Analysis of peptides from known proteins: Clusterization in sequence space

A combinatorial sequence space (CSS) model was introduced to represent sequences as a set of overlapping k-tuples of some fixed length which correspond to points in the CSS. The aim was to analyze clusterization of protein sequences in the CSS and to test various hypotheses about the possible evolutionary basis of this clusterization. The authors developed an easy-to-use technique which can reveal and analyze such a clusterization in a multidimensional CSS. Application of the technique led to an unexpectedly high clusterization of points in the CSS corresponding to k-tuples from known proteins. The clusterization could not be inferred from nonuniform amino acid frequencies or be explained by the influence of homologous data. None of the tested possible evolutionary and structural factors could explain the clusterization observed either. It looked as if certain protein sequence variations occurred and were fixed in the early course of evolution. Subsequent evolution (predominantly neutral) allowed only a limited number of changes and permitted new variants which led to preservation of certain k-tuples during the course of evolution. This was consistent with the theory of exon shuffling and protein block structure evolution. Possible applications of sequence space features found were also discussed.Correspondence to: H.A. Lim     

Kinetics of synonymous codon change for an amino acid of arbitrary degeneracy

The kinetics of synonymous codon change and species divergence is described in a matrix formalism that is equally applicable to all levels of codon degeneracy and all levels of codon or nucleotide bias. Based on the formalism it is possible to calculate the sum of all the synonymous substitution rate constants from the observed sequence differences between two species. This sum, the relaxation rate, is equivalent to the LogDet transformation that has recently been proposed as a new measure of evolutionary distance (Lockhardt et al.Mol. Biol. Evol. 11(4): 605–612, 1994). The relationship between this measure and the average number of base changes per site (K) is discussed. The formalism is tested on some sets of simulated sequence divergence data.     

An Experimental and Computational Evolution-Based Method to Study a Mode of Co-evolution of Overlapping Open Reading Frames in the AAV2 Viral Genome

Overlapping open reading frames (ORFs) in viral genomes undergo co-evolution; however, how individual amino acids coded by overlapping ORFs are structurally, functionally, and co-evolutionarily constrained remains difficult to address by conventional homologous sequence alignment approaches. We report here a new experimental and computational evolution-based methodology to address this question and report its preliminary application to elucidating a mode of co-evolution of the frame-shifted overlapping ORFs in the adeno-associated virus (AAV) serotype 2 viral genome. These ORFs encode both capsid VP protein and non-structural assembly-activating protein (AAP). To show proof of principle of the new method, we focused on the evolutionarily conserved QVKEVTQ and KSKRSRR motifs, a pair of overlapping heptapeptides in VP and AAP, respectively. In the new method, we first identified a large number of capsid-forming VP3 mutants and functionally competent AAP mutants of these motifs from mutant libraries by experimental directed evolution under no co-evolutionary constraints. We used Illumina sequencing to obtain a large dataset and then statistically assessed the viability of VP and AAP heptapeptide mutants. The obtained heptapeptide information was then integrated into an evolutionary algorithm, with which VP and AAP were co-evolved from random or native nucleotide sequences in silico. As a result, we demonstrate that these two heptapeptide motifs could exhibit high degeneracy if coded by separate nucleotide sequences, and elucidate how overlap-evoked co-evolutionary constraints play a role in making the VP and AAP heptapeptide sequences into the present shape. Specifically, we demonstrate that two valine (V) residues and β-strand propensity in QVKEVTQ are structurally important, the strongly negative and hydrophilic nature of KSKRSRR is functionally important, and overlap-evoked co-evolution imposes strong constraints on serine (S) residues in KSKRSRR, despite high degeneracy of the motifs in the absence of co-evolutionary constraints.     

