Environmental risks from the release of genetically modified organisms (GMOs)–the need for molecular ecology

Applications to release genetically modified organisms (GMOs) into the environment, usually the agricultural environment, are increasing exponentially. Many involve crop plants that are also weeds. Studies of biological invasions and of biological control show that the probability that a genetically new organism establishing itself is small; it is also unpredictable and in some cases could have severe ecological effects. GMOs pose risks both because they will be released in large numbers and because the greater the genetic novelty the greater the possibility of ecological novelty. Molecular ecology is an essential ingredient in ensuring that risks are assessed efficiently.     

Using Functional Genetics to Understand Breast Cancer Biology

Genetic screens were for long the prerogative of those that studied model organisms. The discovery in 2001 that gene silencing through RNA interference (RNAi) can also be brought about in mammalian cells paved the way for large scale loss-of-function genetic screens in higher organisms. In this article, we describe how functional genetic studies can help us understand the biology of breast cancer, how it can be used to identify novel targets for breast cancer therapy, and how it can help in the identification of those patients that are most likely to respond to a given therapy.Much remains to be learned regarding the function of mammalian genes. Only some quarter of all human genes have well-described functions. It is likely that quite a few of these currently unannotated genes will turn out to play key parts in cancer biology. For example, a 70-gene gene signature that can discriminate breast tumors of good and poor prognosis contained some 20 genes of currently unknown function (van ‘t Veer et al. 2002). The fact that these genes of unknown function foretell breast cancer prognosis hints at a role for at least some of these genes in breast cancer biology. The unbiased search for genes that contribute to breast cancer development is therefore likely to yield a rich harvest of new insights. RNA interference allows us to suppress genes systematically on a large scale and study the effects of gene suppression on specific cellular processes or signaling pathways. Consequently, RNA interference-based genetic screens have the potential to deepen our understanding of the molecular events that cause breast cancer, to find novel targets for therapy and to find biomarkers of drug responsiveness. In this article, we will describe the technologies available to perform both gain-of-function and loss-of-function genetic screens and will illustrate how such functional genetic screens have been used in the recent past to study a variety of outstanding questions in the biology of breast cancer.     

Prospects for inferring pairwise relationships with single nucleotide polymorphisms

An extraordinarily large number of single nucleotide polymorphisms (SNPs) are now available in humans as well as in other model organisms. Technological advancements may soon make it feasible to assay hundreds of SNPs in virtually any organism of interest. One potential application of SNPs is the determination of pairwise genetic relationships in populations without known pedigrees. Although microsatellites are currently the marker of choice for this purpose, the number of independently segregating microsatellite markers that can be feasibly assayed is limited. Thus, it can be difficult to distinguish reliably some classes of relationship (e.g. full-sibs from half-sibs) with microsatellite data alone. We assess, via Monte Carlo computer simulation, the potential for using a large panel of independently segregating SNPs to infer genetic relationships, following the analytical approach of Blouin et al. (1996). We have explored a 'best case scenario' in which 100 independently segregating SNPs are available. For discrimination among single-generation relationships or for the identification of parent-offspring pairs, it appears that such a panel of moderately polymorphic SNPs (minor allele frequency of 0.20) will provide discrimination power equivalent to only 16-20 independently segregating microsatellites. Although newly available analytical methods that can account for tight genetic linkage between markers will, in theory, allow improved estimation of relationships using thousands of SNPs in highly dense genomic scans, in practice such studies will only be feasible in a handful of model organisms. Given the comparable amount of effort required for the development of both types of markers, it seems that microsatellites will remain the marker of choice for relationship estimation in nonmodel organisms, at least for the foreseeable future.     

Evolutionary principles and the regulation of engineered bacteria

The introduction of engineered bacteria to the environment is being overregulated, on the basis of several assumptions: (i) the danger from deliberate introduction on a large scale is much greater than that from accidental release; (ii) the more distant the source of the DNA the greater the risk; (iii) novel organisms are likely to cause unexpected ecological damage, like that seen with native organisms transplanted to a novel location; (iv) even if the probability of harm is very small, great care must be taken because the harm might be large; (v) products of recombinant DNA must be treated differently from products of classical genetic manipulation; and (vi) our unlimited power to manipulate DNA implies an unlimited power to refashion organisms. Evolutionary principles contradict all these assumptions. Moreover, our increased power of genetic manipulation must be recognized as an expansion of the biotechnology of domestication; and unlike the physical technologies, the long history of domestication has not adventitiously created harmful by-products. I propose that in dealing with such novel and unpredictable developments it would be better to respond with speed and resilience to problems as they arise, rather than to hamper advances by clumsy regulations based on unsubstantiated guesses.     

