Hello List In keeping with other recent non topic posts recently I forward the following .....
Ron Date: 20/12/99 13:39 Greetings friends, associates, and acquaintances, Please forgive the unsolicited mail, but I thought that this communication would be of interest to you all. At least, it answers the question as to why the Soil Association does not include provisions for composted GMO contaminated materials in their standards. Also, I consider that the delete key is a quick and painless solution to unwanted emails! Take care all, and happy Xmas. 20 December, 1999 Dear XXX XXXXXX UK MAFF investigated DNA fragmentation and found that GMO material would need to be heated to 95C for at least 5 min in order to fragment the DNA into sizes of less than 100bp - which would be enough to disrupt the antibiotic resistance genes. But even this treatment would leave short fragments of DNA which do retain some biological activity. You simply must try to ensure the material you compost is GMO free I'm afraid. MAFF scientists have stated in their reports that GMO material must not be used for silage, because DNA is not destroyed during composing and soil micro-organisms can take it up and incorporate it - indeed, they do so regularly. The dispersal of naked GMO DNA in the environment is a major concern here at ISIS and we are planning a number of papers about it next year. Animal feed is a mess I agree and there is nothing easy about this awful GMO situation for any sort of farmer. I attach a critique on the UK MAFF study for your information. Angela Ryan The Institute of Science in Society Critique of MAFF Report CS0116 "Effect of feed processing conditions on DNA fragmentation" By Angela Ryan Molecular Biologist, Open University 19th Nov 1999 Introduction The purpose of this report was to determine the extent to which DNA is fragmented in animal feed. Animal feed is either fed raw or processed under a variety of conditions in commercial mills. The effect of several methods of processing on the fragmentation of DNA in animal feed was analysed. DNA fragmentation in raw foodstuffs from a number of commercial feeds and non-commercial feeds was also investigated. This work set out to address concerns regarding the transfer of foreign genes, especially antibiotic resistance genes introduced in GM crops, to microbes rendering them antibiotic-resistant. The report points to the resurgence of tuberculosis as an example of the significant medical problems that may come about from the transfer of such resistance to micro-organisms and suggests transfer of resistance genes from GM crops would exacerbate the problem of resistant strains of bacteria. In order for gene transfer to occur, a gene would need to be complete and therefore the DNA of which it is composed, stable and relatively intact. The report states that it is unlikely for transfer of such genes from plant to microbe to be proved impossible. It goes on: "Therefore, if GM plants are to be licensed, it is necessary to know what onditions are necessary in installations preparing animal feeds for the prevention of gene transmission, i.e. sufficient disruption of DNA." This report describes the results of a Pilot Study commissioned from Leeds University by MAFF. The study is on a small scale, has limited resources and has been performed on non-GM material. Nevertheless, the results give every indication that further studies are needed, which the scientists concerned were ready to undertake and were expecting further funding for from MAFF. Experimental Details Processing Specifications Three major UK animal feed companies were involved with this study, providing advice on processing specifications and a range of animal feed samples. Ranges of laboratory treatments were carried out to determine the conditions required during processing to sufficiently disrupt the DNA. Wheat samples were subjected to mechanical grinding and milling at different grist sizes in the laboratory. The processing of oil seed samples did not receive the closely monitored processing conditions initially required by the project - processed oil samples were supplied by the industry. Samples Samples of animal feed ingredients were obtained from the collaborating companies and include: Oil seeds, before and after oil extraction by expulsion and compression; Wheat grains of the variety Riband subjected to heating both dry and moist, at a range of temperatures and pressures and for a range of durations. Smaller samples of 20 varieties of wheat were subject to proximate analysis and five of these varieties were subjected to dry heat treatment at 93 degrees centigrade; Fresh maize leaves and maize silage, from the University Farm; fresh maize cobs and maize grains from the local supermarket; Maize gluten and flaked maize; Fresh wet sugar beet and dried sugar beet pulp; Fresh ryegrass and rye silage. Experimental Protocol DNA was extracted from the variety of animal feed samples by standard procedures. Two approaches were taken to monitor the extent of DNA fragmentation. The first allowed visualization of the DNA fragments by electrophoresing them on an agarose gel followed by DNA staining with ethidium bromide. The gel was then photographed and the DNA fragment sizes determined by comparing them with fragments of known length. This approach, whilst determining the sizes of fragments, does not give any indication of their composition and also has limited sensitivity. In the second approach, the polymerase chain reaction (PCR) technique was used to determine whether a specific gene sequence could survive particular feed processing treatments. This was adopted in order to develop a means to increase the precision of the analysis of fragment lengths,down to the level of single genes. The Rubisco small subunit (RbcS) gene from maize was sequenced and primers were designed and made in order to amplify approx. 