Rates of DNA Evolution in Drosophila Depend on Function and Developmental Stage of Expression

DNA-sequence divergence of genes expressed in the embryonic stage was compared with the divergence of genes expressed in adults for 13 species of Drosophila representing various degrees of relatedness. DNA-DNA hybridization experiments were conducted using as tracers complementary DNA (cDNA) reversed transcribed from poly(A)(+) mRNA isolated from different developmental stages. The results indicate: (1) cDNA is less diverged than total single-copy DNA; (2) cDNA sequences are not in the rapidly evolving fraction of the single-copy genome of Drosophila; (3) early in evolutionary divergence embryonic messages are about half as diverged as adult messages; sequence data from some of the species compared indicate this is likely due to differences in rates of silent substitutions in genes expressed at different stages of development; and (4) at greater evolutionary distance, the differences in embryonic and adult messages disappear; this could be due to lineage-specific shifts in codon usage.     

On the Informational Content of Overlapping Genes in Prokaryotic and Eukaryotic Viruses

In genetic language a peculiar arrangement of biological information is provided by overlapping genes in which the same region of DNA can code for functionally unrelated messages. In this work, the informational content of overlapping genes belonging to prokaryotic and eukaryotic viruses was analyzed. Using information theory indices, we identified in the regions of overlap a first pattern, exhibiting a more uniform base composition and more severe constraints in base ordering with respect to the nonoverlapping regions. This pattern was found to be peculiar to coliphage, avian hepatitis B virus, human lentivirus, and plant luteovirus families. A second pattern, characterized by the occurrence of similar compositional constraints in both types of coding regions, was found to be limited to plant tymoviruses. At the level of codon usage, a low degree of correlation between overlapping and nonoverlapping coding regions characterized the first pattern, whereas a close link was found in tymoviruses, indicating a fine adaptation of the overlapping frame to the original codon choice of the virus. As a result of codon usage correlation analysis, deductions concerning the origin and evolution of several overlapping frames were also proposed. Comparison of amino acid composition revealed an increased frequency of amino acid residues with a high level of degeneracy (arginine, leucine, and serine) in the proteins encoded by overlapping genes; this peculiar feature of overlapping genes can be viewed as a way with which they may expand their coding ability and gain new, specialized functions. Received: 28 October 1996 / Accepted: 29 January 1997     

Nucleotide sequence and evolution of a mammalian α-Tubulin messenger RNA

Bacterial clones containing complementary DNA sequences specific for rat brain α-tubulin messenger RNA were constructed. One plasmid, pILαTl, contains >95% of the sequences found in the mRNA: the entire coding sequence as well as extensive 5′ and 3′ untranslated sequences. Comparison of the rat amino acid sequence with the known chicken α-tubulin sequence (Valenzuela et al., 1981) reveals the extraordinary evolutionary stability of α-tubulin protein. The presence of only two interspecies amino acid differences within analogous 411 amino acid sequences predicts that amino acid substitutions in this protein are fixed with a unit evolutionary period (Wilson et al., 1977) of 550 million years (i.e. the time required for a 1% difference to arise within a specific protein in two diverging evolutionary lineages). An analysis of the silent nucleotide differences, permissible because of the degeneracy of the genetic code, demonstrates that these might not occur in a random fashion. The high guanine-cytosine bias in silent codon positions within the chicken α-tubulin sequence, previously noted by Valenzuela et al. (1981), is not conserved within the rat sequence. This decrease in guanine-cytosine bias is accompanied by a selective loss of CpG dinucleotides in the rat sequence.     

