Contrast-FEL—A Test for Differences in Selective Pressures at Individual Sites among Clades and Sets of Branches

A number of evolutionary hypotheses can be tested by comparing selective pressures among sets of branches in a phylogenetic tree. When the question of interest is to identify specific sites within genes that may be evolving differently, a common approach is to perform separate analyses on subsets of sequences and compare parameter estimates in a post hoc fashion. This approach is statistically suboptimal and not always applicable. Here, we develop a simple extension of a popular fixed effects likelihood method in the context of codon-based evolutionary phylogenetic maximum likelihood testing, Contrast-FEL. It is suitable for identifying individual alignment sites where any among the K≥2 sets of branches in a phylogenetic tree have detectably different ω ratios, indicative of different selective regimes. Using extensive simulations, we show that Contrast-FEL delivers good power, exceeding 90% for sufficiently large differences, while maintaining tight control over false positive rates, when the model is correctly specified. We conclude by applying Contrast-FEL to data from five previously published studies spanning a diverse range of organisms and focusing on different evolutionary questions.     

A unique,thermostable dimer linkage structure of RD114 retroviral RNA

Retroviruses package their genome as RNA dimers linked together primarily by base-pairing between palindromic stem–loop (psl) sequences at the 5′ end of genomic RNA. Retroviral RNA dimers usually melt in the range of 55°C–70°C. However, RNA dimers from virions of the feline endogenous gammaretrovirus RD114 were reported to melt only at 87°C. We here report that the high thermal stability of RD114 RNA dimers generated from in vitro synthesized RNA is an effect of multiple dimerization sites located in the 5′ region from the R region to sequences downstream from the splice donor (SD) site. By antisense oligonucleotide probing we were able to map at least five dimerization sites. Computational prediction revealed a possibility to form stems with autocomplementary loops for all of the mapped dimerization sites. Three of them were located upstream of the SD site. Mutant analysis supported a role of all five loop sequences in the formation and thermal stability of RNA dimers. Four of the five psls were also predicted in the RNA of two baboon endogenous retroviruses proposed to be ancestors of RD114. RNA fragments of the 5′ R region or prolonged further downstream could be efficiently dimerized in vitro. However, this was not the case for the 3′ R region linked to upstream U3 sequences, suggesting a specific mechanism of negative regulation of dimerization at the 3′ end of the genome, possibly explained by a long double-stranded RNA region at the U3-R border. Altogether, these data point to determinants of the high thermostability of the dimer linkage structure of the RD114 genome and reveal differences from other retroviruses.     

The Relationship between the Structure of the Tick-Borne Encephalitis Virus Strains and Their Pathogenic Properties

Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates.     

Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes

Summary Osmotic swelling of human and rat erythrocytes does not induce regulatory volume decrease. Regulatory volume increase was observed in shrunken erythrocytes of rats only. This reaction was blocked by the inhibitors of Na⁺/H⁺ exchange. Cytoplasmic acidification in erythrocytes of both species increases the amiloride-inhibited component of²²Na influx by five- to eight-fold. Both the osmotic and isosmotic shrinkage of rat erythrocytes results in the 10- to 30-fold increase of amiloride-inhibited²²Na influx and a two-fold increase of furosemide-inhibited⁸⁶Rb influx. We failed to indicate any significant changes of these ion transport systems in shrunken human erythrocytes. The shrinking of quin 2-loaded human and rat erythrocytes results in the two- to threefold increase of the rate of⁴⁵Ca influx, which is completely blocked by amiloride. The dependence of volume-induced²²Na influx in rat erythrocytes and⁴⁵Ca influx in human erythrocytes on amiloride concentration does not differ. The rate of⁴⁵Ca influx in resealed ghosts was reduced by one order of magnitude when intravesicular potassium and sodium were replaced by choline. It is assumed that the erythrocyte shrinkage increases the rate of a nonselective Ca _o ²⁺ (Na _i ⁺ , K _i ⁺ ) exchange. Erythrocyte shrinking does not induce significant phosphorylation of membrane protein but increases the³²P incorporation in diphosphoinositides. The effect of shrinkage on the³²P labeling of phosphoinositides is diminished after addition of amiloride. It is assumed that volume-induced phosphoinositide response plays an essential role in the mechanism of the activation of transmembrane ion movements.     