600bp of it from maize samples. Should the DNA strand bearing this gene be disrupted between two primer binding sites, then the PCR amplification would simply not take place and no PCR product would be obtained. Treatments which fragment the RbcS gene would also fragment transgenes of comparable size in trangenic plants. NB: The B-lactamase gene used extensively in GM plants is approx. 1200bp in length. Results Table of results showing treatment/sample and state of DNA. SAMPLE/ TREATMENT STATE OF DNA wheat/grinding intact wheat/ milling intact linseed leaves intact linseed grains intact wheat leaves intact wheat grain intact soya leaves intact soya grain intact maize grain intact maize leaves intact maize silage intact rapeseed leaves intact rapeseed grain Intact fresh sugar beet pulp intact ryegrass intact ryegrass silage intact oil seed rape meal (after extraction) degraded oilseed rape cake degraded maize gluten degraded maize flaked degraded wheat, dry heat, 90 C or below for 30 min intact wheat, low pressure steam, 60 C, 30 min intact wheat dry heat, 93C, 4 min intact wheat dry heat, 93C, 5-15 min partially degraded wheat dry heat, 95C, 5min degraded wheat low pressure steam, 85 C, 10 min partially degraded wheat low pressure steam, 95-100 C, 30 sec or more degraded wheat high pressure steam at 100-124 C, 1min or more degraded PCR Following extra purification of template genomic DNA from fresh maize leaves, grains and silage, amplification of specific gene sequences within maize RbcS1 and RbcS2 genome sequences was allowed and generated PCR products of up to 577bp. Conclusion The results of this study clearly show that DNA remains stable in silage and suggests that ensiled GM crops should not be used as animal feed. In small-scale reactions under laboratory conditions, it was found that temperatures of 95 C for at least 5 min were required to completely fragment the DNA. The report admits that most commercial-processed animal feeds are subjected to temperatures that do not exceed 85 C. Extrusion would only cause temperatures on the outside of particles to reach 100 C and only briefly. Where steam is used to condition material for pelleting, temperatures reach at least the mid-80s and the length of time these temperatures are maintained is uncertain. The results show that DNA is only partially fragmenting in material subjected to 85 C for 10 min - maintained by steam. Physical extrusion or chemical extraction of oilseed leaves a residue in which the DNA is highly fragmented. The report concludes that most commercially produced animal feed contains intact DNA fragments of a size greater than 1200bp - comparable to the B-lactamase transgene used in many GM crops. It states, "These findings will be of considerable importance to the feedstuff industry." The report recommends more extensive research to be done. Critique The report states "There is no reason to believe that the conditions necessary to fragment the DNA of GM plant material are any different from unmodified material." Whilst in theory this assumption holds true, this study works only with non-GM plant material. Transgenic DNA in GM plants may possess unknown and as yet un-investigated characteristics, such as enhanced resistance to fragmentation. Therefore, duplicate experiments using actual GM plant material are warranted and required for reliable conclusions regarding GM crops. The report gives no consideration to the transfer of other transgenic DNA used in GM crop plants, such as the CaMV promoter. The CaMV promoter is about 350bp in length and contains a recombination hotspot as well as several modular motifs that have specific molecular function (1). Recombination involves the breaking of one DNA strand and joining of it to another DNA strand and is the molecular mechanism by which gene transfer occurs. CaMV is closely related to human hepatitis B virus, and also has a reverse transcriptase gene related to that in retroviruses such as the AIDS- associated HIV (3). Thus, the CaMV promoter not only enhances horizontal gene transfer but has the potentialto reactivate dormant viruses (which are in all genomes) and to generate new viruses by recombination. The transfer of small DNA fragments or motifs of specific molecular function are also given no consideration in this report. For example, an imperfect 19bp palindromic sequence has been isolated as the recombination hotspot in the CaMV promoter and short poly-purine rich tracts within the promoter act as binding sites