CRISPR/Cas9 and Genome Editing in Drosophila

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.     

Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives

To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner. In the mouse, only about 10% (estimate) of all genes have been knocked out, despite continuous methodologic improvement and extensive effort. Moreover, a large proportion of inactivated genes exhibit no obvious phenotypic alterations. Thus, in order to facilitate analysis of gene function, new genetic tools and strategies are currently under development in these model organisms. Loss of function and gain of function mutagenesis screens based on transposable elements have numerous advantages because they can be applied in vivo and are therefore phenotype driven, and molecular analysis of the mutations is straightforward. At present, laboratory harnessing of transposable elements is more extensive in invertebrate models, mostly because of their earlier discovery in these organisms. Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila. However, transposon based genetic strategies were recently established in vertebrates, and current progress in this field indicates that transposable elements will indeed serve as indispensable tools in the genetic toolkit for vertebrate models. In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems.     

Eukaryotic microbes, species recognition and the geographic limits of species: examples from the kingdom Fungi

The claim that eukaryotic micro-organisms have global geographic ranges, constituting a significant departure from the situation with macro-organisms, has been supported by studies of morphological species from protistan kingdoms. Here, we examine this claim by reviewing examples from another kingdom of eukaryotic microbes, the Fungi. We show that inferred geographic range of a fungal species depends upon the method of species recognition. While some fungal species defined by morphology show global geographic ranges, when fungal species are defined by phylogenetic species recognition they are typically shown to harbour several to many endemic species. We advance two non-exclusive reasons to explain the perceived difference between the size of geographic ranges of microscopic and macroscopic eukaryotic species when morphological methods of species recognition are used. These reasons are that microbial organisms generally have fewer morphological characters, and that the rate of morphological change will be slower for organisms with less elaborate development and fewer cells. Both of these reasons result in fewer discriminatory morphological differences between recently diverged lineages. The rate of genetic change, moreover, is similar for both large and small organisms, which helps to explain why phylogenetic species of large and small organisms show a more similar distribution of geographic ranges. As a consequence of the different rates in fungi of genetic and morphological changes, genetic isolation precedes a recognizable morphological change. The final step in speciation, reproductive isolation, also follows genetic isolation and may precede morphological change.     

A bioinformatics approach to investigating developmental pathways in the kidney and other tissues.

Over the past few years, large amounts of data linking gene-expression (GE) patterns and other genetic data with the development of the mouse kidney have been published, and the next task will be to integrate these data with the molecular networks responsible for the emergence of the kidney phenotype. This paper discusses how a start to this task can be made by using the kidney database and its associated search tools, and shows how the data generated by such an approach can be used as a guide to future experimentation. Many of the events taking place as the kidney develops do, of course, also take place in other tissues and organisms and it will soon be possible to incorporate relevant information from these systems into analyses of kidney data as well as the new information from microarray technology. The key to success here will be the ability to access over the internet data from the textual and graphical databases for the mouse and other organisms now being established. In order to do this, informatic tools will be needed that will allow a user working with one database to query another. This paper also considers both the types of tools that will be necessary and the databases on which they will operate.     

Adapting immunity with subunit vaccines: case studies with group A Streptococcus and malaria

Although vaccines have widely been regarded as the most cost-effective way to improve public health, for some organisms new technological advances in vaccine design and delivery, incurring additional developmental costs, will be essential. These organisms are typically those for which natural immunity is either slow to develop or does not develop at all. Clearly, such organisms have evolved strategies to evade immune responses and innovative approaches will be required to induce a type of immune response which is both different to that which develops naturally and is effective. This article describes some approaches to develop vaccines for two such organisms (malaria parasites and Streptococcus pyogenes (group A Streptococcus)) that are associated with widespread mortality and morbidity, mostly in the poorest countries of the world. At this stage, the challenges are primarily scientific, but if these hurdles are surmounted then the challenges will become financial ones--developing much needed vaccines for people least able to afford them.     

