Systems-biology approaches for predicting genomic evolution

Is evolution predictable at the molecular level? The ambitious goal to answer this question requires an understanding of the mutational effects that govern the complex relationship between genotype and phenotype. In practice, it involves integrating systems-biology modelling, microbial laboratory evolution experiments and large-scale mutational analyses - a feat that is made possible by the recent availability of the necessary computational tools and experimental techniques. This Review investigates recent progresses in mapping evolutionary trajectories and discusses the degree to which these predictions are realistic.     

Kinetic dissection of fundamental processes of eukaryotic translation initiation in vitro

Approaches have been developed for the kinetic dissection of eukaryotic translation initiation in vitro using rabbit reticulocyte ribosomes and a crude preparation of initiation factors. These new approaches have allowed the kinetics of formation of the 43S and 80S ribosomal complexes to be followed and have substantially improved the ability to follow formation of the first peptide bond. The results suggest the existence of a new step on the initiation pathway that appears to require at least one additional factor and the hydrolysis of GTP and may prepare the 80S complex for the formation of the first peptide bond. The initial kinetic framework and methods developed herein will allow the properties of individual species along the initiation pathway to be probed further and will facilitate dissection of the mechanistic roles of individual translation factors and their interplay with RNA structural elements.     

Molecular genetics of the eukaryotic cell

A short review is presented concerning the studies developed in the Soviet Union during the last decade in molecular genetics of higher organisms, especially dealing with mobile genetic elements.     

The origin of the eukaryotic cell. III. Principles of the morphofunctional organization of the eukaryotic cell

The eukaryotic plasmalemma, eukaryotic cytoplasm with its usual cytomembranes, and eukaryotic nucleus are obligatory components of the eukaryotic cell. All other structural elements (organelles) are only derivates of the aforesaid cell components and they may be absent sometimes. There are protozoans having simultaneously no flagelles, mitochondria and chloroplasts (all the representatives of phylum Microspora, amoeba Pelomyxa palustris, and others). The following five general principles play the main role in the morphofunctional organization of the cell. The principle of hierarchy of block organization of living systems. Complex morphofunctional blocks (organelles) specific for the eukaryotic cell are formed. The compartmentalization principle. The main cell organelles (nuclei, flagellae, mitochondria, chloroplasts, etc.) undergo a relative morphological isolation from each other and other cell organelles by means of the total or partial surrounding by membranes; this may ensure the originality of their evolution and function. The principle of poly- and oligomerization of morphofunctional blocks. It permits the cell to enlarge its sizes and to raise the level of integration. The principle of heterochrony, including three subprinciples: conservatism of useful signs; a strong acceleration of evolutionary development of the separate blocks; simplification of the structure, reduction or total disappearance of some blocks. It explains a preservation of prokaryotic signs in the eukaryotic cell or in its organelles. The principle of independent origin of similar morphofunctional blocks in the process of evolution of living systems. The parallelism of the signs in unrelated groups of cells (or protists) arises due to this principle.     

Development of high-throughput phenotyping of metagenomic clones from the human gut microbiome for modulation of eukaryotic cell growth

Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.     

Long-range dielectric aspects of the eukaryotic cell cycle

The phases of the eukaryotic cell cycle are described in terms of H. Fr?hlich's theory of long-range coherence in biological systems. A phonon condensation is believed to initiate the S phase of the cycle. Following this event, an increasing polarization of the cell should occur, resulting in a phase transition to a metastable ferroelectric state at the beginning of mitosis. This polarized state is expected to be dissipated after mitosis is completed. It is believed that malignant cells possess a ferroelectric state throughout their life cycles.     

Effects of novel C-methylated spermidine analogs on cell growth via hypusination of eukaryotic translation initiation factor 5A

The polyamines, putrescine, spermidine, and spermine, are ubiquitous multifunctional cations essential for cellular proliferation. One specific function of spermidine in cell growth is its role as a butylamine donor for hypusine synthesis in the eukaryotic initiation factor 5A (eIF5A). Here, we report the ability of novel mono-methylated spermidine analogs (α-MeSpd, β-MeSpd, γ-MeSpd, and ω-MeSpd) to function in the hypusination of eIF5A and in supporting the growth of DFMO-treated DU145 cells. We also tested them as substrates and inhibitors for deoxyhypusine synthase (DHS) in vitro. Of these compounds, α-MeSpd, β-MeSpd, and γ-MeSpd (but not ω-MeSpd) were substrates for DHS in vitro, while they all inhibited the enzyme reaction. As racemic mixtures, only α-MeSpd and β-MeSpd supported long-term growth (9-18 days) of spermidine-depleted DU145 cells, whereas γ-MeSpd and ω-MeSpd did not. The S-enantiomer of α-MeSpd, which supported long-term growth, was a good substrate for DHS in vitro, whereas the R-isomer was not. The long-term growth of DFMO-treated cells correlated with the hypusine modification of eIF5A by intracellular methylated spermidine analogs. These results underscore the critical requirement for hypusine modification in mammalian cell proliferation and provide new insights into the specificity of the deoxyhypusine synthase reaction.     

Enzymatic dissection of embryonic cell adhesive mechanisms

In this paper we describe a kinetic assay for cell adhesion which measures the formation of cell clusters. Cluster formation is dependent on both calcium and protein synthesis, two parameters essential for the formation of histotypic aggregates. We also describe modifications of the stndard method for trypsinization of tissues which result in populations of single cells that appear to bear intact and functional cell surface adhesive systems. These modifications involve the use of chymotrypsin and the inclusion of calcium during enzyme digestion of tissues with trypsin and chymotrypsin. Using the cluster formation assay and the modified tissue dissociation techniques, we demonstrate the presence of two functionally distinct adhesive systems operating among embryonic chick neural retina cells. These two systems differ in proteolytic sensitivity, protection by calcium against proteolysis, dependence on calcium for function and morphogenetic potential. Cells possessing one of these intact adhesive systems are capable of extensive morphogenetic interactions in the absence of protein synthesis.     

Zinc-buffering capacity of a eukaryotic cell at physiological pZn

In spite of the paramount importance of zinc in biology, dynamic aspects of cellular zinc metabolism remain poorly defined at the molecular level. Investigations with human colon cancer (HT-29) cells establish a total cellular zinc concentration of 264 microM. Remarkably, about 10% of the potential high-affinity zinc-binding sites are not occupied by zinc, resulting in a surplus of 28 muM ligands (average Kd(c) = 83 pM) that ascertain cellular zinc-buffering capacity and maintain the "free" zinc concentration in proliferating cells at picomolar levels (784 pM, pZn = 9.1). This zinc-buffering capacity allows zinc to fluctuate only with relatively small amplitudes (DeltapZn = 0.3; below 1 nM) without significantly perturbing physiological pZn. Thus, the "free" zinc concentrations in resting and differentiated HT-29 cells are 614 pM and 1.25 nM, respectively. The calculation of these "free" zinc concentrations is based on measurements at different concentrations of the fluorogenic zinc-chelating agent and extrapolation to a zero concentration of the agent. It depends on the state of the cell, its buffering capacity, and the zinc dissociation constant of the chelating agent. Zinc induction of thionein (apometallothionein) ensures a surplus of unbound ligands, increases zinc-buffering capacity and the availability of zinc (DeltapZn = 0.8), but preserves the zinc-buffering capacity of the unoccupied high-affinity zinc-binding sites, perhaps for crucial physiological functions. Jointly, metallothionein and thionein function as the major zinc buffer under conditions of increased cellular zinc.