a site in the middle of the p-35S sequence and therefore DNA amplified by iPCR from ligation products with primer pairs matching the right and left parts of the p-35S sequence should contain one restored EcoRV cleavage site.Eight sequenced iPCR products were presented in Fig. 2 of Ref. 1 (sequences AF434754 to AF434761). None of them contains the EcoRV site (see box in our Fig.) .This casts doubts on the authors assumption that restriction by EcoRV and ligation had created the circular DNA products necessary for iPCR.

2. Next we examined whether the nucleotides directly ahead of the four applied primers and expected to be identical to the p-35S sequence were present in the eight sequenced iPCR amplification products. As shown in our Fig. the primers iCMV2 and iCMV1 were used for iPCR on the left side of the 35S promoter sequence,the primers iCMV4 and iCMV3 on the right side.[At this point two details of the authors experimental setup must be critizised. First, the binding sites of iCMV2 and iCMV3 are located outside of the p-35S region initially amplified by primers cm01 and cm02 from the DNA samples and therefore the presence of these binding sites in the sample DNA was not certain. Second, iCMV2 has 10 nucleotides at the 3' end which do not match the 35S promoter region (waved line in iCMV2 in our Fig.) and therefore is not expected to allow specific amplification of p-35S sequences.]

In the sequences of the amplification products AF434754, -55, -56, -57 in which the iPCR primer sequences can be identified the nucleotide sequences ahead of the primers are not from p-35S.The expected p-35S sequence is only partially present ahead of iCVM1 in AF434758. In five cases the primer sequences were not discernible (AF434758, -59, -60). In one case (AF434761) the expected p-35S nucleotides were present. These data indicate that perhaps with the exception of AF434761 the template of the PCR amplifications was not the 35S promoter region.

Three other inconsistencies between the sequences AF434754 to AF434761 and the Fig. 2 are apparent (1) . First, the sequences described as "downstream" relative to the p-35S by Quist and Chapela are in fact "upstream" sequences and correspondingly the "upstream" sequences of Quist and Chapela are in fact "downstream" (compare our Fig. with Fig. 2 of  1) . Second, the vertical lines in Fig. 2 which according to Quist and Chapela indicate the ends of CMV sequences are misleading since as outlined above they only mark the 3' ends of the primers employed (except for the sequence AF434761; the source of this sequence is termed B3 in the Fig.2 ,B2 in the sequence deposited in GenBank,and A2 in the supplement to Ref. 1). Thirdly, the thin lines in Fig. 2 of Ref. 1 supposedly indicating the parts of CMV DNA in the amplified sequences are unduely overstretched (they essentially always represent only the PCR primers) and several of them should not be there at all because primer sequences can not be identified. This is the case at one side of A2 (AF434758) and both sides of B3 (AF434759).

3. We characterized with the help of BLAST searches those parts of the sequences of the iPCR amplification products that were denoted by Quist and Chapela in their Fig.2 as regions flanking the CMV p-35S sequence.We find that the sequence of AF434754 denoted adh1 in the K1 source of Fig. 2 does not match with the maize adh1 gene. Rather, it matches with a sequence located about 40.000 nucleotides away from the adh1 gene (but still within the database entry of about 160.000 bp termed adh1).A corresponding BLAST search result was also obtained with AF434755 from the A3 quence. Therefore, the conclusion that "sequences adjacent to the p-35S DNA were diverse" in the maize genome cannot be drawn. 4 . We examined whether the identified regions in the maize genomic DNA from which PCR amplification products were obtained by the authors would perhaps be flanked by primer binding sites. For this we performed pairwise BLAST alignments of the iPCR sequences with the five matching maize genomic sequences. By adjusting the alignment parameters we were able to identify putative primer binding sites at the expected distances in the maize genome target sequences corresponding to AF434754 to AF434757 and also one binding site for the single primer sequence identified in AF434758. This indicates that these five assumed iPCR amplification products were most likely obtained by normal PCR amplification directly from continuous sequences of the maize genome having accidentally flanking regions with similarity to the primers. No primer binding sites were found in sequences AF434759 and AF434760. These findings support our conclusion from section 2 that the template for at least seven of the eight iPCR products were not p-35S sequences. Rather, the templates were sequences of the maize genome related to retroviral sequences which frequently had reasonably matching primer binding sites.

