Self-organizing proto-replicators and the origin of life

Standard theories suggest that the first informational replicator involved RNA molecules (or a more primitive analogue) and that a preliminary step for the development of such replicating systems may have been the emergence of subunits capable of forming chains and interchain pairings. Following these hypotheses, this discussion describes various abstract simulations designed to investigate the structures resulting from such interactions for generalised subunits. Three classes of pairing strategy were considered for a range of subunit concentrations. The resulting dynamic self-organization of the systems produced high levels of structural complexity (some at low subunit concentrations and in the presence of disruptive subunits) and a significantly increased percentage of complementary base pairing (particularly in the more substantial structures). These properties of the systems, which did not require pre-existing replicators, templates or functional catalysts, were shown to be sensitive to the form of pairing strategy, subunit concentrations and various conditions that could theoretically be altered by products of the systems. Though no systems behaved as a replicator, some possessed collections of properties from which a replicating system might theoretically be constructed without requiring the introduction of additional classes of properties. The implications of such systems were considered with respect to the origin of life.     

Computer modelling of DNA structures involved in chromosome maintenance.

Sequence-dependent DNA bending of synthetic and natural molecules was studied by computer analysis. Modelling of synthetic oligonucleotides and of 107 kb of natural sequences gave results which closely resembled published electrophoretic data, demonstrating the powerful predictive capacity of the procedure. The analysis was extended to the study of DNA structures involved in chromosome maintenance. Centromeric DNAs from yeast were found to have sequences in their functional elements which cause them to be unusually straight. Autonomous replicating sequences were found to have two structural domains, one consisting of unusually straight sequences surrounding the consensus and the other of bending elements in flanking DNA. In addition to a structural homology, centromeric and autonomous replicating sequences share common sequence elements. These observations show that computer modelling of natural sequences is a viable approach to the study of the biological implications of alternative DNA structures.     

Innate immune recognition of viral infection

Toll-like receptors (TLRs) are key molecules of the innate immune systems, which detect conserved structures found in a broad range of pathogens and triggers innate immune responses. A subset of TLRs recognize viral components and induce antiviral responses by producing type I interferons. Whereas TLR2 and TLR4 recognize viral components at the cell surface, TLR3, TLR7, TLR8 and TLR9 are exclusively expressed in endosomal compartments. After phagocytes internalize viruses or virus-infected apoptotic cells, viral nucleic acids are released in phagolysosomes and are recognized by these TLRs. Recent reports have shown that hosts also have a mechanism to detect replicating viruses in the cytoplasm in a TLR-independent manner. In this review, we focus on the viral recognition by innate immunity and the signaling pathways.     

Olfactory receptor neuron profiling using sandalwood odorants

The mammalian olfactory system can discriminate between volatile molecules with subtle differences in their molecular structures. Efforts in synthetic chemistry have delivered a myriad of smelling compounds of different qualities as well as many molecules with very similar olfactive properties. One important class of molecules in the fragrance industry are sandalwood odorants. Sandalwood oil and four synthetic sandalwood molecules were selected to study the activation profile of endogenous olfactory receptors when exposed to compounds from the same odorant family. Dissociated rat olfactory receptor neurons were exposed to the sandalwood molecules and the receptor activation studied by monitoring fluxes in the internal calcium concentration. Olfactory receptor neurons were identified that were specifically stimulated by sandalwood compounds. These neurons expressed olfactory receptors that can discriminate between sandalwood odorants with slight differences in their molecular structures. This is the first study in which an important class of perfume compounds was analyzed for its ability to activate endogenous olfactory receptors in olfactory receptor neurons.     

Molecular recognition: model studies with convergent functional groups

Model studies of molecular recognition are reviewed with emphasis on contributions from macrocyclic chemistry. Recent developments concerning new molecular shapes are discussed. The advantages of a molecular cleft are presented. In these structures functional groups converge to create a microenvironment complementary to substrates. Specifically, di-, tri- and tetracarboxylic acids of varying sizes are shown to recognize smaller molecules of complementary shape. The new receptors function by a combination of hydrogen bonding and aryl stacking interactions. Substrates include amines, acids, amino acids, metal ions and nucleotide components. The relationship between functional group orientation and catalysis is explored and two systems capable of concerted acid/base catalysis are introduced.     

Molecular and cellular basis of human olfaction

The human olfactory systems recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein in the ciliary membrane of olfactory neurons. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed, and characterized some of the human olfactory receptors from chromosome 17. Our results show that a receptor protein is capable of recognizing the particular chemical substructure of an odor molecule and, therefore, is able to respond only to odorants that have a defined molecular structure. These findings represent the beginning of the molecular understanding of odorant recognition in humans. In the future, this knowledge could be used for the design of synthetic ideal receptors for specific odors (biosensors), or the perfect odor molecule for a given receptor.     

