The promise of behavioural biology

The studies of behaviour that were strongly rooted in biology have had a long tradition of bringing together the ‘how’ and the ‘why’ questions. This integrative approach will serve the subject well in the postgenomic era as the long trend towards analysis at lower and lower levels starts to reverse. The new studies will make use of the resources uncovered by molecular biology and the neurosciences but will use the behaviour of the whole animal to measure outcomes and the context in which behaviour occurs to frame analytical questions. Two examples are given of how movement between levels of analysis is being used with increasing power and promise. The first is the study of filial imprinting in birds where many of the molecular and neural mechanisms involved have been uncovered and are now being integrated to explain the behaviour of the whole animal. The second is the triggering during sensitive periods in early life by environmental events of one of several alternative modes of development leading to different phenotypes. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Quantitative Analyses of Cell Division in Plants

At the molecular level regulatory interactions between cell cycle genes are being uncovered rapidly, but less progress is made in unravelling how these molecular events regulate growth processes at the level of cells and of the whole organism. The main obstacle is the absence of a set of analytical tools that are powerful enough to determine pertinent parameters and, at the same time, relatively easy to use by non-specialized laboratories. Appropriate methodology to obtain this type of data has been pioneered in the first half of the last century and is now commonly defined as ‘kinematic analysis’. Unfortunately, the laborious nature of these analyses and the relatively complex numerical methods used, have limited their use to only a handful of specialized research groups. In this article we attempt to present an accessible entry to this methodology, particularly in terms of the mathematical framework. We start describing the simplest possible system, i.e., a virtually homogenous cell suspension culture. Then, we outline the analysis of dicotyledonous leaves, root tips, monocotyledonous leaves, and finally shoot apical meristems. For each of these systems we discuss the details of the calculation of cell division parameters such as cell cycle duration, size of the meristem and number of cells contained in it, which enables answering fundamental questions about the relative contribution of differences in cell production and cell size to variation in growth. In addition, we discuss the assumptions and limitations of these and alternative methodologies with the aim to facilitate the choice of appropriate analyses depending on the specific research question.     

Anomalies of the anaerobic tricarboxylic acid cycle in Shewanella oneidensis revealed by Tn‐seq

The availability of increasingly inexpensive sequencing combined with an ever‐expanding molecular biology toolbox has transported classical bacterial genetics into the 21st century. Whole genome genetic fitness analysis using transposon mutagenesis combined with next‐generation high‐throughput sequencing (Tn‐seq) promises to revolutionize systems level analysis of microbial metabolism. Tn‐seq measures the frequency of actual members of a heterogeneous mutant pool undergoing purifying selection to determine the contribution of every non‐essential gene in the genome to the fitness of an organism under a given condition. Here we use Tn‐seq to assess gene function in the Gram negative γ‐proteobacterium Shewanella oneidensis strain MR‐1. In addition to being a model environmental organism, there is considerable interest in using S. oneidensis as a platform organism for bioremediation and biotechnology, necessitating a complete understanding of the metabolic pathways that may be utilized. Our analysis reveals unique aspects of S. oneidensis metabolism overlooked by over 30 years of classical genetic and systems level analysis. We report the utilization of an alternative citrate synthase and describe a dynamic branching of the S. oneidensis anaerobic tricarboxylic acid cycle, unreported in any other organism, which may be a widespread strategy for microbes adept at dissipating reducing equivalents via anaerobic respiration.     

Listeria monocytogenes, a food-borne pathogen.

The gram-positive bacterium Listeria monocytogenes is an ubiquitous, intracellular pathogen which has been implicated within the past decade as the causative organism in several outbreaks of foodborne disease. Listeriosis, with a mortality rate of about 24%, is found mainly among pregnant women, their fetuses, and immunocompromised persons, with symptoms of abortion, neonatal death, septicemia, and meningitis. Epidemiological investigations can make use of strain-typing procedures such as DNA restriction enzyme analysis or electrophoretic enzyme typing. The organism has a multifactorial virulence system, with the thiol-activated hemolysin, listeriolysin O, being identified as playing a crucial role in the organism's ability to multiply within host phagocytic cells and to spread from cell to cell. The organism occurs widely in food, with the highest incidences being found in meat, poultry, and seafood products. Improved methods for detecting and enumerating the organism in foodstuffs are now available, including those based on the use of monoclonal antibodies, DNA probes, or the polymerase chain reaction. As knowledge of the molecular and applied biology of L. monocytogenes increases, progress can be made in the prevention and control of human infection.     

