Ecological and evolutionary genomics of Saccharomyces cerevisiae

Saccharomyces cerevisiae, the budding yeast, is the most thoroughly studied eukaryote at the cellular, molecular, and genetic levels. Yet, until recently, we knew very little about its ecology or population and evolutionary genetics. In recent years, it has been recognized that S. cerevisiae occupies numerous habitats and that populations harbour important genetic variation. There is therefore an increasing interest in understanding the evolutionary forces acting on the yeast genome. Several researchers have used the tools of functional genomics to study natural isolates of this unicellular fungus. Here, we review some of these studies, and show not only that budding yeast is a prime model system to address fundamental molecular and cellular biology questions, but also that it is becoming a powerful model species for ecological and evolutionary genomics studies as well.     

The art and design of genetic screens: Arabidopsis thaliana

Molecular genetic studies rely on well-characterized organisms that can be easily manipulated. Arabidopsis thaliana--the model system of choice for plant biologists--allows efficient analysis of plant function, combining classical genetics with molecular biology. Although the complete sequence of the Arabidopsis genome allows the rapid discovery of the molecular basis of a characterized mutant, functional characterization of the Arabidopsis genome depends on well-designed forward genetic screens, which remain a powerful strategy to identify genes that are involved in many aspects of the plant life cycle.     

The genetic proteome: Using genetics to inform the proteome of mycobacterial pathogens

Mycobacterial pathogens pose a sustained threat to human health. There is a critical need for new diagnostics, therapeutics, and vaccines targeting both tuberculous and nontuberculous mycobacterial species. Understanding the basic mechanisms used by diverse mycobacterial species to cause disease will facilitate efforts to design new approaches toward detection, treatment, and prevention of mycobacterial disease. Molecular, genetic, and biochemical approaches have been widely employed to define fundamental aspects of mycobacterial physiology and virulence. The recent expansion of genetic tools in mycobacteria has further increased the accessibility of forward genetic approaches. Proteomics has also emerged as a powerful approach to further our understanding of diverse mycobacterial species. Detection of large numbers of proteins and their modifications from complex mixtures of mycobacterial proteins is now routine, with efforts of quantification of these datasets becoming more robust. In this review, we discuss the “genetic proteome,” how the power of genetics, molecular biology, and biochemistry informs and amplifies the quality of subsequent analytical approaches and maximizes the potential of hypothesis-driven mycobacterial research. Published proteomics datasets can be used for hypothesis generation and effective post hoc supplementation to experimental data. Overall, we highlight how the integration of proteomics, genetic, molecular, and biochemical approaches can be employed successfully to define fundamental aspects of mycobacterial pathobiology.     

Technicolour transgenics: imaging tools for functional genomics in the mouse

Over the past decade, a battery of powerful tools that encompass forward and reverse genetic approaches have been developed to dissect the molecular and cellular processes that regulate development and disease. The advent of genetically-encoded fluorescent proteins that are expressed in wild type and mutant mice, together with advances in imaging technology, make it possible to study these biological processes in many dimensions. Importantly, these technologies allow direct visual access to complex events as they happen in their native environment, which provides greater insights into mammalian biology than ever before.     

Discovering susceptibility genes for asthma and allergy

Asthma and asthma-related traits are complex diseases with strong genetic and environmental components. Rapid progress in asthma genetics has led to the identification of several candidate genes that are associated with asthma-related traits. Typically the phenotypic impact of each of these genes, including the ones most often replicated in association studies, is mild, but larger effects may occur when multiple variants synergize within a permissive environmental context. Despite the achievements made in asthma genetics formidable challenges remain. The development of novel, powerful tools for gene discovery, and a closer integration of genetics and biology, should help to overcome these challenges.     

The art and design of genetic screens: yeast

Understanding the biology of complex systems is facilitated by comparing them with simpler organisms. Budding and fission yeasts provide ideal model systems for eukaryotic cell biology. Although they differ from one another in terms of a range of features, these yeasts share powerful genetic and genomic tools. Classical yeast genetics remains an essential element in discovering and characterizing the genes that make up a eukaryotic cell.     