Evidence for the integration of p-35S sequences into the 'criollo' genome was not obtained because in none of the eight cases studied a linkage of p-35S sequences (aside from the primers used for PCR) to maize DNA could be demonstrated. In one case p-35S sequences were linked to a non-identified sequence. This can be a consequence of the use of iPCR in which the essential ligation step always bears the risk of fusing (restriction) fragments that were not naturally contiguous in the sample DNA. In this case the authors did not perform the necessary PCR control experiment using primers from p-35S and the unknown sequence to show that these sequences are in fact contiguously present in the 'criollo' genome. The fact that the p-35S specific primers used by the authors had considerable similarity to retroviral sequences explains the formation of PCR products from 'criollo' DNA under conditions when the the hybridization stringency is not sufficiently controlled. Five of the eight amplified sequences gave in fact matches with retroviral or retrotransposon elements.The claim of the authors that two of their sequences were related to sequences present in the transgenic construct of Novartis event Bt11 corn was disproven by careful analysis of the sequence and its target.The low amounts of p-35S sequences detected in the 'criollo' DNA preparation can easily be explained by contamination of the samples with CMV. If the samples had been tested for other CMV sequences they would probably be there in the equivalent amounts as the p-35S sequence.

Achter Bericht nach Inkrafttreten des Gentechnikgesetzes (GenTG) für den Zeitraum vom 1.1.1997 bis 31.12

Gentechnisch veränderte Pflanzen der

Elfter Bericht nach Inkrafttreten des Gentechnikgesetzes (GenTG) für den Zeitraum vom 1.1.2000 bis 31

Yellow head complex viruses: Transmission cycles and topographical distribution in the Asia-pacific region

Observation of measles virus cell-to-cell spread in astrocytoma cells by using a green fluorescent protein-expressing recombinant virus

Neunter Bericht nach Inkrafttreten des Gentechnikgesetzes (GenTG) für den Zeitraum vom 1.1.1998 bis 31

Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexiko

Further tests at CIMMYT find no presence of promoter associated with transgenes in Mexican landraces in gene bank or from recent field collections

No credible scientific evidence is presented to support claims that transgenic DNA was introgressed into traditional maize landraces in Oaxaca

Doubts linger over Mexican corn analysis

Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico

A method of detecting recombinant DNAs from four lines of genetically modified maize

 Fig.2 .These two sequences thus incorrectly associated with maize adh1 gene were a strong argument for the authors that a transgenic construct was identified in the 'criollo' because adh1 sequences are in fact present in the transgenic construct of the Bt11 event of Novartis. However, in the Bt11 construct the adh1-related sequences are the introns IVS6 and IVS2 of the adh1 gene and are located downstream of p-35S (2). Different from the Bt11 construct the so called adh1 sequences in AF434755 and AF434756 are located upstream of p-35S (see previous section).Thus,the adh1 hits of two iPCR products presented by the authors as evidence for "the integrity as an unaltered construct" retained in the 'criollo' genomes are wrong in two ways: (i) the sequences are not from the adh1 gene and (ii) they are located on the wrong side of p-35S. The sequence AF434758 (A2 sample) was denoted as zea mays alpha zein gene, although the matching region in GenBank sequence AF031569 is not an alpha zein gene. Instead, the target sequence is part of a region denoted as "similar to retrovirus-related POL polyprotein sequence".Similarly, our BLAST search also identified the previously discussed "adh1" sequences of AF434754 and AF434755 as being highly similar to a putative gag-pol precurser, e.g. in the GenBank sequence AF464738. In case of the sequence AF434757 (A3 sample) the similarity (bit score 44) with the DULL1 gene could not be reproduced. A match of only 14 identical nucleotides (bit score 28) was obtained when using decreased stringency parameters in a pairwise alignment with "BLAST 2 sequences". In summary, five of the eight iPCR sequences are retro element sequences, the other three are not (AF434760,-61) or not closely (AF434757) related to any known maize DNA se-