238

Exploitation of molecular proling techniques for GM food safety assessment

Harry A Kuipery, Esther J Kok and Karl-Heinz Engelz
Several strategies have been developed to identify unintended alterations in the composition of genetically modied (GM) food crops that may occur as a result of the genetic modication process. These include comparative chemical analysis of single compounds in GM food crops and their conventional non-GM counterparts, and proling methods such as DNA/RNA microarray technologies, proteomics and metabolite proling. The potential of proling methods is obvious, but further exploration of specicity, sensitivity and validation is needed. Moreover, the successful application of proling techniques to the safety evaluation of GM foods will require linked databases to be built that contain information on variations in proles associated with differences in developmental stages and environmental conditions.
Addresses RIKILT, Institute of Food Safety, PO Box 230, 6700 AE Wageningen, The Netherlands y e-mail: h.a.kuiper@rikilt.wag-ur.nl z Technische Universitat Munchen, Lehrstuhl fur Allgemeine Lebensmitteltechnologie, Am Forum 2, 85350 Freising-Weihenstephan, Germany

food crops, but it is emphasised that this phenomenon is not unique to GM organisms, it happens frequently in conventional plant breeding via point mutations as well as through chromosomal recombination mechanisms [1] (K-H Engel et al., unpublished results). Food safety evaluation strategies for GM crops have been designed and are internationally broadly accepted [24]. The concept of substantial equivalence was formulated as a comparative tool to identify similarities and differences between the GM food crop and its non-GM counterpart, which should be further assessed with respect to their potential impact on human and animal health. Kuiper et al. [5] have recently reviewed the issues involved in the safety evaluation of GM foods. This review deals with methods to identify unintended alterations that may occur in GM organisms as a result of the genetic modication. It highlights the activities of the European Thematic Network Safety Assessment of Genetically Modied Food Crops (ENTRANSFOOD), which among others is focussed on the development of new methods for safety evaluation of GM foods, including methods for the identication of unintended effects (Figure 1) [6,7].

Current Opinion in Biotechnology 2003, 14:238243 This review comes from a themed section on Food biotechnology Edited by David Archer and Mike Gasson 0958-1669/03/$ see front matter 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0958-1669(03)00021-1

Strategies for the identication of unintended effects

Different strategies may be adopted to identify unintended effects in GM food crops resulting from the genetic modication (Figure 2). The most direct way to predict unintended effects is by analysis of the transgene anking regions, to establish whether the insertion has taken place within or in proximity to an endogenous gene. Although information on genomes and the regulation of gene expression is still limited, sequencing of the place of insertion(s) will become more important as a tool to predict phenotypic changes in the modied organism. Possible alterations in the phenotype may be identied through a comparative analysis of growth performance, yield, disease resistance, chemical composition and so on. For spotting alterations in the composition of a GM organism compared with the parent, normally a targeted approach is used (i.e. measurements of single known compounds such as macronutrients, micronutrients, toxins or anti-nutrients, that is compounds that negatively affect the digestion of macronutrients like proteins or inhibit the gastro-intestinal uptake of essential elements). Analysis of the chemical composition represents an important part within the safety assessment framework of GM foods, as was demonstrated in the case of tubers
www.current-opinion.com

Abbreviations 2DGE two-dimensional gel electrophoresis FSA Foods Standards Agency GC gas chromatography GM genetically modied MS mass spectrometry NMR nuclear magnetic resonance

Introduction
The potential occurrence of unanticipated alterations in the composition of genetically modied (GM) food crops as a result of the genetic modication process is one of the key elements of the safety assessment procedure. Random insertion of genes into the genomic DNA of a host organism may, in addition to the intended effects, result in unexpected shifts in metabolic pathways leading to alterations in concentrations of nutrients and secondary metabolites or, in theory, even to the formation of new toxins. Unintended effects are known to occur in GM
Current Opinion in Biotechnology 2003, 14:238243

Molecular profiling techniques for GM food safety assessment Kuiper, Kok and Engel 239

Figure 1

New safety testing (SAFOTEST)

Detection of unintended effects (GMOCARE)

GMO detection (QPCRGMOFOOD, GMOCHIPS)

