DNA microarray to detect and identify trichothecene- and moniliformin-producing Fusarium species.

AIMS: To develop a DNA microarray for easy and fast detection of trichothecene- and moniliformin-producing Fusarium species. METHOD AND RESULTS: A DNA microarray was developed for detection and identification of 14 trichothecene- and moniliformin-producing species of the fungal genus Fusarium. The array could also differentiate between four species groups. Capture probes were designed based on recent phylogenetic analyses of translation elongation factor-1 alpha (TEF-1alpha) sequences. Particular emphasis was put on designing capture probes corresponding to groups or species with particular mycotoxigenic synthetic abilities. A consensus PCR amplification of a part of the TEF-1alpha is followed by hybridization to the Fusarium chip and the results are visualized by a colorimetric Silverquant detection method. We validated the Fusarium chip against five naturally infected cereal samples for which we also have morphological and chemical data. The limit of detection was estimated to be less than 16 copies of genomic DNA in spiked commercial wheat flour. CONCLUSIONS: The current Fusarium chip proved to be a highly sensitive and fast microarray for detection and identification of Fusarium species. We postulate that the method also has potential for (semi-)quantification. SIGNIFICANCE AND IMPACT OF THE STUDY: The Fusarium chip may prove to be a very valuable tool for screening of cereal samples in the food and feed production chain, and may facilitate detection of new or introduced Fusarium spp.

Simultaneous detection and identification of trichothecene- and moniliformin-producing Fusarium species based on multiplex SNP analysis.

AIM: To develop a multiplex identification method for trichothecene- and moniliformin-producing Fusarium species. METHOD AND RESULTS: In this article, we present a single nucleotide polymorphism (SNP) assay to simultaneously detect and identify 16 trichothecene- and moniliformin-producing Fusarium species. A number of SNP primers are designed to detect clades of species with particular mycotoxigenic synthetic abilities. The assay is based on minisequencing using SNaPshot reactions and the SNP primers are designed based on motifs derived from phylogenetic analyses of translation elongation factor-1alpha sequences. The present version of the Fusarium SNP assay can distinguish major groups of trichothecene producers; the strict-type-A, the strict-type-B, the type-A and type-B trichothecene producers and the putative moniliformin producers. The SNP assay was validated against five naturally infected cereal samples that previously had been analysed morphologically, chemically and by a multiplex DNA array hybridization. CONCLUSIONS: The Fusarium SNP assay reveals the advantages of using SNPs for multiplex species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The current assay may qualify as a high-throughput screening method for small-grain cereals in the feed and food chain, and may facilitate detection of new or introduced Fusarium species.

Detection and traceability of genetically modified organisms in the food production chain.

Both labelling and traceability of genetically modified organisms are current issues that are considered in trade and regulation. Currently, labelling of genetically modified foods containing detectable transgenic material is required by EU legislation. A proposed package of legislation would extend this labelling to foods without any traces of transgenics. These new legislations would also impose labelling and a traceability system based on documentation throughout the food and feed manufacture system. The regulatory issues of risk analysis and labelling are currently harmonised by Codex Alimentarius. The implementation and maintenance of the regulations necessitates sampling protocols and analytical methodologies that allow for accurate determination of the content of genetically modified organisms within a food and feed sample. Current methodologies for the analysis of genetically modified organisms are focused on either one of two targets, the transgenic DNA inserted- or the novel protein(s) expressed- in a genetically modified product. For most DNA-based detection methods, the polymerase chain reaction is employed. Items that need consideration in the use of DNA-based detection methods include the specificity, sensitivity, matrix effects, internal reference DNA, availability of external reference materials, hemizygosity versus homozygosity, extrachromosomal DNA, and international harmonisation. For most protein-based methods, enzyme-linked immunosorbent assays with antibodies binding the novel protein are employed. Consideration should be given to the selection of the antigen bound by the antibody, accuracy, validation, and matrix effects. Currently, validation of detection methods for analysis of genetically modified organisms is taking place. In addition, new methodologies are developed, including the use of microarrays, mass spectrometry, and surface plasmon resonance. Challenges for GMO detection include the detection of transgenic material in materials with varying chromosome numbers. The existing and proposed regulatory EU requirements for traceability of genetically modified products fit within a broader tendency towards traceability of foods in general and, commercially, towards products that can be distinguished from each other. Traceability systems document the history of a product and may serve the purpose of both marketing and health protection. In this framework, segregation and identity preservation systems allow for the separation of genetically modified and non-modified products from "farm to fork". Implementation of these systems comes with specific technical requirements for each particular step of the food processing chain. In addition, the feasibility of traceability systems depends on a number of factors, including unique identifiers for each genetically modified product, detection methods, permissible levels of contamination, and financial costs. In conclusion, progress has been achieved in the field of sampling, detection, and traceability of genetically modified products, while some issues remain to be solved. For success, much will depend on the threshold level for adventitious contamination set by legislation.

Societal aspects of genetically modified foods.

