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The anoxygenic phototrophic bacterium Heliobacterium modesticaldum contains a photochemical reaction center protein complex (called the HbRC) consisting of a homodimer of the PshA polypeptide and two copies of a newly discovered polypeptide called PshX, which is a single transmembrane helix that binds two bacteriochlorophyll g molecules. To assess the function of PshX, we produced a ∆pshX strain of Hbt. modesticaldum by leveraging the endogenous Hbt. modesticaldum Type I-A CRISPR-Cas system to aid in mutant selection. We optimized this system by separating the homologous recombination and CRISPR-based selection steps into two plasmid transformations, allowing for markerless gene replacement. Fluorescence and low-temperature absorbance of the purified HbRC from the wild-type and ∆pshX strains showed that the bacteriochlorophylls bound by PshX have the lowest site energies in the entire HbRC. This indicates that PshX acts as a low-energy antenna subunit, participating in entropy-assisted uphill energy transfer toward the P800 special bacteriochlorophyll g pair. We further discuss the role that PshX may play in stability of the HbRC, its conservation in other heliobacterial species, and the evolutionary pressure to produce and maintain single-TMH subunits in similar locations in other reaction centers.

Of Chargaff's four "rules" on DNA base frequencies, the functional interpretation of his second parity rule (PR2) is the most contentious. Thermophile base compositions (GC%) were taken by Galtier and Lobry (1997) as favoring Sueoka's neutral PR2 hypothesis over Forsdyke's selective PR2 hypothesis, namely that mutations improving local within-species recombination efficiency had generated a genome-wide potential for the strands of duplex DNA to separate and initiate recombination through the "kissing" of the tips of stem-loops. However, following Chargaff's GC rule, base composition mainly reflects a species-specific, genome-wide, evolutionary pressure. GC% could not have consistently followed the dictates of temperature, since it plays fundamental roles in both sustaining species integrity and, through primarily neutral genome-wide mutation, fostering speciation. Evidence for a local within-species recombination-initiating role of base order was obtained with a novel technology that masked the contribution of base composition to nucleic acid folding energy. Forsdyke's results were consistent with his PR2 hypothesis, appeared to resolve some root problems in biology and provided a theoretical underpinning for alignment-free taxonomic analyses using relative oligonucleotide frequencies (k-mer analysis). Moreover, consistent with Chargaff's cluster rule, discovery of the thermoadaptive role of the "purine-loading" of open reading frames made less tenable the Galtier-Lobry anti-selectionist arguments. Supplementary Information The online version contains supplementary material available at 10.1007/s10709-021-00119-5.

Differences in elemental stoichiometry among consumers can regulate fundamental ecological processes such as animal production or nutrient cycling within ecosystems. Research to date has established that consumer stoichiometry differs within and across taxa, but the degree to which organismal stoichiometry ranges ontogenetically across life stages or body sizes is poorly understood. To understand patterns of variation in organismal C:N:P stoichiometry, we analyzed the elemental carbon (C), nitrogen (N) and phosphorus (P) composition of 436 aquatic macroinvertebrates across the ontogeny of 13 taxa representing different life history strategies and developmental modes in a high mountain stream. Our results showed that C:N:P stoichiometry varied among taxonomic groups, macroinvertebrate taxa, but not among functional feeding groups. However, stoichiometric variability within taxa was comparable or exceeded those among taxa and was primarily caused by changes in P content. We found undocumented positive relationships between P content and body mass in hemimetabolous taxa, with %P increments ranging from five to eightfold across ontogeny. Overall, findings lend evidence to the idea that phylogenetic relationships may be an important but secondary determinant of C:N:P stoichiometry relative to ontogeny. Elucidating the mechanistic basis for the positive relationship between body mass and P content in hemimetabolous taxa may reveal fundamental differences in the ecology and evolutionary pressure behind hemi- versus holometabolous developmental modes in nature.

Lower Miocene outcrops from Patagonia (Gaiman Formation, Burdigalian) may reveal more clues for the yet unknown aspects for this period in the evolution of odontocetes. Here, we present the first toothless platanistoid dolphin from the lower Miocene of Patagonia, Dolgopolis kinchikafiforo, gen. et sp. nov. The specimen includes an incomplete skull, with no mandibles or earbones, but sufficiently different from other named odontocetes to propose a new genus and species. Phylogenetic analyses indicated it is a platanistoid of uncertain position within the group, and that it shares some homoplastic characters with physeteroids and ziphioids. Given the absence of defined alveoli and teeth and an inferred moderately short and wide rostrum, we interpreted this new species as most likely a capture suction feeder. Based on our phylogenetic hypothesis, the optimization of feeding strategies recovered raptorial feeding as the plesiomorphic method, and convergent evolution of capture suction feeders in at least four lineages. Platanistoids recorded all feeding strategies during the late Oligocene-early Miocene, although raptorial is the predominant method. This suggests a partitioning of the ecological niches in the early phases of platanistoid evolution, as well as a high diversification of feeding methods previously underestimated for this period. Thus, ecological adaptations have a strong evolutionary pressure in odontocetes communities and should be further explored Fil: Viglino, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico de Geología y Paleontología; Argentina Fil: Gaetán, Carlos Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico de Geología y Paleontología; Argentina Fil: Cuitiño, José Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico de Geología y Paleontología; Argentina Fil: Buono, Mónica Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico de Geología y Paleontología; Argentina