EUROPEAN THEMATIC NETWORK SAFETY ASSESSMENT OF GENETICALLY MODIFIED FOOD CROPS 'ENTRANSFOOD'

Gene transfer (GMOBILITY)

Working group safety testing of transgenic foods

www.entransfood.com

Working group W traceability and T quality assurance Working group consumer involvement

Working group detection of unintended effects

Working group gene transfer

Current Opinion in Biotechnology

Organisation of the EU funded Thematic Network ENTRANSFOOD in research projects (green ovals) and working groups (blue ovals).

from insect- and virus-resistant potato plants [8]. This study is a good example of an extensive analysis of major constituents (e.g. fats, proteins and carbohydrates) and of
Figure 2

Safety assessment of GM food

Identification of unintended effects

minor components (e.g. minerals, vitamins and specic toxins such as glycoalkaloids) and of protein composition. The aim of this work was to demonstrate that the composition of the GM potatoes is not different from conventional varieties. Statistically signicant differences between parental and GM lines that went beyond the intended effects of the genetic modication have been observed in two cases: an increased content of glycoalkaloids was seen in a GM potato variety expressing soybean glycinin [9] and an increase of vitamin B content was identied in GM rice expressing soybean glycinin [10]. This targeted approach has its limitations with respect to a restricted and biased selection of compounds that can be analysed. Furthermore, the detection of unknown toxicants or anti-nutrients is not possible using this method. To increase the chances of detecting unintended effects, proling methods have been proposed as a tool for characterising changes in the composition of GM plants [3,11]. This may be of particular relevance for GM food crops with improved nutritional or health-protecting properties, obtained through the insertion of multiple genes. This non-targeted approach using DNA/RNA microarray technology, proteomics and hyphenated (i.e. coupled) analytical techniques allows unbiased proling of possible changes in the physiology and metabolism of the modied host organism at different cellular integration levels. The potential of these methods for studying
Current Opinion in Biotechnology 2003, 14:238243

Targeted approach Analysis of: macronutrients micronutrients anti-nutrients toxins secondary metabolites

Non-targeted approach DNA microarray Proteomics Metabolomics

Current Opinion in Biotechnology

The identification of unintended affects resulting from genetic modification. The two different approaches and the techniques they employ are depicted. www.current-opinion.com

240 Food biotechnology

molecular genetics and physiology of plants has been discussed in a recent review [12].

Proling methods
Detection of altered gene expression

The development of microarray technology marks the most recent step in the history of gene expression analysis. This technology makes it feasible to monitor the expression of thousands of different genes simultaneously and to link detected differences directly to the underlying gene(s) [13,14]. The technology is now being applied [15] and tested in the medical area [16], plant biology [17,18], and human nutrition [19]. The most crucial part in the detection of altered gene expression using microarray technology is the construction of the array. Several array systems are available commercially that comprise large numbers of the expressed genes in specic organisms, but their number is still very limited especially in the area of food plants. Therefore, research projects have been initiated to develop informative arrays for the tomato and potato as model systems for food plants (GMOCARE project in progress [6], UK Foods Standards Agency [FSA] project in progress [20]). In the case of the tomato, cDNA libraries for use in construction of the array have been obtained that contain specic cDNAs for the green and red stages of ripening (EJ Kok, unpublished data). The green-specic library is likely to contain cDNAs that are related to the formation of natural toxins, such as tomatin, whereas the red-specic library will contain cDNAs that are associated with the metabolism of the positive factors in the tomato, such as vitamins and avonoids. The idea behind the construction of these specic cDNA libraries is that genetic modication may affect key metabolic pathways involved in the production of natural toxins and/or health-benecial compounds. Similarly, a potato library has been constructed that aims to further
Figure 3

