Pedro H. Oliveira

The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts

The roles of restriction-modification (R-M) systems in providing immunity against horizontal gene transfer (HGT) and in stabilizing mobile genetic elements (MGEs) have been much debated. However, few studies have precisely addressed the distribution of these systems in light of HGT, its mechanisms and its vectors. We analyzed the distribution of R-M systems in 2261 prokaryote genomes and found their frequency to be strongly dependent on the presence of MGEs, CRISPR-Cas systems, integrons and natural transformation. Yet R-M systems are rare in plasmids, in prophages and nearly absent from other phages. Their abundance depends on genome size for small genomes where it relates with HGT but saturates at two occurrences per genome. Chromosomal R-M systems might evolve under cycles of purifying and relaxed selection, where sequence conservation depends on the biochemical activity and complexity of the system and total gene loss is frequent. Surprisingly, analysis of 43 pan-genomes suggests that solitary R-M genes rarely arise from the degradation of R-M systems. Solitary genes are transferred by large MGEs, whereas complete systems are more frequently transferred autonomously or in small MGEs. Our results suggest means of testing the roles for R-M systems and their associations with MGEs.

Marker-free plasmids for biotechnological applications – implications and perspectives

Nonviral gene therapy and DNA vaccines have become the first promising approaches to treat, cure, or ultimately prevent disease by providing genetic information encoded on a plasmid. Since 1989, more than 1800 clinical trials have been approved worldwide, and approximately 20% of them are using plasmid DNA (pDNA) as a vector system. Although much safer than viral approaches, DNA vectors generally do encode antibiotic resistance genes in the plasmid backbone. These antibiotic resistance markers constitute a possible safety risk, and they are associated with structural plasmid instabilities and decreased gene delivery efficiency. These drawbacks have initiated the development of various antibiotic marker-free selection approaches. We provide an overview on the potential implications of marker-free plasmids and perspectives for their successful biotechnological use in the future.

Structural instability of plasmid biopharmaceuticals: challenges and implications

The global increase in the number of applications involving therapeutic plasmid DNA (pDNA) is creating a need for large amounts of highly stable and purified molecules. One of the main obstacles during the developmental stages of a new therapeutic DNA molecule involves tackling a wide array of structural instability events occurring in/with pDNA and therefore assuring its structural integrity. This review focuses on major instability determinants in pDNA. Their elimination could be considered an important step towards the design of safer and more efficient plasmid molecules. Particular emphasis is given to mutations triggered by the presence of repeated sequences, instability events occurring during plasmid intracellular routing, instability mediated by insertion sequences and host genome integration.

Impact of hypoxia and long-term cultivation on the genomic stability and mitochondrial performance of ex vivo expanded human stem/stromal cells.

Recent studies have described the occurrence of chromosomal abnormalities and mitochondrial dysfunction in human stem/stromal cells (SCs), particularly after extensive passaging in vitro and/or expansion under low oxygen tensions. To deepen this knowledge we investigated the influence of hypoxia (2% O(2)) and prolonged passaging (>P10) of human bone marrow stromal cells (BMSCs) and adipose-derived stromal cells (ASCs) on the expression of genes involved in DNA repair and cell-cycle regulation pathways, as well as on the occurrence of microsatellite instability and changes in telomere length. Our results show that hypoxic conditions induce an immediate and concerted down-regulation of genes involved in DNA repair and damage response pathways (MLH1, RAD51, BRCA1, and Ku80), concomitantly with the occurrence of microsatellite instability while maintaining telomere length. We further searched for mutations occurring in the mitochondrial genome, and monitored changes in intracellular ATP content, membrane potential and mitochondrial DNA content. Hypoxia led to a simultaneous decrease in ATP content and in the number of mitochondrial genomes, whereas the opposite effect was observed after prolonged passaging. Moreover, we show that neither hypoxia nor prolonged passaging significantly affected the integrity of the mitochondrial genome. Ultimately, we present evidence on how hypoxia selectively impacts the cellular response of BMSCs and ASCs, thus pointing for the need to optimize oxygen tension according to the cell source.

Regulation of genetic flux between bacteria by restriction–modification systems

Significance The role of restriction–modification (R-M) as bacterias innate immune system, and a barrier to sexual exchange, has often been challenged. Recent works suggested that the diversification of these systems might have driven the evolution of highly virulent bacterial lineages. Here, we showed that R-M systems were more abundant in species enduring more DNA exchanges and that within-species flux of genetic material was higher when cognate systems were present. Presumably, bacteria enduring frequent infections by mobile elements select for the presence of more numerous R-M systems, but rapid diversification of R-M systems leads to varying patterns of sexual exchanges between bacterial lineages. Restriction–modification (R-M) systems are often regarded as bacterias innate immune systems, protecting cells from infection by mobile genetic elements (MGEs). Their diversification has been recently associated with the emergence of particularly virulent lineages. However, we have previously found more R-M systems in genomes carrying more MGEs. Furthermore, it has been suggested that R-M systems might favor genetic transfer by producing recombinogenic double-stranded DNA ends. To test whether R-M systems favor or disfavor genetic exchanges, we analyzed their frequency with respect to the inferred events of homologous recombination and horizontal gene transfer within 79 bacterial species. Genetic exchanges were more frequent in bacteria with larger genomes and in those encoding more R-M systems. We created a recognition target motif predictor for Type II R-M systems that identifies genomes encoding systems with similar restriction sites. We found more genetic exchanges between these genomes, independently of their evolutionary distance. Our results reconcile previous studies by showing that R-M systems are more abundant in promiscuous species, wherein they establish preferential paths of genetic exchange within and between lineages with cognate R-M systems. Because the repertoire and/or specificity of R-M systems in bacterial lineages vary quickly, the preferential fluxes of genetic transfer within species are expected to constantly change, producing time-dependent networks of gene transfer.