Genetics and molecular physiology of the yeast Kluyveromyces lactis

With the recent development of powerful molecular genetic tools, Kluyveromyces lactis has become an excellent alternative yeast model organism for studying the relationships between genetics and physiology. In particular, comparative yeast research has been providing insights into the strikingly different physiological strategies that are reflected by dominance of respiration over fermentation in K. lactis versus Saccharomyces cerevisiae. Other than S. cerevisiae, whose physiology is exceptionally affected by the so-called glucose effect, K. lactis is adapted to aerobiosis and its respiratory system does not underlie glucose repression. As a consequence, K. lactis has been successfully established in biomass-directed industrial applications and large-scale expression of biotechnically relevant gene products. In addition, K. lactis maintains species-specific phenomena such as the "DNA-killer system, " analyses of which are promising to extend our knowledge about microbial competition and the fundamentals of plasmid biology.     

Transposable elements as tools for genomics and genetics in Drosophila.

The P-element has been the workhorse of Drosophila genetics since it was developed as a tool for transgenesis in 1982; the subsequent development of a variety of systems based on the transposon have provided a range of powerful and flexible tools for genetics and genomics applications. P-element insertions are frequently used as starting-points for generating chromosomal deletions to remove flanking genes, either by screening for imprecise excision events or by selecting for male recombination events. Elements that utilise the yeast FLP/FLP recombination target (FRT) site-specific recombination system have been widely used to generate molecularly marked mitotic clones for mosaic analysis, extending the reach of this powerful genetic tool to virtually all areas of developmental biology. P-elements are still widely used as traditional mutagenesis reagents and form the backbone of projects aimed at generating insertions in every predicted gene in the fly genome. In addition, vectors based on the FLP/FRT system are being used for genome-wide applications, including the development of molecularly-mapped deletion and duplication kits. In addition to these 'traditional' genetic approaches, a variety of engineered elements have been developed for a wide range of transgenic applications, including enhancer trapping, gene-tagging, targeted misexpression, RNA interference (RNAi) delivery and homologous recombination/gene replacement. To complement the use of P-elements, alternative transposon vectors have been developed. The most widely used of these are the lepidopteran element piggyBac and a Drosophila hydei transposon, Minos. In total, a range of transposon vectors offers the Drosophila biologist considerable flexibility and sophistication in manipulating the genome of the fly and has allowed rapid advances in all areas of developmental biology and genome science.     

A bird's-eye view on the modern genetics workflow and its potential applicability to the locust problem

Genetics is an immense science and the current developments in its methods and techniques as well as the fast emerging tools make it one of the most powerful biological sciences. Indeed, from taxonomy and ecology to physiology and molecular biology, every biological science makes use of genetics techniques and methods at one time or another. In fact, development in genetics is such that it is now possible to characterize and analyze the expression of the whole set of genes of virtually every living organism, even if it is a non-model one. Locusts are notorious for the damage they cause to the ecosystems and economies of the areas affected by their recurrent population outbreaks. To prevent and deal with these outbreaks, we now count on both biological as well as chemical agents that are proving to be successful in reducing the damage that otherwise locust population outbreaks might cause. However, a better, efficient and environmentally friendly solution is still a hoped-for target. In my opinion, the ideal future pesticide should be both environmentally friendly, risk free and species-specific. To reach the knowledge needed for the development of such species-specific anti-locust agent, deep and accurate knowledge of the locusts’ genetics and molecular biology is a must. Since genes and their expression levels lie at the bottom of every biological phenomenon, any species-specific solution to the locust problem requires a good knowledge of these organisms’ genes as well as the quantitative and spatio-temporal dynamics of their expression. To reach such knowledge, collaborative work is needed as well as a clear workflow that, given the fast development in the genetics tools, is not always clear to all research groups. For this reason, here I describe a genetics workflow that should allow taking advantage of the most recent genetics tools and techniques to answer question relating to locust biology. My hope is that the adoption of this and other work strategies by different research groups, especially when the work is a collaborative one, would provide precious information on the biology and the biological phenomena that these economically important organisms exhibit.     

Molecular systematics and population genetics of biological invasions: towards a better understanding of invasive species management

The study of population genetics of invasive species offers opportunities to investigate rapid evolutionary processes at work, and while the ecology of biological invasions has enjoyed extensive attention in the past, the recentness of molecular techniques makes their application in invasion ecology a fairly new approach. Despite this, molecular biology has already proved powerful in inferring aspects not only relevant to the evolutionary biologist but also to those concerned with invasive species management. Here, we review the different molecular markers routinely used in such studies and their application(s) in addressing different questions in invasion ecology. We then review the current literature on molecular genetic studies aimed at improving management and the understanding of invasive species by resolving of taxonomic issues, elucidating geographical sources of invaders, detecting hybridisation and introgression, tracking dispersal and spread and assessing the importance of genetic diversity in invasion success. Finally, we make some suggestions for future research efforts in molecular ecology of biological invasions.     