elucidate the metabolic pathways of the potato natural toxins, especially the glycoalkaloids such as chaconine and solanine. Moreover, more elaborate potato libraries are being developed for both the tuber as well as for the potato plant. In this way the resulting array will not only be able to screen for altered gene expression in the tuber in pathways that are active in traditionally bred potato varieties, but it will also be able to detect potential activation of plant-part-related metabolic routes in the tuber. Proper selection of the mRNA populations to be hybridised is of key importance. It is necessary to sample in a reproducible way comparable tissues of plants that are in the same stage of development and have been grown under (near) identical environmental conditions. To evaluate the microarray uorescent patterns adequately it will be necessary to gain sufcient insight into the natural variation in gene expression during the different stages of development of the tissues of interest and under different environmental conditions. Detected differences in two plant lines (e.g. a GMO variety versus the traditionally bred parent line) can thus be evaluated against known background patterns. Work is now ongoing to document the natural background variation in specic tissues (Figure 3). The outcome of this analysis will largely determine the usefulness of microarray technology to screen for unintended effects in GM varieties associated with the genetic modication itself. Preliminary results show that, at the least, this methodology can effectively monitor the intended effects in GM tomato varieties. On this basis it seems likely that it will be possible to extend this approach to screen for unintended side-effects of the breeding process (EJ Kok, unpublished data).
Proteomics

Proteomics, that is, the study of the entire set of proteins present in a cell, organism or tissue under dened

(a)

(b)

(c)

(d)

Current Opinion in Biotechnology

Part of a microarray with expressed sequence tag sequences that are specific for the red stage of tomato ripening. The array has been hybridised with total RNA samples that were isolated from tomatoes in (a) green, (b) breaker, (c) light red and (d) red stages of ripening. The results reflect the increased expression of ripening-related genes towards the final red-ripe stage. Current Opinion in Biotechnology 2003, 14:238243 www.current-opinion.com

Molecular profiling techniques for GM food safety assessment Kuiper, Kok and Engel 241

conditions, is a well-established technique enabling analysis after transcript proling. The main approach currently applied involves two-dimensional gel electrophoresis (2DGE) followed by excision of protein spots from the gel, digestion into fragments by specic proteases, analysis by mass spectrometry (MS) and subsequent computerassisted identication of the fragments using databases [21]. The potential of proteomics to perform comparative analyses of protein patterns may be of importance for food safety assessment. This type of differential display proteomics has been applied to follow changes in concentrations of proteins or post-translational modications upon stimulation by environmental factors or genetic mutations [2224]. One of the major challenges is the quantication of proteins only highly expressed proteins are detected by 2DGE techniques [25] and the dynamic range of quantication is limited. Alternatives may be provided by quantication on the basis of isotope-coded afnity tags [26] or the use of multidimensional liquid chromatography coupled to tandem mass spectrometry [2729]. At present, the applicability of proteomic techniques is being studied within European multidisciplinary projects for the food safety evaluation of GM crops [7,20]. 2DGE protein proling is being tested on GM potato and tomato varieties (GMOCARE project in progress) and the potential of polypeptide fractionation and proling using multidimensional column chromatography and quantitative analysis of fractionated peptides using isotope-coded afnity tags and mass spectrometry are under investigation (British FSA project, in progress). The detection of differences related to the genetic modication might prove to be very difcult, taking into account the large number of proteins not connected to such changes and given the natural variations in protein patterns owing to different environmental conditions. The limited knowledge of the natural variability of plant protein patterns demands the development of validated databases and further validation of the methods. It might be useful to focus on proteins involved in important metabolic pathways and a combination of immunoblotting and protein microarrays may offer interesting possibilities in this respect.
Metabolite proling

in a single sample. Application is totally unbiased or targeted to metabolites in key metabolic pathways [33]. A metabolite proling methodology has been developed using rice as a model crop [34]. It involves fractionation of total rice extracts, thus enabling analysis of a broad spectrum of major and minor constituents. Proles of silylated/methylated compounds are obtained by means of GC coupled to ame ionization detection, and identication can be achieved by GC-MS. The applicability of GC-MS to the simultaneous analysis of a broad variety of polar and apolar metabolites in Arabidopsis thaliana leaves and potato tubers has been elegantly demonstrated [35,36]. This method was also used to characterise the genotypes of potatoes modied in sucrose metabolism [37]. The approach revealed the appearance of novel unexpected metabolites in chromatograms from transgenic tubers. Although GC results in highly resolved chromatograms and MS provides valuable information on the structural identity of compounds, the de novo identication of unknown metabolites is difcult. The necessity to derivatise compounds to make them volatile and the limited opportunities to collect them from the eluate of the GC column for further analysis are the main drawbacks of this approach. Metabolite proling methods applying liquid chromatography may help to overcome these limitations [38]. Off-line coupling of liquid chromatography to NMR was applied to the analysis of GM tomato varieties (with slow ripening characteristics achieved through antisense RNA exogalactanase modication) and to their non-modied counterpart [39,40]. 1 H-NMR spectra of pre-fractionated extracts revealed that a-lycopene was present in the antisense fruit at a concentration two to four times higher than that found in its parental line. This change is not an intended target of the modication, but presumably a consequence of the slow ripening process. NMR and different MS approaches are also being used in the GMOCARE and FSA projects already mentioned on the genetically modied varieties under study. The approaches applied so far for metabolite proling are powerful tools for unbiased analysis of a broad spectrum of metabolites and are a good compromise between comprehensiveness and specicity.
Data analysis