The ribosomal peptidyl transferase center: structure, function, evolution, inhibition

The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring ribozymes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.     

Regulation of antioxidant enzymes.

Free radicals generated by a partial reduction of O2 pose a serious hazard to tissues and vital organs, especially membrane lipids, connective tissues, and the nucleic acids of cells. For protection, enzymes have evolved that specifically attack O2-, hydrogen, and organic peroxides, and repair any damage incurred to DNA. With few exceptions, antioxidant enzymes are found in all aerobic and aerotolerant anaerobic organisms. Logic assumes that a basal level of antioxidant enzyme activity is maintained at all times. This may be true. Yet cells must have ways to amplify antioxidant enzyme activity to counter sudden increases in oxygen metabolites. The full details of that regulation are slowly coming to light. Bacteria possess a series of elaborate and interacting genes that can sense specific increases in intracellular H2O2 and O2-. In higher organisms, hormones and metal ion cofactors impose pre- and posttranslational control over the genetic expression of antioxidant enzymes. Furthermore, aging, cellular differentiation, and organ specificity must also be factored into the final equation in higher organisms. This review will discuss some of the more recent findings relevant to antioxidant enzyme regulation in bacteria and higher organisms.     

Key questions in the genetics and genomics of eco-evolutionary dynamics

Increasing acceptance that evolution can be ‘rapid'' (or ‘contemporary'') has generated growing interest in the consequences for ecology. The genetics and genomics of these ‘eco-evolutionary dynamics'' will be—to a large extent—the genetics and genomics of organismal phenotypes. In the hope of stimulating research in this area, I review empirical data from natural populations and draw the following conclusions. (1) Considerable additive genetic variance is present for most traits in most populations. (2) Trait correlations do not consistently oppose selection. (3) Adaptive differences between populations often involve dominance and epistasis. (4) Most adaptation is the result of genes of small-to-modest effect, although (5) some genes certainly have larger effects than the others. (6) Adaptation by independent lineages to similar environments is mostly driven by different alleles/genes. (7) Adaptation to new environments is mostly driven by standing genetic variation, although new mutations can be important in some instances. (8) Adaptation is driven by both structural and regulatory genetic variation, with recent studies emphasizing the latter. (9) The ecological effects of organisms, considered as extended phenotypes, are often heritable. Overall, the study of eco-evolutionary dynamics will benefit from perspectives and approaches that emphasize standing genetic variation in many genes of small-to-modest effect acting across multiple traits and that analyze overall adaptation or ‘fitness''. In addition, increasing attention should be paid to dominance, epistasis and regulatory variation.     

Drosophila melanogaster: a model for the study of DNA damage checkpoint response

The cells of metazoans respond to DNA damage by either arresting their cell cycle in order to repair the DNA, or by undergoing apoptosis. This response is highly conserved across species, and many of the genes involved in this DNA damage response have been shown to be inactivated in human cancers. This suggests the importance of DNA damage response with regard to the prevention of cancer. The DNA damage checkpoint responses vary greatly depending on the developmental context, cell type, gene expression profile, and the degree and nature of the DNA lesions. More valuable information can be obtained from studies utilizing whole organisms in which the molecular basis of development has been well established, such as Drosophila. Since the discovery of the Drosophila p53 orthologue, various aspects of DNA damage responses have been studied in Drosophila. In this review, I will summarize the current knowledge on the DNA damage checkpoint response in Drosophila. With the ease of genetic, cellular, and cytological approaches, Drosophila will become an increasingly valuable model organism for the study of mechanisms inherent to cancer formation associated with defects in the DNA damage pathway.     

Genes in new environments: genetics and evolution in biological control

The availability of new genetic technologies has positioned the field of biological control as a test bed for theories in evolutionary biology and for understanding practical aspects of the release of genetically manipulated material. Purposeful introductions of pathogens, parasites, predators and herbivores, when considered as replicated semi-natural field experiments, show the unpredictable nature of biological colonization. The characteristics of organisms and their environments that determine this variation in the establishment and success of biological control can now be explored using genetic tools. Lessons from studies of classical biological control can help inform researchers and policy makers about the risks that are associated with the release of genetically modified organisms, particularly with respect to long-term evolutionary changes.     