This paper aims to examine some of the reasons behind public controversy associated with the introduction of genetically modified foods in Europe the 1990s. The historical background to the controversy is provided to give context. The issue of public acceptance of genetically modified foods, and indeed the emerging biosciences more generally, is considered in the context of risk perceptions and attitudes, public trust in regulatory institutions, scientists, and industry, and the need to develop communication strategies that explicitly include public concerns rather than exclude them. Increased public participation has been promoted as a way of increasing trust in institutional practices associated with the biosciences, although questions still arise as to how to best utilise the outputs of such exercises in policy development. This issue will become more of a priority as decision-making systems become more transparent and open to public scrutiny. The results are discussed in the context of risk assessment and risk management, and recommendations for future research are made. In particular, it is recommended that new methods are developed in order to integrate public values more efficaciously into risk analysis processes, specifically with respect to the biosciences and to technology implementation in general.

Release of normal bases from intact DNA by a native DNA repair enzyme.

Base excision repair is initiated by DNA glycosylases removing inappropriate bases from DNA. One group of these enzymes, comprising 3-methyladenine DNA glycosylase II (AlkA) from Escherichia coli and related enzymes from other organisms, has been found to have an unusual broad specificity towards quite different base structures. We tested whether such enzymes might also be capable of removing normal base residues from DNA. The native enzymes from E.coli, Saccharomyces cerevisiae and human cells promoted release of intact guanines with significant frequencies, and further analysis of AlkA showed that all the normal bases can be removed. Transformation of E. coli with plasmids expressing different levels of AlkA produced an increased spontaneous mutation frequency correlated with the expression levels, indicating that excision of normal bases occurs at biologically significant rates. We propose that the broad specificity 3-methyladenine DNA glycosylases represent a general type of repair enzyme 'pulling' bases in DNA largely at random, without much preference for a specific structure. The specificity for release of damaged bases occurs because base structure alterations cause instability of the base-sugar bonds. Damaged bases are therefore released more readily than normal bases once the bond activation energy is reduced further by the enzyme. Qualitatively, the model correlates quite well with the relative rate of excision observed for most, if not all, of the substrates described for AlkA and analogues.

Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli.

One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We have analyzed the function of this gene, now assigned NTG1 (endonuclease three-like glycosylase 1), by cloning, mutant analysis, and gene expression in E. coli. Targeted gene disruption of NTG1 produces a mutant that is sensitive to H2O2 and menadione, indicating that NTG1 is required for repair of oxidative DNA damage in vivo. Northern blot analysis and expression studies of a NTG1-lacZ gene fusion showed that NTG1 is induced by cell exposure to different DNA damaging agents, particularly menadione, and hence belongs to the DNA damage-inducible regulon in S. cerevisiae. When expressed in E. coli, the NTG1 gene product cleaves plasmid DNA damaged by osmium tetroxide, thus, indicating specificity for thymine glycols in DNA similarly as is the case for EndoIII. However, NTG1 also releases formamidopyrimidines from DNA with high efficiency and, hence, represents a glycosylase with a novel range of substrate recognition. Sequences similar to NTG1 from other eukaryotes, including Caenorhabditis elegans, Schizosaccharomyces pombe, and mammals, have recently been entered in the GenBank suggesting the universal presence of NTG1-like genes in higher organisms. S. cerevisiae NTG1 does not have the [4Fe-4S] cluster DNA binding domain characteristic of the other members of this family.

Cloning and expression in Escherichia coli of a gene for an alkylbase DNA glycosylase from Saccharomyces cerevisiae; a homologue to the bacterial alkA gene.

An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase activity for removal of alkylated bases, was transformed by a genomic yeast DNA library and clones selected which survived plating on medium containing the alkylating agent methylmethane sulphonate. Three distinct yeast clones were identified which were able to suppress the alkylation sensitive phenotype of the bacterial mutant. Restriction enzyme analysis revealed common DNA fragments present in all three clones spanning 2 kb of yeast DNA. DNA from this region was sequenced and analysed for possible translation of polypeptides with any homology to either the Tag or the AlkA DNA glycosylases of E. coli. One open reading frame of 296 amino acids was identified encoding a putative protein with significant homology to AlkA. DNA containing the open reading frame was subcloned in E. coli expression vectors and cell extracts assayed for alkylbase DNA glycosylase activity. It appeared that such activity was expressed at levels sufficiently high for enzyme purification. The molecular weight of the purified protein was determined by SDS-PAGE to be 35,000 daltons, in good agreement with the 34,340 value calculated from the sequence. The yeast enzyme was able to excise 7-methylguanine as well as 3-methyladenine from dimethyl sulphate treated DNA, confirming the related nature of this enzyme to the AlkA DNA glycosylase from E. coli.

Chédiak-Higashi syndrome. Neurologic appearance.

A 25-year-old man was first seen with a neurologic disorder that resembled a spinocerebellar degeneration and parkinsonism. A peripheral smear revealed the characteristic peroxidase-positive panleukocytic granules associated with the Chédiak-Higashi syndrome. He did not have any associated oculocutaneous abnormalities. The Chédiak-Higashi syndrome may appear primarily with neurologic dysfunction and should be considered in a differential diagnosis of children and young adults first seen with a spinocerebellar degeneration or movement disorder.