In order to identify in a GM food crop alterations in the content of cellular compounds such as sugars, fats, acids, and other metabolites that play an essential role in the metabolism, it is preferable to measure as many individual compounds as possible. To this end so-called metabolite proling approaches are being developed based on gas chromatography (GC), high-performance liquid chromatography, MS, nuclear magnetic resonance (NMR) or Fourier-transform (near) infrared spectroscopy [3032]. These methods are capable of detecting, identifying, resolving and quantifying a wide range of compounds
www.current-opinion.com

The application of proling techniques even to a limited number of samples results in a huge amount of data. A meaningful analysis of proles from a GM food and its non-GM counterpart, with respect to safety implications, should be based on the entirety of potential differences. For this reason, multivariate techniques, for example, principal component analysis (PCA) or hierarchical cluster analysis (HCA) are frequently applied [35,37]. The
Current Opinion in Biotechnology 2003, 14:238243

242 Food biotechnology

application of multivariate methods is useful, but discrimination between intended and unintended effects may not always be possible. The wealth of data generated through the application of proling methods demands a rigorous analysis with respect to their biological relevance. To this end a set up of linked databases containing gene expression, protein and metabolite proles, reecting different developmental stages and environmental conditions, is essential. Different approaches to establish such databases have been described [41,42]. Furthermore, standardisation of sampling procedures and inter-laboratory testing and validation of these methods is needed.

agriculture Organisation of the United Nations. URL: http:// www.fao.org/es/esn/gm/biotec-e.htm 5. Kuiper HA, Kleter GA, Noteborn HPJM, Kok EJ: Assessment of the food safety issues related to genetically modied foods. Plant J 2001, 27:503-528. ENTRANSFOOD: European network safety assessment of genetically modied food crops. URL: http://www. ENTRANSFOOD.com GMOCARE: European project new methodologies for assessing the potential of unintended effects in genetically modied food crops. URL: www.GMOCARE.com Rogan GJ, Bookout JT, Duncan DR, Fuchs RL, Lavrik PB, Love SL, Mueth M, Olson T, Owens ED, Raymond PJ, Zalewski J: Compositional analysis of tubers from insect and virus resistant potato plants. J Agric Food Chem 2000, 48:5936-5945. Hashimoto W, Momma K, Katsube T, Ohkawa Y, Ishige T, Kito M, Utsumi S, Murata K: Safety assessment of genetically engineered potatoes with designed soybean glycinin: compositional analyses of the potato tubers and digestibility of the newly expressed protein in transgenic potatoes. J Sci Food Agric 1999, 79:1607-1612.

6.

7.

8.

Conclusions
The application of molecular proling techniques for the safety assessment of GM foods may, in principle, provide relevant information regarding alterations in gene expression and associated metabolic consequences as a result of genetic modication. An unbiased, non-selective comparison of GM organisms and their traditional counterparts offers, in theory, almost unlimited possibilities for tracing differences at various integration levels of cells and tissues. However, a bottleneck for the successful application of these techniques is the generation of massive amounts of data for the evaluation of individual GM lines and the inherent difculties in making a meaningful interpretation. Also very important in this respect is the lack of validated interconnected databases containing information on variations in proles associated with relevant developmental stages and environmental conditions. The potential of proling methods to screen for unintended effects possibly associated with genetic modication are obvious, but further exploration with respect to specicity and sensitivity of the methods and validation is needed to demonstrate their usefulness for the safety evaluation of GM foods.