Mathematical modelling of runoff and material transport from drainage areas into recipient water bodies

This paper describes a set of mathematical models to calculate runoff, soil erosion and chemical substance yields for drainage areas. The models represent different degrees of complexity and details of describing hydrological and hydrochemical processes. The availability of initial information and the requirements of the task determine the choice of a model for each particular case. These models have been written as PC/AT programs, to enable their use by a wide range of specialists. The models have been tested in different physical and geographical conditions, e.g. in some small watersheds on the Karelian Isthmus, west of Lake Ladoga, and in experimental catchment areas located in the Valdai Hills area in the southern part of Lake Ladoga drainage area. In further applications the drainage area models have been combined with models of the recipient water body, to obtain a single model of a watershed-lake system. The results of tests have proved that the models can be used for evaluation, simulation and forecasting of the processes of rainfall and snowmelt runoff, infiltration, evaporation, water erosion and pollutant wash-off from drainage areas.     

Ontogeny of endocrine cells in the respiratory system of Syrian golden hamsters

The ontogeny of protein gene product 9.5 (PGP 9.5), serotonin (5–HT), calcitonin gene-related peptide (CGRP), and calcitonin (CT) immunoreactivity was evaluated in small-granule endocrine cells of hamster laryngotracheal epithelium from fetal day 11 to adulthood. Two centrifugal (proximal-to-distal) patterns of differentiation occur. The first pattern begins during fetal life. Endocrine cells, single and clustered in groups (presumptive- or protoneuroepithelial bodies, pNEBs), initially co-localize immunostaining for PGP 9.5, 5–HT, and CGRP in the larynx and proximal 2/3 of the trachea on day 12 and spread to the caudal trachea on day 13. 5–HT disappears fleetingly during the 24 h preceding birth; otherwise immunoreactivity for all three substances persists into adulthood. The clusters of endocrine cells survive beyond birth but are so diluted by expansion of the nonendocrine epithelium as to become inconspicuous. Since innervation was not actually observed, these clusters may persist as pNEBs, without developing connections to afferent or efferent nerve fibers. The second pattern concerns single small-granule cells stainable for CGRP but not for 5–HT. These cells first appear in the larynx and cartilaginous part of the cranial trachea on postnatal day 3, and in the middle and caudal trachea, on day 5. The cells increase in number on day 7. In adults, they predominate among endocrine cells of the cartilaginous region. A subset of these cells begins to co-express CT proximally on postnatal day 10, reaching the caudal end of the trachea by 3 weeks. A few elements of the older 5–HT-positive population may also become immunoreactive for CT in juvenile hamsters.     

Structure,molecular evolution and maintenance of copy number of extended repeated structures in the X-heterochromatin of Drosophila melanogaster

The 60 kb repeats located in the distal heterochromatin of the X chromosome of Drosophila melanogaster were cloned in overlapping cosmids. These regions, designated as SCLRs, comprised the following types of repeated elements Stellate genes, which are known to be involved in spermatogenesis; copia-like retrotransposons; LINE elements, including amplified Type rDNA insertions; and rDNA fragments. The following steps in SCLR formation were hypothesized: insertion of mobile elements into the rDNA and Stellate gene clusters: internal tandem duplication events; recombination between the rDNA cluster and Stellate tandem repeat; and amplification of the whole SCLR structure. There are about nine SCLR copies per haploid genome, but there is approximately a twofold variation in copy number between fly stocks. The SCLR copy number differences between closely related stocks are suggested to be the result of unequal sister chromatid exchange (USCE). The restricted variation in SCLR copy number between unrelated stocks and the absence of chromosomes free of SCLRs suggests that natural selection is active in copy number maintenance.