Xenopus: an ideal system for chemical genetics

Chemical genetics, or chemical biology, has become an increasingly powerful method for studying biological processes. The main objective of chemical genetics is the identification and use of small molecules that act directly on proteins, allowing rapid and reversible control of activity. These compounds are extremely powerful tools for researchers, particularly in biological systems that are not amenable to genetic methods. In addition, identification of small molecule interactions is an important step in the drug discovery process. Increasingly, the African frog Xenopus is being used for chemical genetic approaches. Here, we highlight the advantages of Xenopus as a first-line in vivo model for chemical screening as well as for testing reverse engineering approaches.     

The human genome project--some implications of extensive "reverse genetic" medicine

Impressive progress has been made during the past several decades in understanding the pathogenesis of human genetic disease. The tools of molecular biology have allowed the isolation of many disease-related genes by forward and a few by reverse genetics, and the imminent completion of a complete human genetic linkage map will accelerate the genetic characterization of many more genetic diseases. The major impacts of the molecular characterization of human genetic diseases will be 1. To increase markedly the number of human diseases that we recognize to have major genetic components. We already understand that genetic diseases are not rare medical curiosities with negligible societal impact, but rather constitute a wide spectrum of both rare and extremely common diseases responsible for an immense amount of suffering in all human societies. The characterization of the human genome will lead to the identification of genetic factors in many more human diseases, even those that now seem too multifactorial or polygenic for ready understanding. 2. To allow the development of powerful new approaches to diagnosis, detection, screening and even therapy of these disorders aimed directly at the mutant genes rather than at the gene products. This should eventually allow much more accurate and specific management of human genetic disease and the genetic factors in many human maladies. The preparation of a fine-structure physical map of the entire human genome together with an overlapping contiguous set of clones spanning entire chromosomes or large portions of chromosomes is rapidly becoming feasible, and the information that will flow from this effort promises eventually to affect the management of many important genetic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fundamentals of fungal molecular population genetic analyses

The last two decades have seen tremendous growth in the development and application of molecular methods in the analyses of fungal species and populations. In this paper, I provide an overview of the molecular techniques and the basic analytical tools used to address various fundamental population and evolutionary genetic questions in fungi. With increasing availability and decreasing cost, DNA sequencing is becoming a mainstream data acquisition method in fungal evolutionary genetic studies. However, other methods, especially those based on the polymerase chain reaction, remain powerful in addressing specific questions for certain groups of taxa. These developments are bringing fungal population and evolutionary genetics into mainstream ecology and evolutionary biology.     

Physical mapping of the rice genome with BACs

The development of genetics in this century has been catapulted forward by several milestones: rediscovery of Mendel's laws, determination of DNA as the genetic material, discovery of the double-helix structure of DNA and its implications for genetic behavior, and most recently, analysis of restriction fragment length polymorphisms (RFLPs). Each of these milestones has generated a huge wave of progress in genetics. Consequently, our understanding of organismal genetics now extends from phenotypes to their molecular genetic basis. It is now clear that the next wave of progress in genetics will hinge on genome molecular physical mapping, since a genome physical map will provide an invaluable, readily accessible system for many detailed genetic studies and isolation of many genes of economic or biological importance. Recent development of large-DNA fragment (>100 kb) manipulation and cloning technologies, such as pulsed-field gel electrophoresis (PFGE), and yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) cloning, has provided the powerful tools needed to generate molecular physical maps for higher-organism genomes. This chapter will discuss (1) an ideal physical map of plant genome and its applications in plant genetic and biological studies, (2) reviews on physical mapping of the genomes of Caenorhabditis elegans, Arabidopsis thaliana, and man, (3) large-insert DNA libraries: cosmid, YAC and BAC, and genome physical mapping, (4) physical mapping of the rice genome with BACs, and (5) perspectives on the physical mapping of the rice genome with BACs.     

Recent advances in plasmid-based tools for establishing novel microbial chassis

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with “build-your-own” microbial host.     

