Insertion Of Foreign DNA Research Articles

Disrupted mitochondrial response to nutrients is a presymptomatic event in the cortex of the APPSAA knock-in mouse model of Alzheimer's disease.

Reduced brain energy metabolism, mammalian target of rapamycin (mTOR) dysregulation, and extracellular amyloid beta (Aβ) oligomer (xcAβO) buildup are some well-known Alzheimer's disease (AD) features; how they promote neurodegeneration is poorly understood. We previously reported that xcAβOs inhibit nutrient-induced mitochondrial activity (NiMA) in cultured neurons. We now report NiMA disruption in vivo. Brain energy metabolism and oxygen consumption were recorded in heterozygous amyloid precursor protein knock-in (APPSAA) mice using two-photon fluorescence lifetime imaging and multiparametric photoacoustic microscopy. NiMA is inhibited in APPSAA mice before other defects are detected in these Aβ-producing animals that do not overexpress APP or contain foreign DNA inserts into genomic DNA. Glycogen synthase kinase 3 (GSK3β) signals through mTORC1 to regulate NiMA independently of mitochondrial biogenesis. Inhibition of GSK3β with TWS119 stimulates NiMA in cultured human neurons, and mitochondrial activity and oxygen consumption in APPSAA mice. NiMA disruption in vivo occurs before plaques, neuroinflammation, and cognitive decline in APPSAA mice, and may represent an early stage in human AD. Amyloid beta blocks communication between lysosomes and mitochondria in vivo. Nutrient-induced mitochondrial activity (NiMA) is disrupted long before the appearance of Alzheimer's disease (AD) histopathology in heterozygous amyloid precursor protein knock-in (APPSAA/+) mice. NiMA is disrupted long before learning and memory deficits in APPSAA/+ mice. Pharmacological interventions can rescue AD-related NiMA disruption in vivo.

Sequencing of the complete plastome of the thermal adder’s-tongue fern, Ophioglossum thermale Kom. (Ophioglossaceae)

The Ophioglossaceae family, one of the oldest orders of extant ferns, exhibits diverse morphological and chromosomal characteristics. This study presents the first complete plastome sequence of thermal adder’s-tongue fern (Ophioglossum thermale), a species renowned for its antioxidant properties in traditional Chinese medicine. Our analyses revealed 27 simple sequence repeats (SSRs) in the plastome, with variations in SSR frequencies compared to related genera. Our phylogenetic analyses placed O. thermale within the Ophioglossum s.s. clade, supporting previous studies and suggesting polyphyly within the genus Ophioglossum based on the sensu PPG I system. The enlarged noncoding regions in fern organelles (ENRFOs) resulting from foreign DNA insertions in O. thermale were identified in the ycf2-trnH and trnT-trnfM regions, similar to other Ophioglossum species. ENRFOs were found at the LSC and SSC, but not in IRs in Ophioglossaceae. Consequently, foreign DNA insertions and lineage-specific SSRs shed light on plastome evolution in the Ophioglossaceae family.

A comprehensive exploration of restriction enzymes and their applications in molecular biology: A review

Restriction enzymes, derived from bacteria, serve as indispensable tools in molecular biology, enabling precise manipulation of DNA with specificity. These enzymes recognize specific DNA sequences, cleaving the DNA strand at those designated sites. Two types, blunt-end cutters, and sticky-end producers, offer distinct advantages and disadvantages. In molecular biology, restriction enzymes find diverse applications, prominently in gene cloning, facilitating the insertion of foreign DNA into host organisms. Additionally, they play a vital role in DNA sequencing, DNA fingerprinting, and other techniques crucial for studying gene function and regulation. A groundbreaking development is witnessed in gene editing, where engineered enzymes recognize specific DNA sequences, allowing scientists to target and modify genes with unprecedented precision. This breakthrough holds the potential to revolutionize medicine, paving the way for treating genetic diseases and creating personalized therapies. Research efforts are focused on discovering new restriction enzymes with novel specificities, expanding the range of manipulable DNA sequences. This opens avenues for innovative applications in synthetic biology and biotechnology, further advancing the field. Despite their numerous applications, challenges persist, including the potential for off-target effects and the quest for enzymes with specific recognition sequences. Ongoing research and development continue to push the boundaries of what is achievable with restriction enzymes. In conclusion, restriction enzymes have significantly impacted molecular biology and biotechnology, facilitating the precise manipulation and study of genetic material. Ongoing research promises to unveil new applications and discoveries in this dynamic and promising field.