9.

10. Momma K, Hashimoto W, Ozawa S, Kawai S, Katsube T, Takaiwa F, Kito M, Utsumi S, Murata K: Quality and safety evaluation of genetically engineered rice with soybean glycinin: analyses of the grain composition and digestability of glycinin in transgenic rice. Biosci Biotechnol Biochem 1999, 63:314-318. 11. Kuiper HA, Noteborn HPJM, Pejinenburg AACM: Adequacy of methods for testing the safety of genetically modied foods. Lancet 1999, 354:1315-1316. 12. Fiehn O, Kloska S, Altmann T: Integrated studies on plant biology  using multiparallel techniques. Curr Opin Biotechnol 2001, 12:82-86. An excellent review on the possibilities and limitations of molecular proling methods for studies on the molecular genetics and physiology of plants. 13. Schena M, Shalon D, Heller R, Chai A, Brown PO, Davis RW: Parallel human genome analyis: microarray based expression monitoring of 1000 genes. Proc Natl Acad Sci USA 1996, 93:10614-10619. 14. Watson A, Mazumder A, Stewart M, Balasubramanian S: Technology for microarray analysis of gene expression. Curr Opin Biotechnol 1998, 9:609-614. 15. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC, Sabet H, Tran T, Yu X et al.: Distinct types of diffuse large B-cell lymphoma identied by gene expression proling. Nature 2000, 403:503-511. 16. Ziauddin J, Sabatini DM: Microarrays of cells expressing dened cDNAs. Nature 2001, 411:107-110. 17. Baldwin D, Crane V, Rice D: A comparison of gel-based, nylon lter and microarray techniques to detect differential RNA expression in plants. Curr Opin Plant Biol 1999, 2:96-103. 18. Aharoni A, Vorst O: DNA microarrays for functional plant  genomics. Plant Mol Biol 2001, 48:99-118. This article provides a good overview of the current situation in plant functional genomics. 19. Boeuf S, Klingenspor M, Van Hal NLW, Schneider T, Keijer J, Klaus S: Differential gene expression in white and brown preadipocytes. Physiol Genomics 2001, 7:15-25. 20. FSA project (Food Standards Agency): Transcriptome, proteome and metabolome analysis to detect unintended effects in genetically modied potato. URL: http://www. foodstandards.gov.uk/science/research/NovelFoodsResearch/ g02programme/g02projectlist/g02001/#re. 21. Anderson JS, Mann M: Functional genomics by mass spectrometry. FEBS Lett 2000, 480:25-31. 22. Thiellement H, Bahrman N, Damerval C, Plomion C, Rossignol M, Santoni M, de Vienne D, Zivy M: Proteomics of genetic and physiological studies in plants. Electrophoresis 1999, 20:2013-2026. www.current-opinion.com

Acknowledgements
The authors want to acknowledge the European Commission (DG Research) for nancial support of ENTRANSFOOD and GMOCARE, and the Dutch Ministry of Agriculture, Nature Conservation and Fisheries for nancial support of the research programme Food and Feed Safety (LNV 390).

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. 2. Mohan Jain S: Tissue culture-derived variation in crop improvement. Euphytica 2001, 118:153-166. OECD: Safety evaluation of foods derived by modern biotechnology. Paris, 1993. Organisation for Economic Cooperation and Development. URL: http://www.oecd.org/dsti/sti/ s_t/biotech/prod/modern.htm. FAO/WHO: Safety aspects of genetically modied foods of plant origin. Report of a joint FAO/WHO expert consultation on foods derived from biotechnology. World Health Organization: Geneva 2000. URL: http://www.fao.org/es/esn/gm/gm/biotec-e.htm FAO/WHO: Allergenicity of genetically modied foods. Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology, Rome, 2225 January 2001. Rome: Food and

3.

4.