Chemical biology approaches in plant stress research

In addition to their importance as essential agrochemicals and life-saving drugs, small molecules serve as powerful research tools to address questions at all levels of biological complexity from protein function to plant biotic interactions. In certain contexts, chemical tools are complementary or even preferred to genetic analysis, since not all experimental systems are amenable for genetic dissection. For example, mutants impaired in oxygen sensing cannot easily be recovered. Pharmacological and chemical genetics approaches have come to the rescue of biologists in unraveling such genetically intractable systems. In this review, I have discussed my own efforts to analyze oxygen deprivation signaling in plants to illustrate the validity of small molecular approaches in elucidating an essential pathway such as oxygen sensing. Chemical biology is also a potent approach to tease out genetically redundant biological processes. The recent breakthrough in identifying the elusive abscisic acid receptors has clearly demonstrated the power of chemical tools in dissecting redundant pathways and led to the blossoming of this area as a distinct discipline of plant biology research. I present a summary of this work and conclude the review with potential challenges in using chemical tools.     

Zebrafish as a model organism for nutrition and growth: towards comparative studies of nutritional genomics applied to aquacultured fishes

Zebrafish (Danio rerio) is a common research model in fish studies of toxicology, developmental biology, neurobiology and molecular genetics; it has been proposed as a possible model organism for nutrition and growth studies in fish. The advantages of working with zebrafish in these areas are their small size, short generation time (12–14 weeks) and their capacity to produce numerous eggs (100–200 eggs/clutch). Since a wide variety of molecular tools and information are available for genomic analysis, zebrafish has also been proposed as a model for nutritional genomic studies in fish. The detailed study of every species employed as a model organism is important because these species are used to generalize how several biological processes occur in related organisms, and contribute considerably toward improving our understanding of the mechanisms involved in nutrition and growth. The objective of this review is to show the relevant aspects of the nutrition and growth in zebrafish that support its utility as a model organism for nutritional genomics studies. We made a particular emphasis that gene expression and genetic variants in response to zebrafish nutrition will shed light on similar processes in aquacultured fish.     

Bacillus subtilis and its relatives: molecular biological and industrial workhorses.

The non-pathogenic bacterium Bacillus subtilis, since its first reported genetic transformation in 1959, has become a model system for the study of many aspects of the biochemistry, genetics and physiology of Gram-positive bacteria, and particularly of sporulation and associated metabolism. Extensive knowledge of the molecular biology of B. subtilis has led to the recent development of this bacterium as a host for the industrial production of heterologous proteins. Although difficulties have been encountered, these are being systematically addressed and overcome.     

Molecular Analysis of In Vitro Shoot Organogenesis

Shoot organogenesis is one of the in vitro plant regeneration pathways. It has been widely employed in plant biotechnology for in vitro micropropagation and genetic transformation, as well as in study of plant development. Morphological and physiological aspects of in vitro shoot organogenesis have already been extensively studied in plant tissue culture for more than 50 years. Within the last ten years, given the research progress in plant genetics and molecular biology, our understanding of in vivo plant shoot meristem development, plant cell cycle, and cytokinin signal transduction has advanced significantly. These research advances have provided useful molecular tools and resources for the recent studies on the genetic and molecular aspects of in vitro shoot organogenesis. A few key molecular markers, genes, and probable pathways have been identified from these studies that are shown to be critically involved in in vitro shoot organogenesis. Furthermore, these studies have also indicated that in vitro shoot organogenesis, just as in in vivo shoot development, is a complex, well-coordinated developmental process, and induction of a single molecular event may not be sufficient to induce the occurrence of the entire process. Further study is needed to identify the early molecular event(s) that triggers dedifferentiation of somatic cells and serves as the developmental switch for de novo shoot development.     

Techniques for preservation of mammalian germ plasm

Abstract

The preservation of mammalian germ plasm by freezing has become an integral part of animal breeding, medicine, agriculture, reproductive biology and embryology. Considerable understanding of the physical‐chemical and physiological phenomena involved in cryopreservation of sperm, eggs and embryos has been achieved. This understanding has resulted in substantial improvements in the efficiency and efficacy of methods used to cryopreserve germ plasm. In addition, many of these methods have become integrated directly into the practice of animal breeding, and have contributed directly to the international trade in animal genetics. Development of these methods has been derived from close cooperation and interaction between the research and industrial communities. As the powerful techniques of molecular biology are focused on fundamental and applied aspects of embryology and reproductive biology, there are new problems regarding the cryobiology of germ cells to be solved.