Bacteria are under immense evolutionary pressure from their viral invaders-bacteriophages. Bacteria have evolved numerous immune mechanisms, both innate and adaptive, to cope with this pressure. The discovery and exploitation of CRISPR-Cas systems have stimulated a resurgence in the identification and characterization of anti-phage mechanisms. Bacteriophages use an extensive battery of counter-defence strategies to co-exist in the presence of these diverse phage defence mechanisms. Understanding the dynamics of the interactions between these microorganisms has implications for phage-based therapies, microbial ecology and evolution, and the development of new biotechnological tools. Here we review the spectrum of anti-phage systems and highlight their evasion by bacteriophages.

The strong biological rationale to pursue challenging drug targets such as protein-protein interactions has stimulated the development of novel screening strategies, such as DNA-encoded libraries, to allow broader areas of chemical space to be searched. There has also been renewed interest in screening natural products, which are the result of evolutionary selection for a function, such as interference with a key signalling pathway of a competing organism. However, recent advances in several areas, such as understanding of the biosynthetic pathways for natural products, synthetic biology and the development of biosensors to detect target molecules, are now providing new opportunities to directly harness evolutionary pressure to identify and optimize compounds with desired bioactivities. Here, we describe innovations in the key components of such strategies and highlight pioneering examples that indicate the potential of the directed-evolution concept. We also discuss the scientific gaps and challenges that remain to be addressed to realize this potential more broadly in drug discovery.

Many high-throughput experimental technologies have been developed to assess the effects of large numbers of mutations (variation) on phenotypes. However, designing functional assays for these methods is challenging, and systematic testing of all combinations is impossible, so robust methods to predict the effects of genetic variation are needed. Most prediction methods exploit evolutionary sequence conservation but do not consider the interdependencies of residues or bases. We present EVmutation, an unsupervised statistical method for predicting the effects of mutations that explicitly captures residue dependencies between positions. We validate EVmutation by comparing its predictions with outcomes of high-throughput mutagenesis experiments and measurements of human disease mutations and show that it outperforms methods that do not account for epistasis. EVmutation can be used to assess the quantitative effects of mutations in genes of any organism. We provide pre-computed predictions for ∼7,000 human proteins at http://evmutation.org/.

Evolutionarily conserved virulence factors can be candidate therapeutic targets or vaccine antigens. Here, we investigated the evolutionary selective pressures on 16 pneumococcal choline-binding cell-surface proteins since Streptococcus pneumoniae is one of the pathogens posing the greatest threats to human health. Phylogenetic and molecular analyses revealed that cbpJ had the highest codon rates to total numbers of codons under considerable negative selection among those examined. Our in vitro and in vivo assays indicated that CbpJ functions as a virulence factor in pneumococcal pneumonia by contributing to evasion of neutrophil killing. Deficiency of cbpL under relaxed selective pressure also caused a similar tendency but showed no significant difference in mouse intranasal infection. Thus, molecular evolutionary analysis is a powerful tool that reveals the importance of virulence factors in real-world infection and transmission, since calculations are performed based on bacterial genome diversity following transmission of infection in an uncontrolled population., Yamaguchi et al. investigated evolutionary pressure of 16 pneumococcal choline-binding cell-surface proteins (Cbp) and identified CbpJ as a virulence factor that contributes to pneumococcal evasion of neutrophil killing. This study highlights the utility of molecular evolutionary analysis in discovering virulence factors.

The histone-like DNA-binding proteins (HU) serve as model molecules for protein thermostability studies, as they function in different bacteria that grow in a wide range of temperatures and show sequence diversity under a common fold. In this work, we report the cloning of the hutth gene from Thermus thermophilus, the purification and crystallization of the recombinant HUTth protein, as well as its X-ray structure determination at 1.7 Å. Detailed structural and thermodynamic analyses were performed towards the understanding of the thermostability mechanism. The interaction of HUTth protein with plasmid DNA in solution has been determined for the first time with MST. Sequence conservation of an exclusively thermophilic order like Thermales, when compared to a predominantly mesophilic order (Deinococcales), should be subject, to some extent, to thermostability-related evolutionary pressure. This hypothesis was used to guide our bioinformatics and evolutionary studies. We discuss the impact of thermostability adaptation on the structure of HU proteins, based on the detailed evolutionary analysis of the Deinococcus-Thermus phylum, where HUTth belongs. Furthermore, we propose a novel method of engineering thermostable proteins, by combining consensus-based design with ancestral sequence reconstruction. Finally, through the structure of HUTth, we are able to examine the validity of these predictions. Our approach represents a significant advancement, as it explores for the first time the potential of ancestral sequence reconstruction in the divergence between a thermophilic and a mainly mesophilic taxon, combined with consensus-based engineering.

