Chromatin: constructing the big picture

Chromatin is the ensemble of genomic DNA and a large number of proteins. Various genome-wide mapping techniques have begun to reveal that, despite the tremendous complexity, chromatin organization is governed by simple principles. This review discusses the principles that drive the spatial architecture of chromatin, as well as genome-wide-binding patterns of chromatin proteins.     

Microstudies versus big picture accounts?

Microstudies and big picture accounts are often counterposed. This paper investigates the supposed dichotomy between the two historiographical approaches. In particular it investigates how the discussions are reflected in the historiography of molecular biology and the special questions posed by the disciplinary context. Taking inspiration from the microhistory tradition as exemplified by the works of Carlo Ginzburg, Jacques Revel, and David Sabean among others, the paper highlights the heuristic value of microstudies to reconstruct the multiple contexts that link apparently small events with broader structures. In a parallel fashion, the paper argues for using microstudies to open up the history of molecular biology to other fields of study and thus moving beyond the confines of the disciplinary framework. Such an approach does not dismiss the search for big pictures. Yet rather than opposing big pictures to microstudies, it sees microstudies as a valid way to gain new and broad vistas.     

Darwin Medal presentation: Corals-seeking the big picture

Recipients of Darwin Medals from the International Society of Reef Studies are requested to write an overview of the work that led to their award. This account is a personal perspective of thirty-five years work on corals. The fields of taxonomy, biogeography, palaeontology, molecular biology, and evolution are presented in an historical context. Emphasis is given to the changing relevance of these fields to today’s world of information technology and the ever-increasing conservation needs.     

Bacterial cell cycle: seeing the big picture with microarrays

Global assays of gene expression and protein stability during the Caulobacter crescentus cell cycle reveal that a surprisingly large fraction of the genome and proteome is affected as cells grow and divide. These studies are an important step toward understanding how the cell cycle is controlled in prokaryotes.     

Purification and visualization of native spliceosomes

Mammalian spliceosomes were purified in preparative amounts by gel filtration chromatography and shown to be functional by in vitro complementation experiments. The column fractions containing spliceosomes are enriched in the snRNAs U1, U2, U4, U5, and U6 and a subset of proteins present in the nuclear extract. Splicing intermediates, the entire set of snRNAs, and the enriched proteins can be immunoprecipitated with three different monoclonal antibodies that recognize snRNP determinants. At least one U1 snRNP is present in each spliceosome since the particles are quantitatively immunoprecipitated by an anti-U1 snRNP monoclonal antibody. Examination of the spliceosome fractions by EM revealed a relatively homogeneous population of 40-60 nm particles with a striking morphology. Evidence that these particles are spliceosomes is their sensitivity to micrococcal nuclease, their ATP-dependent assembly, and their immunoprecipitation with a trimethyl cap monoclonal antibody. In addition, pre-mRNA was visualized in the particles by EM.     

Selective forces for the origin of spliceosomes

It has been proposed that eukaryotic spliceosomes evolved from bacterial group II introns via constructive neutral changes. However, a more likely interpretation is that spliceosomes and group II introns share a common undefined RNA ancestor--a proto-spliceosome. Although, the constructive neutral evolution may have probably played some roles in the development of complexity including the evolution of modern spliceosomes, in fact, the origin, losses and the retention of spliceosomes can be explained straight-forwardly mainly by positive and negative selection: (1) proto-spliceosomes evolved in the RNA world as a mechanism to excise functional RNAs from an RNA genome and to join non-coding information (ancestral to exons) possibly designed to be degraded. (2) The complexity of proto-spliceosomes increased with the invention of protein synthesis in the RNP world and they were adopted for (a) the addition of translation signal to RNAs via trans-splicing, and for (b) the exon-shuffling such as to join together exons coding separate protein domains, to translate them as a single unit and thus to facilitate the molecular interaction of protein domains needed to be assembled to functional catalytic complexes. (3) Finally, the spliceosomes were adopted for cis-splicing of (mainly) non-coding information (contemporary introns) to yield translatable mRNAs. (4) Spliceosome-negative organisms (i.e., prokaryotes) have been selected in the DNA-protein world to save a lot of energy. (5) Spliceosome-positive organisms (i.e., eukaryotes) have been selected, because they have been completely spliceosome-dependent.     

Complementation of in vitro-assembled spliceosomes

We describe the development and application of a system of in vitro-assembled splicing complexes that can be used for the identification of protein splicing factors which become associated with the spliceosome at the end of the assembly process ("late" splicing components). A splicing reaction performed in the presence of polyvinyl alcohol is interrupted after 15 to 20 minutes, before the appearance of splicing intermediates and products in significant amounts. Following low-speed centrifugation, a pellet is obtained containing splicing complexes that can be solubilized with 0.6 M-KCl. These complexes can be rapidly complemented for splicing in the presence of ATP and Mg2+ with protein factors that are present in HeLa cell nuclear extracts or in chromatographic extract fractions. Biochemical features of the complementation reactions, and conditions for reversible uncoupling of the two splicing steps, are described and discussed. These conditions are used to generate fully assembled spliceosomes in which splicing of the pre-mRNA can occur in the presence of ATP and Mg2+, but in the absence of nuclear extract ("autonomous splicing").     

Endocannabinoids in nervous system health and disease: the big picture in a nutshell

The psychoactive component of the cannabis resin and flowers, delta9-tetrahydrocannabinol (THC), was first isolated in 1964, and at least 70 other structurally related ‘phytocannabinoid’ compounds have since been identified. The serendipitous identification of a G-protein-coupled cannabinoid receptor at which THC is active in the brain heralded an explosion in cannabinoid research. Elements of the endocannabinoid system (ECS) comprise the cannabinoid receptors, a family of nascent lipid ligands, the ‘endocannabinoids’ and the machinery for their biosynthesis and metabolism. The function of the ECS is thus defined by modulation of these receptors, in particular, by two of the best-described ligands, 2-arachidonoyl glycerol and anandamide (arachidonylethanolamide). Research on the ECS has recently aroused enormous interest not only for the physiological functions, but also for the promising therapeutic potentials of drugs interfering with the activity of cannabinoid receptors. Many of the former relate to stress-recovery systems and to the maintenance of homeostatic balance. Among other functions, the ECS is involved in neuroprotection, modulation of nociception, regulation of motor activity, neurogenesis, synaptic plasticity and the control of certain phases of memory processing. In addition, the ECS acts to modulate the immune and inflammatory responses and to maintain a positive energy balance. This theme issue aims to provide the reader with an overview of ECS pharmacology, followed by discussions on the pivotal role of this system in the modulation of neurogenesis in the developing and adult organism, memory processes and synaptic plasticity, as well as in pathological pain and brain ageing. The volume will conclude with discussions that address the proposed therapeutic applications of targeting the ECS for the treatment of neurodegeneration, pain and mental illness.