Enrichment and visualization of small replication units from cultured mammalian cells

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.     

Recognition of neutral species with synthetic receptors

The design of synthetic receptors for neutral molecules is an area of intense current interest. The area has grown from early work on cyclodextrin or single crown ether complexation to encompass a wide array of receptor shapes and structures. Furthermore, the range of substrate selectivities has increased from simple aromatic or metal ion substrates to include key biological components such as peptides and carbohydrates. Recent advances have included the application of split bead combinatorial methods for the identification of receptors for oligopeptides, the design of self-assembling spherical receptors, the use of multiple hydrogen bonding groups for carboxylic acid and carbohydrate recognition and the achievement of impressive catalytic effects with synthetic receptors.     

RNA synthetic biology

RNA molecules play important and diverse regulatory roles in the cell by virtue of their interaction with other nucleic acids, proteins and small molecules. Inspired by this natural versatility, researchers have engineered RNA molecules with new biological functions. In the last two years efforts in synthetic biology have produced novel, synthetic RNA components capable of regulating gene expression in vivo largely in bacteria and yeast, setting the stage for scalable and programmable cellular behavior. Immediate challenges for this emerging field include determining how computational and directed-evolution techniques can be implemented to increase the complexity of engineered RNA systems, as well as determining how such systems can be broadly extended to mammalian systems. Further challenges include designing RNA molecules to be sensors of intracellular and environmental stimuli, probes to explore the behavior of biological networks and components of engineered cellular control systems.     

Ricin antitoxins based on lyotropic mesophases containing galactose amphiphiles

Lyotropic mesophases of the inverse hexagonal or cubic type are nanostructured materials that result from the self-assembly of amphiphilic surfactant molecules in water. The extremely large area of the surfactant-water interface inherent within these structures makes them attractive media for sorbent or encapsulant systems. Here, we report on the development of a new class of polyvalent materials that are based on the incorporation of bioactive ligands within lyotropic mesophases. In particular, we have studied the potential for these materials to behave as polyvalent antitoxins by incorporating synthetic galactose amphiphiles, which mimic the natural cell surface ligand for the protein toxin ricin. The study demonstrates that cubic morphology lyotropic mesophases containing galactose amphiphiles exhibit high specificity ricin uptake, with favorably high dissociation constants and high capacities. We suggest that lyotropic mesophase polyvalent ligands are thus promising materials for the incorporation of a broad range of cell surface recognition moieties and hence may have wide applicability as materials capable of partaking in biological recognition processes.     

Polyoma virus minichromosomes: associated DNA molecules

Electron microscopy was used to identify and quantitate DNA molecules associated with 3H-labeled polyoma minichromosomes which had been fractionated on a sucrose gradient. The percentage of replicating DNA molecules observed in the fractions of the gradient normally designated the replicative intermediate region was up to ninefold higher than in fractions from the mature region. Nevertheless, because of the higher overall concentration of polyoma DNA molecules in the mature region, nearly as many replicating DNA molecules were computed to be in the mature region as in the replicative intermediate region. The replicating molecules in the mature region was predominantly early replicative intermediates. Almost all late replicative intermediates were found in the replicative intermediate region. Under aqueous spreading conditions, a substantial fraction of the replicating DNA structures appeared to be asymmetrical or otherwise unusual, suggesting that extensive single-stranded regions may exist in replicating polyoma minichromosomes.     

Strand switching during rolling circle replication of plasmid-like DNA circles in the mitochondria of the higher plant Chenopodium album (L.)

The structure of sigma-like mitochondrial DNA molecules prepared from suspension cultured cells of Chenopodium album (L.) was studied by electron microscopy. These molecules were highly variable in size, ranging from about 1 to 104 kb, and had single- and double-stranded regions typical for rolling circle replicating intermediates. Partial denaturation studies confirmed that these structures constitute rolling circles. Close inspection of the circle-tail junctions of the replication fork at high magnification suggests that in circles with a double-stranded tail, both strands of the tail seem to be covalently attached to the circle in about 27% of the molecules. This observation can be explained by a phenomenon called strand switching or strand splippage during rolling circle replication, similar to a mechanism proposed for bacterial replicons or in vitro replicating constructs harboring bacteriophage T4 replication origins.     