Physical constrints on the evolution of ctenophore size and shape

Summary Cilia bundled into combs or ctenes are an evolutionary innovation that allow comb jellies (animals in the phylum Ctenophora) to swim faster and grow to sizes at least two orders of magnitude larger than animals that propel themselves by beating single cilia. Ctenophore size, shape and swimming behaviors, however, may be constrained by the mechanisms that coordinate comb plate oscillations.Oscillations of comb plates onPleurobrachia bachei (a cydippid comb jelly), are coupled by fluid interactions between combs. Ctenes beat metachronously (in sequence) and the flows generated byP. bachei are retarded by the amount of time it takes a wave to pass down a group of ctenes. Our model predicts thatP. bachei size is constrained by the maximum thrust that can be produced by ctenes that beat in sequence and our flow visualization studies suggest that swimming via metachronous comb oscillations may constrainP. bachei to spherical shapes.In contrast, comb plate oscillations onMnemiopsis leidyi, a lobate comb jelly, are neurally coordinated and groups of ctenes beat in synchrony. As a result, fluid flows generated byM. leidyi are not retarded by the passage of metachronal waves down each comb row.M. leidyi reach sizes 15 times larger, but swim relatively slower (body lengths per second) thanP. bachei.We propose that propulsion via metachronous or synchronous comb plate oscillations has played an important role in the evolution of ctenophore shape and size and may have divided comb jellies into two evolutionary lineages.     

SimRAD: an R package for simulation‐based prediction of the number of loci expected in RADseq and similar genotyping by sequencing approaches

Application of high‐throughput sequencing platforms in the field of ecology and evolutionary biology is developing quickly with the introduction of efficient methods to reduce genome complexity. Numerous approaches for genome complexity reduction have been developed using different combinations of restriction enzymes, library construction strategies and fragment size selection. As a result, the choice of which techniques to use may become cumbersome, because it is difficult to anticipate the number of loci resulting from each method. We developed SimRAD, an R package that performs in silico restriction enzyme digests and fragment size selection as implemented in most restriction site associated DNA polymorphism and genotyping by sequencing methods. In silico digestion is performed on a reference genome or on a randomly generated DNA sequence when no reference genome sequence is available. SimRAD accurately predicts the number of loci under alternative protocols when a reference genome sequence is available for the targeted species (or a close relative) but may be unreliable when no reference genome is available. SimRAD is also useful for fine‐tuning a given protocol to adjust the number of targeted loci. Here, we outline the functionality of SimRAD and provide an illustrative example of the use of the package (available on the CRAN at http://cran.r-project.org/web/packages/SimRAD ).     

Sex allocation in Telenomus busseolae, a solitary parasitoid of concealed eggs: the influence of host patch size

Telenomus busseolae Gahan (Hymenoptera: Scelionidae) is an important egg parasitoid of noctuid stem borers of gramineous crops, attacking egg masses of Sesamia spp. Under natural conditions, and whatever the host species attacked, these egg masses are generally concealed under the leaf sheaths or other narrow spaces, and vary greatly in size. In the work presented here, the influence of host patch size (4, 8, 16, 32, 64, or 128 eggs per mass) on the sex ratio and sex sequence pattern of ovipositing T. busseolae was investigated in the laboratory using Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae) as host. The results are similar to those described for other parasitoids of aggregated hosts, and are in accordance with the Local Mate Competition model. With increasing egg mass size, the overall sex ratio (proportion of males) decreased, although additional males were laid at the end of the sequence in the larger masses (64 and 128 eggs). Sex sequence pattern always followed a males‐first strategy, i.e., with a higher proportion of males at the beginning, but the whole sex ratio sequence was influenced by the size of the egg mass. Such results in a parasitoid of concealed eggs are compared to those observed in parasitoids of exposed eggs and discussed in terms of parasitoid reproductive strategies and evolutionary adaptations.     

Mo-MuLV nucleotide sequence exhibits three levels of oligomeric repetitions,suggesting a stepwise molecular evolution

Summary An exhaustive computer-assisted analysis of the Moloney murine leukemia virus nucleotide sequence shows numerous deviations in the oligomeric distribution, suggesting three overlapping levels of a stepwise duplicative evolution. (1) The sequence fits the universal rule of TG/CT excess which has been proposed as the construction principle of all sequences, and maintains some degree of symmetry between the two complementary strands. (2) Oligomeric repeating units share a core consensus regularly scattered throughout the sequence. This consensus is not merely predictable from the doublet frequencies and codon usage, but could correspond to an intermediary stage in a so-called periodic-to-chaotic transition. (3) Probable stepwise local duplications could be accounted for by slippagelike mechanisms. Comparison with the human spumaretrovirus (HSRV) shows similar segments in the overrepresented oligomers of the two sequences. The intermediary stage of transition oligomeric repeating units is not so clearly suggested in HSRV, perhaps because of numerous stepwise local duplications. In any case, a common evolutionary origin for the two viruses is not ruled out.     