Molecular Characterization of Chimeric Staphylococcus aureus Strains from Waterfowl.

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain's genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying "bird-specific" virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA.

Knockout of the tomato translational elongation factor using CRISPR-Cas9 technology

Pathogenic viruses cause severe tomato losses around the world despite the development of both classical breeding and biotechnological methods. Since replication of phytoviruses involves the interaction between viral components and host plant factors, therefore loss-of-function mutations in the latters can confer viral resistance in plants. There are evidences that eukaryotic translation elongation factor 1 (eEF1) proteins are involved in the replication of some plant viruses. However, the involvement of individual subunits of the eEF1B in the viral cycle is still poorly understood.
 This work is devoted to the study of the role of the eEF1B factor in the development of tomato virus infection. The contribution of each of the α, β and γ subunits of the eEF1B factor to tomato viral resistance will be determined by CRISPR-Cas9-induced targeted mutagenesis of corresponding gene sequences. As an applied aspect, we expect to find ways to create tomato plants with increased resistance to certain viral diseases. A series of binary vectors contained sequences encoded different RNAs targeting the eEF1B subunit genes was constructed. As a result of Agrobacterium-mediated transformation of tomato, more than 300 independent transgenic lines were obtained. The presence of expression cassettes with functional genes (Cas9 and sgRNAs) was confirmed by PCR. The presence of mutations in target sequences was detected using T7E1 analysis and sequencing. It turned out that the majority of transgenic lines carrying mutations have a chimeric genotype, and mutations of the target genes in the homozygous state were not detected. The propagation of self-pollinated transgenic plants under greenhouse condition and following analyses of target genes to segregate the insertion of foreign DNA and obtain homozygous mutations in eEF1B subunit sequences are in progress.

Loss of Foreign DNA Inserts from Barley Stripe Mosaic Virus Vectors—Potential Consequences for Use in Functional Genomics Studies

Barley stripe mosaic virus (BSMV)-based vectors are frequently used in virus-induced gene silencing (VIGS) and, more recently, viral overexpression (VOX) studies in wheat. Two general strategies are employed to initiate infection in wheat plants with BSMV in VIGS and VOX studies. One method involves the direct infection of wheat using viral RNA produced via the in vitro transcription of BSMV constructs. The second class utilizes viral replication in an intermediate host plant to produce large amounts of BSMV viral particles that are then used to inoculate wheat plants. This study was designed to examine the potential for BSMV-VIGS constructs to rearrange during replication in the intermediate host and result in initiating the VIGS studies with a virus that is significantly different from the original experimental construct. It is shown that in the case of BSMV-VIGS constructs harboring a PDS-silencing fragment, significant rearrangement can occur during replication in the intermediate host that has the potential to introduce artifactual experimental outcomes.

Considerations in engineering viral vectors for genome editing in plants

Plant viruses have been engineered to express proteins and induce gene silencing for decades. Recently, plant viruses have also been used to deliver components into plant cells for genome editing, a technique called virus-induced genome editing (VIGE). Although more than a dozen plant viruses have been engineered into VIGE vectors and VIGE has been successfully accomplished in some plant species, application of VIGE to crops that are difficult to tissue culture and/or have low regeneration efficiency is still tough. This paper discusses factors to consider for an ideal VIGE vector, including insertion capacity for foreign DNA, vertical transmission ability, expression level of the target gene, stability of foreign DNA insertion, and biosafety. We also proposed a step-by-step schedule for excavating the suitable viral vector for VIGE.