Concise Review: Genomic Instability in Human Stem Cells: Current Status and Future Challenges

Genomic instability is recognized as one of the most important hurdles in the expanding field of stem cell‐based therapies. In the recent years, an accumulating body of evidence has shown that human stem cells undergo a diverse program of biological changes upon ex vivo cultivation that include numerical and structural chromosomal abnormalities, point mutations, variation of telomere length, and epigenetic instability. As the field moves forward, the growing awareness of the risk factors associated with human genome plasticity strongly advocates for the use of extensive genetic screening as part of a quality control platform to attest to the safety of stem cell‐based products. Here we present a timely and comprehensive review that addresses the current status and emerging trends of the field, ultimately underscoring the need to implement new regulatory standards able to streamline the route to therapeutic applications. Stem Cells 2014;32:2824–2832

Recombination frequency in plasmid DNA containing direct repeats--predictive correlation with repeat and intervening sequence length.

In this study, a simple non-linear mathematical function is proposed to accurately predict recombination frequencies in bacterial plasmid DNA harbouring directly repeated sequences. The mathematical function, which was developed on the basis of published data on deletion-formation in multicopy plasmids containing direct-repeats (14-856 bp) and intervening sequences (0-3872 bp), also accounts for the strain genotype in terms of its recA function. A bootstrap resampling technique was used to estimate confidence intervals for the correlation parameters. More than 92% of the predicted values were found to be within a pre-established +/-5-fold interval of deviation from experimental data. The correlation does not only provide a way to predict, with good accuracy, the recombination frequency, but also opens the way to improve insight into these processes.

Deletion formation mutations in plasmid expression vectors are unfavored by runaway amplification conditions and differentially selected under kanamycin stress

Plasmid pCIneo is a ColE1-like mammalian expression vector also used as backbone for DNA vaccine development. We have recently shown that pCIneo spontaneously recombines due to the presence of two 28bp direct repeats. The persistence of low-frequency recombinants led us to evaluate the impact of environmental stresses typically found during plasmid production on plasmid copy number and recombination frequency. We observed an increase in pCIneo amplification (2.6-4.3-fold) in Escherichia coli cultures grown at 42 degrees C and also in minimal medium (at both 37 degrees C and 42 degrees C). These conditions fit to the smallest ratio between recombinant molecules and total plasmids. Conversely, increasing the dissolved oxygen tension from 20% to 40% in rich media did not have a significant impact on both plasmid copy number and recombination frequency, independently of the temperature used. We have also shown recently that the neomycin resistance (neo(r)) gene of pCIneo becomes actively transcribed as a result of recombination between the repeats. This prompted us to gain some insight into plasmid adaptation and competition by evaluating the impact of distinct concentrations of kanamycin on the differential selection of plasmid recombinant forms: monomer and heterodimers (1+2 and 1+3). We found the monomeric form to be predominantly recovered at lower concentrations of antibiotic whilst higher concentrations led to an increase in the percentage of the 1+2 form. The 1+3 heterodimeric form was invariably found at low percentages, independently of the concentration used.

An Appraisal of Human Mitochondrial DNA Instability: New Insights into the Role of Non-Canonical DNA Structures and Sequence Motifs

Mitochondrial DNA (mtDNA) deletion mutations are frequently observed in aged postmitotic tissues and are the cause of a wide range of human disorders. Presently, the molecular bases underlying mtDNA deletion formation remain a matter of intense debate, and it is commonly accepted that several mechanisms contribute to the spectra of mutations in the mitochondrial genome. In this work we performed an extensive screening of human mtDNA deletions and evaluated the association between breakpoint density and presence of non-canonical DNA elements and over-represented sequence motifs. Our observations support the involvement of helix-distorting intrinsically curved regions and long G-tetrads in eliciting instability events. In addition, higher breakpoint densities were consistently observed within GC-skewed regions and in the close vicinity of the degenerate sequence motif YMMYMNNMMHM. A parallelism is also established with hot spot motifs previously identified in the nuclear genome, as well as with the minimal binding site for the mitochondrial transcription termination factor mTERF. This study extends the current knowledge on the mechanisms driving mitochondrial rearrangements and opens up exciting avenues for further research.

Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events

Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.