for DNA processing enzymes involved in recombination (2). The approaches adopted by this study would not be sensitive enough to detect such short DNA fragments. In addition, the report defines 'completely degraded DNA' equal to DNA fragments of 100bp or less. Motifs bearing important molecular function may therefore be present in samples said to contain completely fragmented DNA. It was agreed early in the project that it would be better to process the feed materials under closely monitored conditions at a semi-commercial scale feed plant in the Roslin Institute, rather than in commercial mills. However, there was considerable delay in commissioning the equipment at the Roslin Institute, which meant that the samples came instead from commercial processing of oil seeds and laboratory processing of wheat. Research to detect "The effects of commercial scale processing on the integrity of DNA in animal feed" has been commissioned by the Chief Sciences Group and it has been confirmed that the results of this study will be available by the end of March next year. These results may provide further confirmation of the conclusions drawn in this study. The scientists at Leeds are now working on an "Assessment of the risks of transferring antibiotic resistance determinants from transgenic plants to micro-organisms" which has also0 been commissioned by MAFF and is due to be completed in April 2001. They are feeding GM plant material to animals and using the PCR technique, with primers specific for antibiotic resistance genes, to detect the transfer of these genes in the animal gut. The fact that this important study has been commissioned by MAFF suggests there is still real concern about the consequences of using GM animal feed. After this report was written, MAFF produced a second report "GM Materials in Animal Feed", which makes reference to this report as a 'first look' study. However, it does not refer to the very important conclusions presented clearly in this report - that most processed animal feed contains intact DNA. (It is admitted by the scientists that processed animal feed is subjected to temperatures that do not exceed 85C and that at least 95C for 5 min is required to completely degrade DNA. See p 8). The follow on report does present important data regarding the level of GM material in British animal feed. It is clear that British animal feed is presently comprised of at least 20% GM material obtained from feed suppliers abroad, who do not segregate GM varieties from non-GM varieties (p5). It is also clear (p6) that 45% of raw materials used in compound feed in GB in 1997/98 contained processed cereals, peas, beans and vegetable residues containing farm wastes after harvest, which may have contained intact DNA from genetically modified organisms. This report gives no reference to other research pertaining to the safety of using GM material as feed. It has been shown by another MAFF study that the human mouth contains bacteria capable of taking up and expressing free DNA containing antibiotic resistance marker genes and similar transformable bacteria are also present in the respiratory tract (4). In addition, in a letter from UK MAFF to the US FDA dated Dec. 1998, it was pointed out that transgenic DNA may be transferred not just by ingestion, but by contact with plant dust and air-borne pollen during farm work and food processing(5). In general the experiments conducted in this study were performed rigorously and the conclusions drawn by the scientists are right and of considerable importance to the animal feed industry. We recommend that British animal feed companies seek sources of GM free material for animal feed destined for this country. This approach alone would eliminate the significant risks posed to human and animal health and well being, associated with the transfer of transgenic DNA from GM material. The report highlights the fact that the necessary safety assessments regarding the consumption of GM material have not yet been carried out. We suggest the need for an immediate withdrawal of all GM material, from every point in the human food chain, until such time as the results from these very important ongoing studies are at least complete and satisfactory. References 1. HO. M.W., Ryan, A., and Cummins. J. (1999). The cauliflower mosaic viral promoter - A recipe for disaster? Micorbial Ecology Health and Disease: 11 (4). 2. Kohli, A., Griffiths, S., Palacios, N,. Twyman, R.M., Vain, P., Laurie, D.D. and Christou, P. (1999). Molecular characterisation of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. The plant Journal 17, 591-601. 3. Xiong, Y and Eickbush, T.H. (1990). Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 9, 3353-3362. 4. Mercer, D.K., Scott, K.P., Bruce-Johnson, W.A. Glover, L.A. and Flint, H.J. (1999). Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plansmid DNA in human saliva, Applied and Environmental Maicrobiology 65, 6-10. 5. See letter from N. Tomlinson, Joint Food Safety and Standards Group, MAFF, to US FDA, 4th Dec, 1998. -- Ron Cuthbertson -- The silver-list is a moderated forum for discussion of colloidal silver. 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