Current Opinion in Biotechnology 2003, 14:238243

Molecular profiling techniques for GM food safety assessment Kuiper, Kok and Engel 243

23. Komatsu S, Muhammad A, Rakwal R: Separation and characterization of proteins from green and etiolated shoots of rice (Oryza sativa L.): towards a rice proteome. Electrophoresis 1999, 20:630-636. 24. Kehr J, Haebel S, Blechschmidt-Schneider S, Willmitzer L, Steup M, Fisahn J: Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. By matrix-assisted laser desorption/ionization time-of-ight mass spectrometry combined with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Planta 2001, 207:612-619. 25. Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R: Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci USA 2000, 97:9390-9395. 26. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R: Quantitative analysis of complex protein mixtures using isotope-coded afnity tags. Nat Biotechnol 1999, 17:994-999. 27. Jensen PK, Pasa-Tolic L, Peden KK, Martinovic S, Lipton MS, Anderson GA, Tolic N, Wong KK, Smith RD: Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures. Electrophoresis 2000, 21:1372-1380. 28. Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM, Yates JR: Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 1999, 17:676-682. 29. Zhang Z, McElvain JS: De novo peptide sequencing by twodimensional fragment correlation mass spectrometry. Anal Chem 2000, 72:2337-2350. 30. Raamsdonk LM, Teusink B, Broadhurst D, Zhang N, Hayes A, Walsh MC, Berden JA, Brindle KM, Kell DB, Rowland JJ et al.: A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations. Nat Biotechnol 2001, 19:45-50. 31. Johnson HE, Gilbert RJ, Winson MK, Goodacre R, Smith AR, Rowland JJ, Hall MA, Kell DB: Explanatory analysis of the metabolome using genetic programming of single interpretable rules. Genet Program Evolv Mach 2000, 1:243-258. 32. Taylor J, Goodacre R, Wade WG, Rowland JJ, Kell DB: The deconvolution of pyrolysis mass spectra using genetic programming: application to the identication of some Eubacterium species. FEMS Microbiol Lett 1998, 160:237-246. 33. Fraser PD, Pinto MES, Holloway DE, Bramley PM: Application of  high performance liquid chromatography with photodiode

array detection to the metabolic proling of plant isoprenoids. Plant J 2000, 24:551-558. An interesting example of HPLC for metabolic proling of isoprenoids in tomatoes and Arabidopsis. 34. Frenzel Th, Miller A, Engel KH: Metabolite proling a fractionation method for analysis of major and minor compounds in rice grains. Cereal Chemistry 2002, 79:215-221. 35. Fiehn O, Kopka J, Dormann P, Altmann T, Trethewey RN, Willmitzer L: Metabolite proling for plant functional genomics. Nat Biotechnol 2000, 18:1157-1161. 36. Roessner U, Wagner C, Kopka J, Trethewey RN, Willmitzer L: Simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry. Plant J 2000, 23:131-142. 37. Roessner U, Luedemann A, Brust D, Fiehn O, Linke T, Willmitzer L,  Fernie AR: Metabolic proling allows comprehensive phenotyping of genetically or environmentally modied plant systems. Plant Cell 2001, 13:11-29. Excellent example of metabolic proling using GC-MS for the phenotype analysis of genetically modied potatoes. 38. Tolstikov VV, Fiehn O: Analysis of highly polar compounds of plant origin: combination of hydrophilic interaction chromatography and electrospray ion trap mass spectrometry. Anal Biochem 2002, 301:298-307. 39. Noteborn HPJM, Lommen A, Weseman JM, van der Jagt RCM, Groenendijk FPF: Chemical ngerprinting and in vitro toxicological proling for the safety evaluation of transgenic food crops. In Report of the Demonstration Programme on Food Safety Evaluation of Genetically Modied Foods as a Basis for Market Introduction. Edited by Horning M. Ministry of Economic Affairs, The Hague, Netherlands, 1998:51-79. 40. Noteborn HPJM, Lommen A, van der Jagt RC, Weseman JM: Chemical ngerprinting for the evaluation of unintended secondary metabolic changes in transgenic food crops. J Biotechnol 2000, 77:103-114. 41. Mendes P: Emerging bioinformatics for the metabolome. Brief Bioinform 2002, 3:134-145. 42. Baxevanis AD: The molecular biology database collection. An  updated complication of biological database resources. Nucleic Acids Res 2001, 29:1-10. This paper contains an extensive list of over 200 different (functional) genomics databases.

www.current-opinion.com

Current Opinion in Biotechnology 2003, 14:238243