High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley

An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Using Markov model to improve word normalization algorithm for biological sequence comparison

There are two crucial problems with statistical measures for sequence comparison: overlapping structures and background information of words in biological sequences. Word normalization in improved composition vector method took into account these problems and achieved better performance in evolutionary analysis. The word normalization is desirable, but not sufficient, because it assumes that the four bases A, C, T, and G occur randomly with equal chance. This paper proposed an improved word normalization which uses Markov model to estimate exact k-word distribution according to observed biological sequence and thus has the ability to adjust the background information of the k-word frequencies in biological sequences. The improved word normalization was tested with three experiments and compared with the existing word normalization. The experiment results confirm that the improved word normalization using Markov model to estimate the exact k-word distribution in biological sequences is more efficient.     

WHY DO PARASITIC CUCKOOS HAVE SMALL BRAINS? INSIGHTS FROM EVOLUTIONARY SEQUENCE ANALYSES

Brain size is under many opposing selection pressures. Estimating their relative influence and reconstructing the brain's evolutionary history have, however, proved difficult. Here, we confirm the suggestion that the brain of brood parasitic cuckoos is smaller in relation to their body weight than that of nonparasitic cuckoo species. Two hypotheses explaining reductions in brain size are tested, using phylogenetically controlled correlations and evolutionary pathway analyses. In a novel approach, the pathway models are combined to build the most likely evolutionary sequence of trait changes correlating with changes in brain size. Brain size changed before brood parasitism, followed by a shift toward less-productive habitats and an increase in migration. This sequence shows that brain size was not reduced as a consequence of a loss of cognitive skills related to chick provisioning, and it offers no support for the hypothesis that an increase in energetic demands or a reduction in energy availability selected for a reduction of brain size. Instead, the sequence suggests that the reduction in energetic demands due to the smaller brain size and parasitic breeding strategy may have enabled parasitic cuckoos to colonize new niches.     

Phylogenetic and ecological structure of Mediterranean caddisfly communities at various spatio‐temporal scales

Aim The evolutionary processes structuring the composition of communities remain unclear due to the complexity of factors active at various spatial and temporal scales. Here, we conducted ecological and evolutionary analyses of communities of caddisflies in the genus Hydropsyche (Insecta: Trichoptera) composed of ecomorphologically differentiated species. Location River ecosystems in the Iberian Peninsula and northern Morocco. Methods Nineteen environmental variables were assessed at 180 local study sites and species presence/absence at these sites was used to determine their ecological niche. The evolutionary framework for all 19 species of Hydropsyche encountered was generated by phylogenetic analysis of the mitochondrial cytochrome c oxidase subunit I gene and three nuclear genes: wingless, elongation factor 1‐alpha and 28S RNA. The phylogenetic tree was used: (1) to assess evolutionary niche conservatism by ecological trait correlation with the tree; and (2) to analyse the phylogenetic relatedness of community member species, at three spatial scales (local stream reaches, drainage basins, biogeographical regions). Results Ecological measurements grouped most species into either headwater, mid‐stream or lowland specialists, and traits presumably relevant to river zonation were found to be phylogenetically conservative. Species assemblages at local stream reaches were mostly mono‐ or dispecific. Species diversity increased at larger spatial scales, by adding species with non‐overlapping ecological niches at the level of river basins and by turnover of anciently differentiated lineages at the level of biogeographical regions. This indicates the effects of competition and niche filtering on community structure locally, and ancient ecological diversification and allopatric speciation, respectively, in building up the species pool at basin and biogeographical scales. Main conclusions The study demonstrates the importance of scale (grain size) in studying what determines community composition. Current ecological factors (i.e. competitive exclusion) in Hydropsyche were evident only when studying narrow local sites, while studies of assemblages at larger spatial scales instead demonstrated the roles of ecological niche differentiation, phylogenetic history of trait diversification and allopatric speciation. Increasing the grain size of investigation reveals different portions of correlated spatial and evolutionary processes.     