Synthetic immune signaling

A successful immune response against pathogens requires the activation of different cell types of the immune system. These activation processes are difficult to study by classical biochemical and genetic methods alone. In this review we describe how approaches of synthetic biology, such as rebuilding of minimal functional signaling systems and the design of new molecules acting as signaling switches, can be used to get a deeper insight into the signaling mechanism of immune cells. In particular, the interaction of receptors with signal-transducing elements can be studied in detail with these new methods. In addition, sophisticated synthetic immune receptors are being tested in the clinic for gene therapy against certain cancer types.     

DNA structures associated with autonomously replicating sequences from plants

DNA fragments capable of conferring autonomous replicating ability to plasmids inSaccharomyces cerevisiae were isolated from four different plant genomes and from the Ti plasmid ofAgrobacterium tumefaciens. The DNA structure of these autonomously replicating sequences (ARSs) as well as two from yeast were studied using retardation during polyacrylamide gel electrophoresis and computer analysis as measures of sequence-dependent DNA structures. Bent DNA was found to be associated with the ARS elements. An 11 bp ARS consensus sequence required for ARS function was also identified in the elements examined and was flanked by unusually straight structures which were rich in A+T content. These results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals.Supported by Grant 1RO1-GM41708-O1 from the National Institute of Health.     

Hormonal control of inflammatory responses

Almost any stage of inflammatory and immunological responses is affected by hormone actions. This provides the basis for the suggestion that hormones act as modulators of the host reaction against trauma and infection. Specific hormone receptors are detected in the reactive structures in inflamed areas and binding of hormone molecules to such receptors results in the generation of signals that influence cell functions relevant for the development of inflammatory responses. Diversity of hormonal functions accounts for recognized pro- and anti-inflammatory effects exerted by these substances. Most hormone systems are capable of influencing inflammatory events. Insulin and glucocorticoids, however, exert direct regulatory effects at concentrations usually found in plasma. Insulin is endowed with facilitatory actions on vascular reactivity to inflammatory mediators and inflammatory cell functions. Increased concentrations of circulating glucocorticoids at the early stages of inflammation results in downregulation of inflammatory responses. Oestrogens markedly reduce the response to injury in a variety of experimental models. Glucagon and thyroid hormones exert indirect anti-inflammatory effects mediated by the activity of the adrenal cortex. Accordingly, inflammation is not only merely a local response, but a hormone-controlled process.     

Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry

Just as synthetic organic chemistry once revolutionized the ability of chemists to build molecules (including those that did not exist in nature) following a basic set of design rules, cell-free synthetic biology is beginning to provide an improved toolbox and faster process for not only harnessing but also expanding the chemistry of life. At the interface between chemistry and biology, research in cell-free synthetic systems is proceeding in two different directions: using synthetic biology for synthetic chemistry and using synthetic chemistry to reprogram or mimic biology. In the coming years, the impact of advances inspired by these approaches will make possible the synthesis of nonbiological polymers having new backbone compositions, new chemical properties, new structures, and new functions.     

Structure of Replicating Simian Virus 40 Deoxyribonucleic Acid Molecules

Properties of replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) have been examined by sedimentation analysis and by direct observation during a lytic cycle of infection of African green monkey kidney cells. Two types of replicating DNA molecules were observed in the electron microscope. One was an open structure containing two branch points, three branches, and no free ends whose length measurements were consistent with those expected for replicating SV40 DNA molecules. A second species had the same features as the open structure, but in addition it contained a superhelix in the unreplicated portion of the molecule. Eighty to ninety per cent of the replicative intermediates (RI) were in this latter configuration, and length measurements of these molecules also were consistent with replicating SV40 DNA. Replicating DNA molecules with this configuration have not been described previously. RI, when examined in ethidium bromide-cesium chloride (EB-CsCl) isopycnic gradients, banded in a heterogeneous manner. A fraction of the RI banded at the same density as circular SV40 DNA containing one or more single-strand nicks (component II). The remaining radioactive RI banded at densities higher than that of component II, and material was present at all densities between that of supercoiled double-stranded DNA (component I) and component II. When RI that banded at different densities in EB-CsCl were examined in alkaline gradients, cosedimentation of parental DNA and newly replicated DNA did not occur. All newly replicated DNA sedimented more slowly than did intact single-stranded SV40 DNA, a finding that is inconsistent with the rolling circle model of DNA replication. An inverse correlation exists between the extent of replication of the SV40 DNA and the banding density in EB-CsCl. Under alkaline conditions, the parental DNA strands that were contained in the RI sedimented as covalently closed structures. The sedimentation rates in alkali of the covalently closed parental DNA decreased as replication progressed. Based on these observations, some possible models for replication of SV40 DNA are proposed.