Tissue-Specific Effects of Genetic and Epigenetic Variation on Gene Regulation and Splicing

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans’ lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole.     

Systems Biology in Aging: Linking the Old and the Young

Aging can be defined as a process of progressive decline in the physiological capacity of an organism, manifested by accumulated alteration and destabilization at the whole system level. Systems biology approaches offer a promising new perspective to examine the old problem of aging. We begin this review by introducing the concepts of systems biology, and then illustrate the application of systems biology approaches to aging research, from gene expression profiling to network analysis. We then introduce the network that can be constructed using known lifespan and aging regulators, and conclude with a look forward to the future of systems biology in aging research. In summary, systems biology is not only a young field that may help us understand aging at a higher level, but also an important platform that can link different levels of knowledge on aging, moving us closer to a more comprehensive control of systematic decline during aging.     

Generation of Chlamydomonas strains that efficiently express nuclear transgenes

The unicellular green alga Chlamydomonas reinhardtii is both an invaluable model organism for plant biology and an attractive biotechnological production system. Despite the availability of efficient methods for introduction of foreign genes into the nuclear genome of the alga, transgene expression levels are usually very poor. This is a serious limitation that has severely hampered both post-genomics research in Chlamydomonas and use of the alga in molecular farming. Here we report a solution to this problem. We have designed a genetic screen that facilitates isolation of algal strains that efficiently express introduced transgenes. The levels of accumulation of foreign protein in our expression strains are almost uniformly high in all transgenic clones and are little influenced by position effects. The possibility of expressing transgenes to high levels will greatly facilitate post-genomics research in Chlamydomonas , and will also boost exploitation of the alga as an inexpensive production host for biopharmaceuticals and other valuable compounds.     

The August Krogh principle applies to plants

The Krogh principle refers to the use of a large number of animals to study the large number of physiological problems, rather than limiting study to a particular organism for all problems. There may be organisms that are more suited to study of a particular problem than others. This same principle applies to plants. The authors are concerned with the recent trend in plant biology of using Arabidopsis thaliana as the "organism of choice." Arabidopsis is an excellent organism for molecular genetic research, but other plants are superior models for other research areas of plant biology. The authors present examples of the successful use of the Krogh principle in plant cell biology research, emphasizing the particular characteristics of the selected research organisms that make them the appropriate choice.     

Population Genomics of the Fission Yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe has been widely used as a model eukaryote to study a diverse range of biological processes. However, population genetic studies of this species have been limited to date, and we know very little about the evolutionary processes and selective pressures that are shaping its genome. Here, we sequenced the genomes of 32 worldwide S. pombe strains and examined the pattern of polymorphisms across their genomes. In addition to introns and untranslated regions (UTRs), intergenic regions also exhibited lower levels of nucleotide diversity than synonymous sites, suggesting that a considerable amount of noncoding DNA is under selective constraint and thus likely to be functional. A number of genomic regions showed a reduction of nucleotide diversity probably caused by selective sweeps. We also identified a region close to the end of chromosome 3 where an extremely high level of divergence was observed between 5 of the 32 strains and the remain 27, possibly due to introgression, strong positive selection, or that region being responsible for reproductive isolation. Our study should serve as an important starting point in using a population genomics approach to further elucidate the biology of this important model organism.     

Clocks not winding down: unravelling circadian networks

An intrinsic clock enables an organism to anticipate environmental changes and use energy sources more efficiently, thereby conferring an adaptive advantage. Having an intrinsic clock to orchestrate rhythms is also important for human health. The use of systems biology approaches has advanced our understanding of mechanistic features of circadian oscillators over the past decade. The field is now in a position to develop a multiscale view of circadian systems, from the molecular level to the intact organism, and to apply this information for the development of new therapeutic strategies or for enhancing agricultural productivity in crops.     

