Evolution of eukaryotic cell cycle regulation: stepwise addition of regulatory kinases and late advent of the CDKs

Protein kinases regulate a number of critical events in mitosis and meiosis. A study of the evolution of kinases involved in cell cycle control (CCC) might shed light on the evolution of the eukaryotic cell cycle. In particular, applying quantitative phylogenetic methods to key CCC kinases could provide information on the relative timing of gene duplication events. To investigate the evolution of CCC kinases, we constructed phylogenetic trees for the CDC28 family and performed statistical tests of the tree topology. This family includes the cyclin-dependent kinases (CDKs), which are key regulators of the eukaryotic cell cycle, as well as other CCC kinases. We found that CDKs and, in particular, the principal cell cycle regulator Cdc28p, branch off the phylogenetic tree at a late stage, after several other kinases involved in either mitosis or meiosis regulation. On the basis of this tree topology, it is proposed that, at early stages of evolution, the eukaryotic cell cycle was not controlled by CDKs and that only a subset of extant kinases, notably the DNA damage checkpoint kinase Chk1p, were in place. During subsequent evolution, a series of duplications of kinase genes occurred, gradually adding more kinases to the CCC system, the CDKs being among the last major additions.     

The Role of Horizontal Gene Transfer by Bacteriophages in the Origin of Pathogenic Bacteria

The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer. Examples of phage participation in this process proved to a different extent are described. Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent KZ-like phages) of conditional pathogen Pseudomonas aeruginosa.Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are similar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution. An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches.     

Role of horizontal gene transfer by bacteriophages in the origin of pathogenic bacteria

The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer. Examples of phage participation in this process proved to a different extent are described. Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent phi KZ-like phages) of conditional pathogen Pseudomonas aeruginosa. Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are similar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution. An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches.     

Phenogenetic Characterization of a Group of Giant φKZ-like Bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group ofPseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage KZ genome (288 334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1–19]. By DNA homology, the phages were assigned to three species (represented by phages KZ, Lin68, and EL, respectively) and two new genera (KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the KZ species (KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the KZ genus. The origin and evolution of the KZ-like phages are discussed. Analysis of protein sequences encoded by the phage KZ genome made it possible to assume wide migration of the KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Evolution and phylogeny of coccidia with the eimerian-type and isomeric-type oocysts

The isosporan-type of oocysts is a later evolutionary purchase in comparison with the eimerioran-type construction of oocysts'. Identical number of sporocysts and sporozoits in the oocysts, bivalvular constructions of sporocysts, heteroxenous life cycles and endogenous sporulation in a group series of coccidia have arisen asynchronously and independently. The evolution of RNA genes and a type of oocysts construction was proceeded in different species of the isosporan-type coccidia and with different velocities.     

Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin-2 in renal epithelial cells

The initial response of renal epithelial cells to the antidiuretic hormone arginine vasopressin (AVP) is an increase in cyclic AMP. By applying immunofluorescence, cell membrane capacitance and transepithelial water flux measurements we show that cAMP alone is sufficient to elicit the antidiuretic cellular response in primary cultured epithelial cells from renal inner medulla, namely the transport of aquaporin-2 (AQP2)-bearing vesicles to, and their subsequent fusion with, the plasma membrane (AQP2 shuttle). The AQP2 shuttle is evoked neither by AVP-independent Ca²⁺ increases nor by AVP-induced Ca²⁺ increases. However, clamping cytosolic Ca²⁺ concentrations below resting levels at 25 nM inhibited exocytosis. Exocytosis was confined to a slow monophasic response, and readily releasable vesicles were missing. Analysis of endocytic capacitance steps revealed that cAMP does not decelerate the retrieval of AQP2 from the plasma membrane. Our data suggest that cAMP initiates an early step, namely the transport of AQP2-bearing vesicles towards the plasma membrane, and do not support a regulatory function for Ca²⁺ in the AQP2 shuttle.     

Investigation of the possibility of exceeding the Greenwald density limit during ECRH in T-10

In T-10 experiments, attempts were made to significantly exceed the Greenwald limit $\bar n_{Gr} $ during high-power (P _ab=750 kW) electron-cyclotron resonance heating (ECRH) and gas puffing. Formally, the density limit $(\bar n_e )_{\lim } $ exceeding the Greenwald limit $({{(\bar n_e )_{\lim } } \mathord{\left/ {\vphantom {{(\bar n_e )_{\lim } } {\bar n_{Gr} }}} \right. \kern-0em} {\bar n_{Gr} }} = 1.8)$ was achieved for q _L=8.2. However, as q _L decreased, the ratio ${{(\bar n_e )_{\lim } } \mathord{\left/ {\vphantom {{(\bar n_e )_{\lim } } {\bar n_{Gr} }}} \right. \kern-0em} {\bar n_{Gr} }}$ also decreased, approaching unity at q _L≈3. It was suggested that the “current radius” (i.e., the radius of the magnetic surface enclosing the bulk of the plasma current I _p), rather than the limiter radius, was the parameter governing the value of $(\bar n_e )_{\lim } $ . In the ECRH experiments, no substantial degradation of plasma confinement was observed up to $\bar n_e \sim 0.9(\bar n_e )_{\lim } $ regardless of the ratio ${{(\bar n_e )_{\lim } } \mathord{\left/ {\vphantom {{(\bar n_e )_{\lim } } {\bar n_{Gr} }}} \right. \kern-0em} {\bar n_{Gr} }}$ . In different scenarios of the density growth up to $(\bar n_e )_{\lim } $ , two types of disruptions related to the density limit were observed.     

Stabilization of O-pyromellitylgramicidin channels in bilayer lipid membranes through electrostatic interaction with polylysines of different chain lengths

Functioning of membrane proteins, in particular ionic channels, can be modulated by alteration of their arrangement in membranes. We addressed this issue by studying the effect of different chain length polylysines on the kinetics of ionic channels formed in a bilayer lipid membrane (BLM) by O-pyromellitylgramicidin carrying three negative charges at the C-terminus. The method of sensitized photoinactivation was applied to the analysis of the channel association-dissociation kinetics (characterized by the exponential factor of the curve describing the time course of the flash-induced decrease in the transmembrane current, tau). Addition of polylysine to the bathing solutions of BLM led to the deceleration of the photoinactivation kinetics, i.e. to the increase in tau. It was shown here that for a series of polylysines differing in their chain lengths, the value of tau grew as their concentration increased above a threshold level until at a certain concentration of each polylysine tau reached maximum. At higher polylysine concentrations tau began to decrease and finally became close to the control level observed in the absence of polylysine. With lengthening of the polylysine chain the maximum value of tau increased, the concentration dependence became steeper, and the threshold concentration decreased. The increase in the ionic strength of the medium shifted the concentration dependence of tau to higher polylysine concentrations and decreased the maximum value of tau. It was concluded that the increase in tau was caused by the formation of domains of O-pyromellitylgramicidin molecules induced by binding of polylysines. This can be related to functional aspects of polycation-induced sequestering of negatively charged transmembrane peptides in neutral membranes.