DNA Sequence Analysis of an Inversion Hot Spot in Lobeliaceae Plastomes

The evolution of plastid genomes (plastomes) in land plants is typically conservative, with extensive structural rearrangements present in only a few groups. Early Southern blot analysis identified two Lobelia species that minimally required deletion of the plastid gene accD and five inversions to account for their plastome arrangement relative to the ancestral organization. Sixty alternative 5-step inversion scenarios could account for the observed arrangement, but only one scenario was consistent with the criterion of 'common cause' attributable to a putative rearrangement hot spot at the accD deletion-site. Plastome sequencing demonstrated that this previously hypothesized inversion order is historically accurate. Detailed reconstructions of the ancestral plastome organization before and after each inversion are presented herein. Stem-loop and disruption-rescue models were evaluated for each inversion. One inversion has an obvious stem-loop basis, but the other four inversions were primarily caused by serial insertion of foreign (extra-plastid) DNA bearing large open-reading frames that disrupted plastome organization at the accD deletion-site, and complete plastomes were rescued by seemingly arbitrary ligation or fortuitous recombination at the other inversion endpoint. Transposed copies of DNA segments from elsewhere in the plastome are frequently inserted at inversion junctions, and four junctions are consistent with the stem-loop ligation model.

CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch

Locus-directed DNA cleavage induced by the CRISPR-Cas9 system triggers DNA repair mechanisms allowing gene repair or targeted insertion of foreign DNA. For gene insertion to be successful, availability of a homologous donor template needs to be timed with cleavage of the DNA by the Cas9 endonuclease guided by a target-specific single guide RNA (sgRNA). We present a novel approach for targeted gene insertion based on a single integrase-defective lentiviral vector (IDLV) carrying a Cas9 off switch. Gene insertion using this approach benefits from transposon-based stable Cas9 expression, which is switched off by excision-only transposase protein co-delivered in IDLV particles carrying a combined sgRNA/donor vector. This one-vector approach supports potent (up to >80%) knockin of a full-length EGFP gene sequence. This traceless cell engineering method benefits from high stable levels of Cas9, timed intracellular availability of the molecular tools, and a built-in feature to turn off Cas9 expression after DNA cleavage. The simple technique is based on transduction with a single IDLV, which holds the capacity to transfer larger donor templates, allowing robust gene knockin or tagging of genes in a single step.

Advances in Crop Breeding Through Precision Genome Editing.

The global climate change and unfavourable abiotic and biotic factors are limiting agricultural productivity and therefore intensifying the challenges for crop scientists to meet the rising demand for global food supply. The introduction of applied genetics to agriculture through plant breeding facilitated the development of hybrid varieties with improved crop productivity. However, the development of new varieties with the existing gene pools poses a challenge for crop breeders. Genetic engineering holds the potential to broaden genetic diversity by the introduction of new genes into crops. But the random insertion of foreign DNA into the plant’s nuclear genome often leads to transgene silencing. Recent advances in the field of plant breeding include the development of a new breeding technique called genome editing. Genome editing technologies have emerged as powerful tools to precisely modify the crop genomes at specific sites in the genome, which has been the longstanding goal of plant breeders. The precise modification of the target genome, the absence of foreign DNA in the genome-edited plants, and the faster and cheaper method of genome modification are the remarkable features of the genome-editing technology that have resulted in its widespread application in crop breeding in less than a decade. This review focuses on the advances in crop breeding through precision genome editing. This review includes: an overview of the different breeding approaches for crop improvement; genome editing tools and their mechanism of action and application of the most widely used genome editing technology, CRISPR/Cas9, for crop improvement especially for agronomic traits such as disease resistance, abiotic stress tolerance, herbicide tolerance, yield and quality improvement, reduction of anti-nutrients, and improved shelf life; and an update on the regulatory approval of the genome-edited crops. This review also throws a light on development of high-yielding climate-resilient crops through precision genome editing.