The Dictyostelium genome project: an invitation to species hopping.

Dictyostelium allows some of the general problems of eukaryotic biology to be addressed by using molecular genetic tools that are more normally associated with yeast. The genome project, now underway, marks an important increase in the attractiveness of Dictyostelium as an experimental organism and will invite increased 'species hopping' by experimenters. We provide a brief guide to the problems that are being addressed in Dictyostelium, to the genome project itself and to the molecular genetic tools available for its exploitation.     

In Vivo Analysis of Voltage-Dependent Calcium Channels

The molecular cloning of calcium channel subunits has identified an unexpectedly large number of genes and splicing variants, many of whichhave complex expression patterns: a central problem of calcium channel biology is to understand the functional significance of this genetic complexity. The genetic analysis of voltage-dependent calcium channels (VDCCs) provides an approach to defining channel function that is complimentary to pharmacological, electrophysiological, and other molecular methods. By discovering or creating alleles of VDCC genes, one can gain an understanding of the VDCC function at the whole animal level. Of particular interest are mutations in the alpha1 genes that encode the pore forming subunits, as they define the specific channel subtypes. In fact, a variety of calcium channelopathies and targeted mutations have been described for these genes in the last 6 years. The mutant alleles described below illustrate how phenotype analysis of these alleles has uncovered very specific functional roles that can be localized to specific synapses or cells.     

Stages in the development of a model organism as a platform for mechanistic models in developmental biology: Zebrafish, 1970-2000

Model organisms became an indispensable part of experimental systems in molecular developmental and cell biology, constructed to investigate physiological and pathological processes. They are thought to play a crucial role for the elucidation of gene function, complementing the sequencing of the genomes of humans and other organisms. Accordingly, historians and philosophers paid considerable attention to various issues concerning this aspect of experimental biology. With respect to the representational features of model organisms, that is, their status as models, the main focus was on generalization of phenomena investigated in such experimental systems. Model organisms have been said to be models for other organisms or a higher taxon. This, however, presupposes a representation of the phenomenon in question. I will argue that prior to generalization, model organisms allow researchers to built generative material models of phenomena - structures, processes or the mechanisms that explain them - through their integration in experimental set-ups that carve out the phenomena from the whole organism and thus represent them. I will use the history of zebrafish biology to show how model organism systems, from around 1970 on, were developed to construct material models of molecular mechanisms explaining developmental or physiological processes.     

Metabolic modeling of microbial strains in silico

The large volume of genome-scale data that is being produced and made available in databases on the World Wide Web is demanding the development of integrated mathematical models of cellular processes. The analysis of reconstructed metabolic networks as systems leads to the development of an in silico or computer representation of collections of cellular metabolic constituents, their interactions and their integrated function as a whole. The use of quantitative analysis methods to generate testable hypotheses and drive experimentation at a whole-genome level signals the advent of a systemic modeling approach to cellular and molecular biology.     

Proteogenomics and systems biology: quest for the ultimate missing parts

This review describes how intimately proteogenomics and system biology are imbricated. Quantitative cell-wide monitoring of cellular processes and the analysis of this information is the basis for systems biology. Establishing the most comprehensive protein-parts list is an essential prerequisite prior to analysis of the cell-wide dynamics of proteins, their post-translational modifications, their complex network interactions and interpretation of these data as a whole. High-quality genome annotation is, thus, a crucial basis. Proteogenomics consists of high-throughput identification and characterization of proteins by extra-large shotgun MS/MS approaches and the integration of these data with genomic data. Discovery of the remaining unannotated genes, defining translational start sites, listing signal peptide processing events and post-translational modifications, are tasks that can currently be carried out at a full-genomic scale as soon as the genomic sequence is available. Proteomics is increasingly being used at the primary stage of genome annotation and such an approach may become standard in the near future for genome projects. Advantageously, the same experimental proteomic datasets may be used to characterize the specific metabolic traits of the organism under study. Undoubtedly, comparative genomics will experience a renaissance taking into account this new dimension. Synthetic biology aimed at re-engineering living systems will also benefit from these significant progresses.