The Impact of Message Mutation on the Fitness of a Genetic Code

The Standard Genetic Code is organized such that similar codons encode similar amino acids. One explanation suggested that the Standard Code is the result of natural selection to reduce the fitness ``load' that derives from the mutation and mistranslation of protein-coding genes. We review the arguments against the mutational load-minimizing hypothesis and argue that they need to be reassessed. We review recent analyses of the organization of the Standard Code and conclude that under cautious interpretation they support the mutational load-minimizing hypothesis. We then present a deterministic asexual model with which we study the mode of selection for load minimization. In this model, individual fitness is determined by a protein phenotype resulting from the translation of a mutable set of protein-coding genes. We show that an equilibrium fitness may be associated with a population with the same genetic code and that genetic codes that assign similar codons to similar amino acids have a higher fitness. We also show that the number of mutant codons in each individual at equilibrium, which determines the strength of selection for load minimization, reflects a long-term evolutionary balance between mutations in messages and selection on proteins, rather than the number of mutations that occur in a single generation, as has been assumed by previous authors. We thereby establish that selection for mutational load minimization acts at the level of an individual in a single generation. We conclude with comments on the shortcomings and advantages of load minimization over other hypotheses for the origin of the Standard Code. Received: 4 April 2001 / Accepted: 22 October 2001     

On the inducibility of nitrate transport by tobacco cells

The question as to whether the nitrate transport system is induced by nitrate was addressed using a cell suspension of the XD line of Nicotiana tabacum L. cv. Xanthi as an experimental system. The cells were grown on area as the sole nitrogen source, and tungstate was used to render nitrate reductase non-functional. To avoid shock due to vacuum filtration, the cells, were harvested by gravity filtration. Nitrate uptake by cells, which were harvested, transferred to fresh medium, and immediately exposed to nitrate (freshly harvested cells), displayed a lag period of about 3 h.
In cells which were given incubation periods in fresh medium before exposure to nitrate (preincubated cells), the lag period was considerably shortened. After 3 h of preincubation in the absence of nitrate (recovered cells), the lag period was almost completely eliminated. Cycloheximide inhibited nitrate uptake by recovered cells within minutes, and prevented the development of nitrate uptake in freshly harvested cells. Cycloheximide did not affect uptake of α-aminoisobutyric acid (AIB) within the first 2 h after its addition. Recovery of the membrane potential from a low value just after the harvest of the cells to a maximal value 3 h later, was observed using the lipophilic cation methyltriphenylphosphonium (MTPP⁺), supplied at low concentrations, as a probe. Depolarization of the membrane potential by MTPP⁺, at the millimolar range, caused a rapid inhibition of nitrate uptake by recovered cells. The results indicate that nitrate transport by the XD cells depends on the membrane potential and on protein components with short half life. In addition, it requires a continuous protein synthesis. The effects of physical manipulation on nitrate uptake are discussed.     

Neuroprotective effect of N-acetyl-aspartyl-glutamate in combination with mild hypothermia in the endothelin-1 rat model of focal cerebral ischaemia

Previously we showed that treatment with mild hypothermia (34 degrees C for 2 h) after a focal cerebral infarct was neuroprotective by reducing apoptosis in the penumbra (cortex), but not in the core (striatum) of the infarct. In this study we examined whether administration of N-acetyl-aspartyl-glutamate (NAAG) in combination with mild hypothermia could improve striatal neuroprotection in the endothelin-1 rat model. NAAG (10 mg/kg i.p.) was injected under normothermic (37 degrees C) or mild hypothermic conditions, either 40 min before or 20 min after the insult. NAAG reduced caspase 3 immunoreactivity in the striatum, irrespective of the time of administration and brain temperature. This neuroprotective effect could be explained, at least partially, by decreased nitric oxide synthase activity in the striatum and was blocked by the group II metabotropic glutamate receptor antagonist, LY341495. Hypothermia applied together with NAAG reduced both cortical and striatal caspase 3 immunoreactivity, as well as the overall ischaemic damage in these areas. However, no pronounced improvement was seen in total damaged brain volume. Extracellular glutamate levels did not correlate with the observed protection, whatever treatment protocol was applied. We conclude that treatment with NAAG causes the same degree of neuroprotection as treatment with hypothermia. Combination of the two treatments, although reducing apoptosis, does not considerably improve ischaemic damage.     

Trapping of the thioacylglyceraldehyde-3-phosphate dehydrogenase intermediate from Bacillus stearothermophilus. Direct evidence for a flip-flop mechanism

The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8A resolution. Formation of the intermediate was obtained by diffusion of the natural substrate within the crystal of the holoenzyme in the absence of inorganic phosphate. To define the soaking conditions suitable for the isolation and accumulation of the intermediate, a microspectrophotometric characterization of the reaction of GAPDH in single crystals was carried out, following NADH formation at 340 nm. When compared with the structure of the Michaelis complex (Didierjean, C., Corbier, C., Fatih, M., Favier, F., Boschi-Muller, S., Branlant, G., and Aubry, A. (2003) J. Biol. Chem. 278, 12968-12976) the 206-210 loop is shifted and now forms part of the so-called "new P(i)" site. The locations of both the O1 atom and the C3-phosphate group of the substrate are also changed. Altogether, the results provide evidence for the flipping of the C3-phosphate group occurring concomitantly or after the redox step.     

Can we predict butterfly diversity along an elevation gradient from space?

An important challenge in ecology is to predict patterns of biodiversity across eco‐geographical gradients. This is particularly relevant in areas that are inaccessible, but are of high research and conservation value, such as mountains. Potentially, remotely‐sensed vegetation indices derived from satellite images can help in predicting species diversity in vast and remote areas via their relationship with two of the major factors that are known to affect biodiversity: productivity and spatial heterogeneity in productivity. Here, we examined whether the Normalized Difference Vegetation Index (NDVI) can be used effectively to predict changes in butterfly richness, range size rarity and beta diversity along an elevation gradient. We examined the relationship between butterfly diversity and both the mean NDVI within elevation belts (a surrogate of productivity) and the variability in NDVI within and among elevation belts (surrogates for spatial heterogeneity in productivity). We calculated NDVI at three spatial extents, using a high spatial resolution QuickBird satellite image. We obtained data on butterfly richness, rarity and beta diversity by field sampling 100 m quadrats and transects between 500 and 2200 m in Mt Hermon, Israel. We found that the variability in NDVI, as measured both within and among adjacent elevation belts, was strongly and significantly correlated with butterfly richness. Butterfly range size rarity was strongly correlated with the mean and the standard deviation of NDVI within belts. In our system it appears that it is spatial heterogeneity in productivity rather than productivity per se that explained butterfly richness. These results suggest that remotely‐sensed data can provide a useful tool for assessing spatial patterns of butterfly richness in inaccessible areas. The results further indicate the importance of considering spatial heterogeneity in productivity along elevation gradients, which has no lesser importance than productivity in shaping richness and rarity, especially at the local scale.     

Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy

The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes closest to fulfilling this crucial requirement. The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal “break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape. Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive, easily applicable, widespread technology.