Abstract 3293: In vitro cellular and molecular effects of oncolytic vaccinia virus in clear and non-clear cell renal cell carcinoma

Abstract Oncolytic vaccinia virus has several advantages over other OV platforms, including an efficient life cycle, multiple mechanisms of viral spread, large genome, allowing acceptance of foreign therapeutic DNA inserts, and proven safety in humans, due to its use as smallpox vaccine. JX-594 (Pexavec, SillaJen) is an attenuated (TKdeactivated ) oncolytic vaccinia VV, engineered to express GM-CSF and to selectively target tumor over non-tumor tissues. It is currently being investigated as a novel therapeutic option in patients with advanced Renal Cell Carcinoma (RCC, NCT03294083). The present study was undertaken to investigate the effects of JX-954 in a panel of clear and non-clear cell RCC cell lines and characterize the potential mechanisms of JX-594 oncolysis in vitro. We evaluated the oncolytic potency of JX-594 in human clear cell RCCs (VHL mutant 786-0, A498, VHL wild type Caki-1), non-clear cell RCC (ACHN, papillary RCC, and UOK-262, an FH deficient papillary RCC cell) and murine renal cancer cells (RENCA). Potent and persistent cytotoxic effects were induced by JX-594 in all human RCC cells, regardless of the histological subtype, and by mJX-594 (a Western Reserve strain of vaccinia virus expressing murine GMCSF) in RENCA cells. Moreover, we observed efficient viral replication in human (786-0, ACHN) and murine (RENCA) cells, confirming RCC permissiveness to viral infection. Functional proteomics analysis of JX-594 treated 786-0 cells was performed by reverse phase protein array (RPPA) to gain knowledge on potential mechanisms of viral oncolysis. JX-594 treatment had significant effects on the expression levels of proteins related to inducing cell cycle, apoptosis, necrosis, Akt signaling, MAPK signaling, autophagy and stress induction. In particular, the proteins related to cell cycle (Wee1, Cdc25c, CHK1/2, and Cyclin B1) and Akt signaling (PI3K, mTOR, Rictor and FOXO), MAPK signaling (JAK2, PAK1, FAK and MEK1) were down regulated, while proteins involved in apoptosis and necrosis were upregulated. In summary, our studies show that human and murine renal cancer cells are permissive to infection and replication by JX-594 and mJX-594, which induce potent oncolytic effects in clear and non-clear cell RCC, effects associated with significant modulation of RCC pathways associated with cell cycle, proliferation, and survival and stress responses. Citation Format: Yuqi Jing, F Bustamante Guerrero, Jaime Merchan. In vitro cellular and molecular effects of oncolytic vaccinia virus in clear and non-clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3293.

A Brief Overview for the Development of Herbicide-Resistant Sugarcane Transformation Approaches


 Weeds are the undesirable and unwanted plants in the wrong place in a situation that can significantly decrease the yield of desirable plant such as sugarcane. One solution to tackle this problem is the development of herbicide resistant crops like sugar cane that are highly resistant to several herbicides or broad-spectrum herbicides which is done in most cases by genetic transformation, site-directed mutagenesis, and plant breeding. Sugarcane (Saccharum officinarum) is the 2nd vital cash crop of Pakistan, placed at 6th position in world cane acreage and 15th in sugar production. Hence, sugarcane occupies a crucial position in the economy of Pakistan and acts like a backbone in economic development of country. There are several reasons for the lower sugarcane yield in Pakistan but the most important is the large number of weeds that covered most of the area of sugarcane cultivated fields which results in major loss of sugarcane yield in terms of quality and quantity. Broad spectrum herbicide resistant plants are produced to tackle this issue by the insertion of CP4 EPSPS (Glyphosate tolerant gene; 5-enolpyruvulshikimate-3-phosphate synthase from Agrobacterium tumefaciens strain CP4) into sugarcane which provides them the ability to survive after the spray of glyphosate but weeds do not survive in this condition. The most common herbicide resistant technology is Glyphosate resistant technology which gives broad-spectrum weed control feasibility along with the flexibility in the application time of herbicide. There are numerous methods of transformation which are available now-a-days for insertion of foreign DNA into plant cells like Agrobacterium-mediated transformation, micro-projectile bombardment (gene gun) and protoplast transformation. Glyphosate tolerant gene expression is showed by three sugarcane cultivars (CPF-213, SPF-234 and HSF-240). Transgenic sugarcane plants showed these expressions are most stable against herbicide to control weeds. Several farmers in many countries have rapidly and extensively utilized herbicide-tolerant crops due to low production and labour costs, huge profit, increased weed control and many environmental benefits.