Sperm size evolution in African greenbuls (Passeriformes: Pycnonotidae)

Sperm morphology is highly diversified across the animal kingdom and recent comparative evidence from passerine birds suggests that postcopulatory sexual selection is a significant driver of sperm evolution. In the present study, we describe sperm size variation among 20 species of African greenbuls and one bulbul (Passeriformes: Pycnonotidae) and analyze the evolutionary differentiation of sperm size within a phylogenetic framework. We found significant interspecific variation in sperm size; with some genera exhibiting relatively long sperm (e.g. Eurillas) and others exhibiting short sperm head lengths (e.g. Phyllastrephus). However, our results suggest that contemporary levels of sperm competition are unlikely to explain sperm diversification within this clade: the coefficients of inter‐male variation (CV_bm) in sperm length were generally high, suggesting relatively low and homogeneous rates of extra‐pair paternity. Finally, in a comparison of six evolutionary or tree transformation models, we found support for both the Kappa (evolutionary change primarily at nodes) and Lambda (lineage‐specific evolutionary rates along branches) models in the evolutionary trajectories of sperm size among species. We therefore conclude that African greenbuls have more variable rates of sperm size evolution than expected from a neutral model of genetic drift. Understanding the evolutionary dynamics of sperm diversification remains a future challenge.     

DNA shape, genetic codes, and evolution

Although the three-letter genetic code that maps nucleotide sequence to protein sequence is well known, there must exist other codes that are embedded in the human genome. Recent work points to sequence-dependent variation in DNA shape as one mechanism by which regulatory and other information could be encoded in DNA. Recent advances include the discovery of shape-dependent recognition of DNA that depends on minor groove width and electrostatics, the existence of overlapping codes in protein-coding regions of the genome, and evolutionary selection for compensatory changes in nucleotide composition that facilitate nucleosome occupancy. It is becoming clear that DNA shape is important to biological function, and therefore will be subject to evolutionary constraint.     

Macroevolutionary pattern of sexual size dimorphism in geckos corresponds to intraspecific temperature‐induced variation

Many animal lineages exhibit allometry in sexual size dimorphism (SSD), known as ‘Rensch’s rule’. When applied to the interspecific level, this rule states that males are more evolutionary plastic in body size than females and that male‐biased SSD increases with body size. One of the explanations for the occurrence of Rensch’s rule is the differential‐plasticity hypothesis assuming that higher evolutionary plasticity in males is a consequence of larger sensitivity of male growth to environmental cues. We have confirmed the pattern consistent with Rensch’s rule among species of the gecko genus Paroedura and followed the ontogeny of SSD at three constant temperatures in a male‐larger species (Paroedura picta). In this species, males exhibited larger temperature‐induced phenotypic plasticity in final body size than females, and body size and SSD correlated across temperatures. This result supports the differential‐plasticity hypothesis and points to the role phenotypic plasticity plays in generating of evolutionary novelties.     

Purifying and directional selection in overlapping prokaryotic genes

In overlapping genes, the same DNA sequence codes for two proteins using different reading frames. Analysis of overlapping genes can help in understanding the mode of evolution of a coding region from noncoding DNA. We identified 71 pairs of convergent genes, with overlapping 3' ends longer than 15 nucleotides, that are conserved in at least two prokaryotic genomes. Among the overlap regions, we observed a statistically significant bias towards the 123:132 phase (i.e. the second codon base in one gene facing the degenerate third position in the second gene). This phase ensures the least mutual constraint on nonconservative amino acid replacements in both overlapping coding sequences. The excess of this phase is compatible with directional (positive) selection acting on the overlapping coding regions. This could be a general evolutionary mode for genes emerging from noncoding sequences, in which the protein sequence has not been subject to selection.