DNA damage and repair: consequences on dose-responses

Damage to DNA is considered to be the main initiating event by which genotoxins cause hereditary effects and cancer. Single or double strand breaks, bases modifications or deletions, intra- or interstrand DNA-DNA or DNA-protein cross-links constitute the major lesions formed in different proportions according to agents and to DNA sequence context. They can result in cell death or in mutational events which in turn may initiate malignant transformation. Normal cells are able to repair these lesions with fidelity or by introducing errors. Base excision (BER) and nucleotide excision (NER) repair are error-free processes acting on the simpler forms of DNA damage. A specialized form of BER involves the removal of mismatched DNA bases occurring as errors of DNA replication or from miscoding properties of damaged bases. Severe damage will be repaired according to several types of recombinational processes: homologous, illegitimate and site-specific recombination pathways. The loss of repair capacity as seen in a number of human genetic diseases and mutant cell lines leads to hypersensitivity to environmental agents. Repair-defective cells show qualitative (mutation spectrum) and quantitative alterations in dose-effect relationships. For such repair-deficient systems, direct measurements at low doses are possible and the extrapolation from large to low doses fits well with the linear or the linear-quadratic no-threshold models. Extensive debate still takes place as to the shape of the dose-response relationships in the region at which genetic effects are not directly detectable in repair-proficient normal cells. Comparison of repair mutants and wild-type organisms pragmatically suggests that, for many genotoxins and tissues, very low doses may have no effect at all in normal cells.     

Applications of next generation sequencing in molecular ecology of non-model organisms

As most biologists are probably aware, technological advances in molecular biology during the last few years have opened up possibilities to rapidly generate large-scale sequencing data from non-model organisms at a reasonable cost. In an era when virtually any study organism can 'go genomic', it is worthwhile to review how this may impact molecular ecology. The first studies to put the next generation sequencing (NGS) to the test in ecologically well-characterized species without previous genome information were published in 2007 and the beginning of 2008. Since then several studies have followed in their footsteps, and a large number are undoubtedly under way. This review focuses on how NGS has been, and can be, applied to ecological, population genetic and conservation genetic studies of non-model species, in which there is no (or very limited) genomic resources. Our aim is to draw attention to the various possibilities that are opening up using the new technologies, but we also highlight some of the pitfalls and drawbacks with these methods. We will try to provide a snapshot of the current state of the art for this rapidly advancing and expanding field of research and give some likely directions for future developments.     

The new CRISPR–Cas system: RNA-guided genome engineering to efficiently produce any desired genetic alteration in animals

The CRISPR–Cas system is the newest targeted nuclease for genome engineering. In less than 1 year, the ease, robustness and efficiency of this method have facilitated an immense range of genetic modifications in most model organisms. Full and conditional gene knock-outs, knock-ins, large chromosomal deletions and subtle mutations can be obtained using combinations of clustered regularly interspaced short palindromic repeats (CRISPRs) and DNA donors. In addition, with CRISPR–Cas compounds, multiple genetic modifications can be introduced seamlessly in a single step. CRISPR–Cas not only brings genome engineering capacities to species such as rodents and livestock in which the existing toolbox was already large, but has also enabled precise genetic engineering of organisms with difficult-to-edit genomes such as zebrafish, and of technically challenging species such as non-human primates. The CRISPR–Cas system allows generation of targeted mutations in mice, even in laboratories with limited or no access to the complex, time-consuming standard technology using mouse embryonic stem cells. Here we summarize the distinct applications of CRISPR–Cas technology for obtaining a variety of genetic modifications in different model organisms, underlining their advantages and limitations relative to other genome editing nucleases. We will guide the reader through the many publications that have seen the light in the first year of CRISPR–Cas technology.     

Swatting flies: modelling wound healing and inflammation in Drosophila

Aberrant wound healing can lead to a variety of human pathologies, from non-healing chronic wounds that can become dangerously infected, to exuberant fibrotic healing in which repair is accompanied by excessive inflammation. To guide therapeutic intervention, we need a better understanding of the fundamental mechanisms driving tissue repair; this will require complementary wound-healing studies in several model organisms. Drosophila has been used to model genetic aspects of numerous human pathologies, and is being used increasingly to gain insight into the molecular and genetic aspects of tissue repair and inflammation, which have classically been modelled in mice or cultured cells. This review discusses the advantages and disadvantages of Drosophila as a wound-healing model, as well as some exciting new research opportunities that will be enabled by its use.