Human Adenovirus Serotypes Efficiently Transducing HEK293 Cells: An In Vitro Propagation of HAdv

Generally, a gene which is inserted directly into a cell does not operate independently. Instead, the transmission of the gene is genetically modified by a biological messenger called a vector, consists of a transgene and a large DNA sequence as a backbone. Since they can deliver the new gene by infecting the cell, such viruses are also used as vectors. The adenovirus is a non-enveloped virus that can be tailored to transfer DNA to target cells, and it has sparked a lot of interest in the field, particularly in clinical trial therapy techniques. For the new age production of COVID-19 vaccine, development of different mammalian cell lines like HEK293 (most reliable growth and prosperity for transfection) and recombinant adenoviral vectors have become the first priority for biopharmaceutical giants and globally approved vaccine manufacturers to scale up their vaccine production. Adenoviruses have an icosahedral shape, with a protein coat encasing the viral double-stranded DNA genome. Because the adenovirus genome is relatively small, it's a good candidate for insertion of foreign DNA. The adenovirus E1A gene is deleted, and the virus loses its capacity to replicate. This ability can be restored during cell culture propagation by employing cells that produce the E1A protein, for example. Hence, in this mini research, I have shared an overview of the propagation of adenoviral vectors, i.e. recombinant adenovirus SARS CoV-2 vector in HEK-293 cell suspension culture.

Protoplast-Based Method for Genome Editing in Tetraploid Potato.

The cultivated potato is tetraploid with four probably equivalent loci for each gene. A potato variety is furthermore commonly genetically heterogeneous and selected based on a beneficial genetic context which is maintained by clonal propagation. When introducing genetic changes by genome editing it is then desirable to achieve edits in all four loci for a certain gene target. This is in order to avoid crosses to achieve homozygosity for edited gene loci and at the same time reduce risk of inbreeding depression. In such a context transient transfection of protoplasts for the introduction of mutations, avoiding stable insertion of foreign DNA, would be very attractive. The protocol of this chapter has been shown to be applicable for the introduction of mutations by DNA vectors containing expression cassettes of TALEN, Cas9, and Cas9 deaminase fusions together with sgRNA expression cassettes on either single or separate vectors. Furthermore, the protoplast-based system has been shown to work very efficiently for mutations introduced by in vitro-produced and transfected RNP (ribonucleoprotein) complexes.

Comparative genome analyses of Mycobacteroides immunogenum reveals two potential novel subspecies.

Mycobacteroides immunogenum is an emerging opportunistic pathogen implicated in nosocomial infections. Comparative genome analyses may provide better insights into its genomic structure, functions and evolution. The present analysis showed that M. immunogenum has an open pan-genome. Approximately 36.8% of putative virulence genes were identified in the accessory regions of M. immunogenum . Phylogenetic analyses revealed two potential novel subspecies of M. immunogenum , supported by evidence from ANIb (average nucleotide identity using blast) and GGDC (Genome to Genome Distance Calculator) analyses. We identified 74 genomic islands (GIs) in Subspecies 1 and 23 GIs in Subspecies 2. All Subspecies 2-harboured GIs were not found in Subspecies 1, indicating that they might have been acquired by Subspecies 2 after their divergence. Subspecies 2 has more defence genes than Subspecies 1, suggesting that it might be more resistant to the insertion of foreign DNA and probably explaining why Subspecies 2 has fewer GIs. Positive selection analysis suggest that M. immunogenum has a lower selection pressure compared to non-pathogenic mycobacteria. Thirteen genes were positively selected and many were involved in virulence.

