Verification of analytical methods for GMO testing when implementing interlaboratory validated methods: Version 2

In the EU, method validation is an essential part of the process that regulates the introduction of new GMOs as food and/or feed into the market. When the inter-laboratory validation study is completed, the method is ready to be implemented in routine testing laboratories. When implementing the new method, the laboratory has to verify that the method can be used for its intended purpose (method verification). The scope of this document is to provide guidance on how to carry out the method verification of inter-laboratory validated methods for the qualitative and quantitative detection of GMOs. Considering that the Polymerase Chain Reaction (PCR) is the method of choice in the EU for the identification and quantification of GMOs, this document refers exclusively to real time PCR. However, if novel methods are subsequently developed that fulfil legal requirements, then this document will be amended accordingly.JRC.F.5-Food and Feed Complianc

A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

Traditional chromatin analysis methods only test one locus at the time or use different templates for each locus, making a standardized analysis of large genomic regions or many co-regulated genes at different loci a difficult task. On the other hand, genome-wide high-resolution mapping of chromatin accessibility employing massive parallel sequencing platforms generates an extensive data set laborious to analyse and is a cost-intensive method, only applicable to the analysis of a limited set of biological samples. To close this gap between the traditional and the high-throughput procedures we have developed a method in which a condition-specific, genome-wide chromatin fragment library is produced and then used for locus-specific DNA fragment analysis. To validate the method, we used, as a test locus, the well-studied promoter of the divergently transcribed niiA and niaD genes coding for nitrate assimilation enzymes in Aspergillus. Additionally, we have used the condition-specific libraries to study nucleosomal positioning at two different loci, the promoters of the general nitrogen regulator areA and the regulator of secondary metabolism, aflR

Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length

Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR–protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein

Histone modifications and chromatin dynamics: a focus on filamentous fungi

The readout of the genetic information of eukaryotic organisms is significantly regulated by modifications of DNA and chromatin proteins. Chromatin alterations induce genome-wide and local changes in gene expression and affect a variety of processes in response to internal and external signals during growth, differentiation, development, in metabolic processes, diseases, and abiotic and biotic stresses. This review aims at summarizing the roles of histone H1 and the acetylation and methylation of histones in filamentous fungi and links this knowledge to the huge body of data from other systems. Filamentous fungi show a wide range of morphologies and have developed a complex network of genes that enables them to use a great variety of substrates. This fact, together with the possibility of simple and quick genetic manipulation, highlights these organisms as model systems for the investigation of gene regulation. However, little is still known about regulation at the chromatin level in filamentous fungi. Understanding the role of chromatin in transcriptional regulation would be of utmost importance with respect to the impact of filamentous fungi in human diseases and agriculture. The synthesis of compounds (antibiotics, immunosuppressants, toxins, and compounds with adverse effects) is also likely to be regulated at the chromatin level

Community profiling and gene expression of fungal assimilatory nitrate reductases in agricultural soil

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the ∼100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium

Detection Methods Fit-for-Purpose in Enforcement Control of Genetically Modified Plants Produced with Novel Genomic Techniques (NGTs)

The comprehensive EU regulatory framework regarding GMOs aims at preventing damage to human and animal health and the environment, and foresees labelling and traceability. Genome-edited plants and products fall under these EU GMO regulations, which have to be implemented in enforcement control activities. GMO detection methods currently used by enforcement laboratories are based on real-time PCR, where specificity and sensitivity are important performance parameters. Genome editing allows the targeted modification of nucleotide sequences in organisms, including plants, and often produces single nucleotide variants (SNVs), which are the most challenging class of genome edits to detect. The test method must therefore meet advanced requirements regarding specificity, which can be increased by modifying a PCR method. Digital PCR systems achieve a very high sensitivity and have advantages in quantitative measurement. Sequencing methods may also be used to detect DNA modifications caused by genome editing. Whereas most PCR methods can be carried out in an enforcement laboratory with existing technical equipment and staff, the processing of the sequencing data requires additional resources and the appropriate bioinformatic expertise

The GATA factor AreA is essential for chromatin remodelling in a eukaryotic bidirectional promoter.

The linked niiA and niaD genes of Aspergillus nidulans are transcribed divergently. The expression of these genes is subject to a dual control system. They are induced by nitrate and repressed by ammonium. AreA mediates derepression in the absence of ammonium and NirA supposedly mediates nitrate induction. Out of 10 GATA sites, a central cluster (sites 5-8) is responsible for approximately 80% of the transcriptional activity of the promoter on both genes. We show occupancy in vivo of site 5 by the AreA protein, even under conditions of repression. Sites 5-8 are situated in a pre-set nucleosome-free region. Under conditions of expression, a drastic nucleosomal rearrangement takes place and the positioning of at least five nucleosomes flanking the central region is lost. Remodelling is strictly dependent on the presence of an active areA gene product, and independent from the NirA-specific and essential transcription factor. Thus, nucleosome remodelling is independent from the transcriptional activation of the niiA-niaD promoter. The results presented cast doubts on the role of NirA as the unique transducer of the nitrate induction signal. We demonstrate, for the first time in vivo, that a GATA factor is involved directly in chromatin remodelling