CAPRIB: a user-friendly tool to study amino acid changes and selection for the exploration of intra-genus evolution

BackgroundThe evolution of bacteria is shaped by different mechanisms such as mutation, gene deletion, duplication, or insertion of foreign DNA among others. These genetic changes can accumulate in the descendants as a result of natural selection. Using phylogeny and genome comparisons, evolutionary paths can be somehow retraced, with recent events being much easier to detect than older ones. For this reason, multiple tools are available to study the evolutionary events within genomes of single species, such as gene composition alterations, or subtler mutations such as SNPs. However, these tools are generally designed to compare similar genomes and require advanced skills in bioinformatics. We present CAPRIB, a unique tool developed in Java that allows to determine the amino acid changes, at the genus level, that correlate with phenotypic differences between two groups of organisms.ResultsCAPRIB has a user-friendly graphical interface and uses databases in SQL, making it easy to compare several genomes without the need for programming or thorough knowledge in bioinformatics. This intuitive software narrows down a list of amino acid changes that are concomitant with a given phenotypic divergence at the genus scale. Each permutation found by our software is associated with two already described statistical values that indicate its potential impact on the protein’s function, helping the user decide which promising candidates to further investigate. We show that CAPRIB is able to detect already known mutations and uncovers many more, and that this tool can be used to question molecular phylogeny. Finally, we exemplify the utility of CAPRIB by pinpointing amino acid changes that coincided with the emergence of slow-growing mycobacteria from their fast-growing counterparts. The software is freely available at https://github.com/BactSymEvol/Caprib.ConclusionsCAPRIB is a new bioinformatics software aiming to make genus-scale comparisons accessible to all. With its intuitive graphical interface, this tool identifies key amino acid changes concomitant with a phenotypic divergence. By comparing fast and slow-growing mycobacteria, we shed light on evolutionary hotspots, such as the cytokinin pathway, that are interesting candidates for further experimentations.

Genes A27L and F13L as Genetic Markers for the Isolation of Recombinant Vaccinia Virus

After assembly in the cytosol, some Vaccinia virus particles go through a complex process that leads to virus egress and eventually cell-to-cell transmission. Intracellular particles are fully infectious, and therefore virus mutants lacking essential functions in the exit pathway are unable to form plaques but can multiply intracellularly. We isolated virus mutants in which two of the genes required for virus spread (F13L and A27L) were deleted independently or concurrently. The phenotypes of the mutant viruses were consistent with the need of A27L and F13L for intercellular virus transmission, the effect of the ΔA27L mutation being more severe than that of ΔF13L. Despite their defect in spread, ΔA27L mutant viruses could be expanded by infecting cell cultures at high multiplicity of infection, followed by the release of virions from infected cells by physical means. We developed a novel system for the isolation of recombinant Vaccinia virus in which selection is efficiently achieved by recovering plaque formation capacity after re-introduction of A27L into a ΔA27L virus. This system allowed the insertion of foreign DNA into the viral genome without the use of additional genetic markers. Furthermore, starting with a double mutant (ΔA27L-ΔF13L) virus, A27L selection was used in conjunction with F13L selection to mediate simultaneous dual insertions in the viral genome. This selection system facilitates combined expression of multiple foreign proteins from a single recombinant virus.

The Court of Justice of the European Union’s Judgment on Mutagenesis and International Trade: A Case of GMO, Mutagenesis and International Trade: A Case of GMO, Mutagenesis and International Trad

The Court of Justice of the European Union (CJEU) was requested by the French Conseil d’État to determine, in essence, whether organisms obtained by mutagenesis (i.e. gene editing) are genetically modified organisms (GMOs) and whether they are subject to the obligations laid down by Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. Contrary to the Advocate-General’s opinion of 18 January 2018, the Court of Justice ruled, on 25 July 2018, in case C-528/16 that Directive 2001/18/EC also applies to organisms obtained by mutagenesis techniques that have emerged since its adoption. The article analyses the grounds and implications of the CJEU’s decision. Unlike transgenesis, mutagenesis is a set of techniques, which make it possible to alter the genome of a living species without the insertion of foreign DNA. Indeed, it is virtually impossible to detect whether the DNA of a plant or animal has been edited or not, because the changes involved are indistinguishable from naturally occurring mutations. Mutagenesis techniques have made it possible to develop, inter alia, seed varieties that are resistant to selective herbicides. In response to the CJEU’s judgment, the US Secretary of Agriculture issued a statement criticizing the ruling. The statement reads, in part, that ‘[g]overnment policies should encourage scientific innovation without creating unnecessary barriers or unjustifiably stigmatizing new technologies. Unfortunately, this [...] ruling is a setback in this regard in that it narrowly considers newer genome editing methods to be within the scope of the European Union’s regressive and outdated regulations governing genetically modified organisms’. The article also analyses the possible international trade implications of this decision and of its effects, in light of the applicable WTO law and of the relevant case law, such as the EU – Biotech Products dispute by Argentina, Canada and the US, where the WTO had ultimately condemned a de facto moratorium on the approval of GM products in the EU and in its Member States.

Determination of virulence and fitness genes associated with the pheU, pheV and selC integration sites of LEE-negative food-borne Shiga toxin-producing Escherichia coli strains

BackgroundIn the current study, nine foodborne “Locus of Enterocyte Effacement” (LEE)-negative Shiga toxin-producing Escherichia coli (STEC) strains were selected for whole genome sequencing and analysis for yet unknown genetic elements within the already known LEE integration sites selC, pheU and pheV. Foreign DNA ranging in size from 3.4 to 57 kbp was detected and further analyzed. Five STEC strains contained an insertion of foreign DNA adjacent to the selC tRNA gene and five and seven strains contained foreign DNA adjacent to the pheU and pheV tRNA genes, respectively. We characterized the foreign DNA insertion associated with selC (STEC O91:H21 strain 17584/1), pheU (STEC O8:H4 strain RF1a and O55:Hnt strain K30) and pheV (STEC O91:H21 strain 17584/1 and O113:H21 strain TS18/08) as examples.ResultsIn total, 293 open reading frames partially encoding putative virulence factors such as TonB-dependent receptors, DNA helicases, a hemolysin activator protein precursor, antigen 43, anti-restriction protein KlcA, ShiA, and phosphoethanolamine transferases were detected. A virulence type IV toxin-antitoxin system was detected in three strains. Additionally, the ato system was found in one strain. In strain 17584/1 we were able to define a new genomic island which we designated GIselC17584/1. The island contained integrases and mobile elements in addition to genes for increased fitness and those playing a putative role in pathogenicity.ConclusionThe data presented highlight the important role of the three tRNAs selC, pheU, and pheV for the genomic flexibility of E. coli.

EuGI: a novel resource for studying genomic islands to facilitate horizontal gene transfer detection in eukaryotes

BackgroundGenomic islands (GIs) are inserts of foreign DNA that have potentially arisen through horizontal gene transfer (HGT). There are evidences that GIs can contribute significantly to the evolution of prokaryotes. The acquisition of GIs through HGT in eukaryotes has, however, been largely unexplored. In this study, the previously developed GI prediction tool, SeqWord Gene Island Sniffer (SWGIS), is modified to predict GIs in eukaryotic chromosomes. Artificial simulations are used to estimate ratios of predicting false positive and false negative GIs by inserting GIs into different test chromosomes and performing the SWGIS v2.0 algorithm. Using SWGIS v2.0, GIs are then identified in 36 fungal, 22 protozoan and 8 invertebrate genomes.ResultsSWGIS v2.0 predicts GIs in large eukaryotic chromosomes based on the atypical nucleotide composition of these regions. Averages for predicting false negative and false positive GIs were 20.1% and 11.01% respectively. A total of 10,550 GIs were identified in 66 eukaryotic species with 5299 of these GIs coding for at least one functional protein. The EuGI web-resource, freely accessible at http://eugi.bi.up.ac.za, was developed that allows browsing the database created from identified GIs and genes within GIs through an interactive and visual interface.ConclusionsSWGIS v2.0 along with the EuGI database, which houses GIs identified in 66 different eukaryotic species, and the EuGI web-resource, provide the first comprehensive resource for studying HGT in eukaryotes.