Background Toll-like receptors (TLRs) are the frontline actors in the innate immune response to various pathogens and are expected to be targets of natural selection in species adapted to habitats with contrasting pathogen burdens. The recent publication of genome sequences of giraffe and okapi together afforded the opportunity to examine the evolution of selected TLRs in broad range of terrestrial ungulates and cetaceans during their complex habitat diversification. Through direct sequence comparisons and standard evolutionary approaches, the extent of nucleotide and protein sequence diversity in seven Toll-like receptors (TLR2, TLR3, TLR4, TLR5, TLR7, TLR9 and TLR10) between giraffe and closely related species was determined. In addition, comparison of the patterning of key TLR motifs and domains between giraffe and related species was performed. The quantification of selection pressure and divergence on TLRs among terrestrial ungulates and cetaceans was also performed. Results Sequence analysis shows that giraffe has 94–99% nucleotide identity with okapi and cattle for all TLRs analyzed. Variations in the number of Leucine-rich repeats were observed in some of TLRs between giraffe, okapi and cattle. Patterning of key TLR domains did not reveal any significant differences in the domain architecture among giraffe, okapi and cattle. Molecular evolutionary analysis for selection pressure identifies positive selection on key sites for all TLRs examined suggesting that pervasive evolutionary pressure has taken place during the evolution of terrestrial ungulates and cetaceans. Analysis of positively selected sites showed some site to be part of Leucine-rich motifs suggesting functional relevance in species-specific recognition of pathogen associated molecular patterns. Notably, clade analysis reveals significant selection divergence between terrestrial ungulates and cetaceans in viral sensing TLR3. Mapping of giraffe TLR3 key substitutions to the structure of the receptor indicates that at least one of giraffe altered sites coincides with TLR3 residue known to play a critical role in receptor signaling activity. Conclusion There is overall structural conservation in TLRs among giraffe, okapi and cattle indicating that the mechanism for innate immune response utilizing TLR pathways may not have changed very much during the evolution of these species. However, a broader phylogenetic analysis revealed signatures of adaptive evolution among terrestrial ungulates and cetaceans, including the observed selection divergence in TLR3. This suggests that long term ecological dynamics has led to species-specific innovation and functional variation in the mechanisms mediating innate immunity in terrestrial ungulates and cetaceans. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0901-7) contains supplementary material, which is available to authorized users.

Evolutionary pressure for protein function leads to unavoidable sampling of conformational states that are at risk of misfolding and aggregation. The resulting tension between functional requirements and the risk of misfolding and/or aggregation in the evolution of proteins is becoming more and more apparent. One outcome of this tension is sensitivity to mutation, in which only subtle changes in sequence that may be functionally advantageous can tip the delicate balance toward protein aggregation. Similarly, increasing the concentration of aggregation-prone species by reducing the ability to control protein levels or compromising protein folding capacity engenders increased risk of aggregation and disease. In this Perspective, we describe examples that epitomize the tension between protein functional energy landscapes and aggregation risk. Each case illustrates how the energy landscapes for the at-risk proteins are sculpted to enable them to perform their functions and how the risks of aggregation are minimized under cellular conditions using a variety of compensatory mechanisms.

Hoffman, Singh, and Prakash (2014) observe that perception evolves to serve as an interface between the perceiver and the world and proceed to reason that percepts need not, or even cannot, resemble their objects. I accept their premise, but argue that there are interesting ways in which perception can be truthful, with regard not to “objects” but to relations, and that evolutionary pressure is expected to favor rather than rule out such veridicality.

Eusociality is the form of animal social organization with a reproductive division of labor, most prominently known from ants and bees. Here I ask the question why this enormously successful form of social organization is missing in the largest and most diverse group of vertebrates, the teleost fishes. I first briefly review the phylogenetic distribution and likely evolutionary origins of eusociality. Then, after an equally very brief review of the diverse life history strategies of teleosts, I conclude that it is not the lack of evolutionary pre-adaptations which is keeping teleosts from becoming eusocial. Rather, I argue, that the absence of eusocial fish is caused by a number of differences between aquatic (chiefly marine) and terrestrial ecosystems: (1) Greater offspring dispersal in aquatic ecosystems reduces the role of kin-selection. (2) Lesser predictability of the environment at larger timescales in marine ecosystems disfavors eusociality. (3) A briefer impact of resource pulses in aquatic ecosystems will cause less evolutionary pressure towards cooperation, and eventually eusociality. Finally, I conclude by predicting that the most likely places to find eusocial fishes will be the deep-water regions of the ocean and the African rift lakes.

It is becoming clear that, in addition to gene gain, the loss of genes may be an important evolutionary mechanism for many organisms. However, gene loss is often associated with an increased mutation rate, thus quickly erasing evidence from the genome. The analysis of evolutionarily related sequences can provide empirical evidence for gene loss events. This paper analyzes the sequences of over 300 genetically distinct papillomaviruses and provides evidence for a role of gene loss during the evolution of certain papillomavirus genomes. Phylogenetic analysis suggests that the viral E6 gene was lost at least twice. Despite belonging to distant papillomaviral genera, these viruses lacking a canonical E6 protein may potentially encode a highly hydrophobic protein from an overlapping open reading frame, which we designate E10. Evolutionary pressure working on this alternative frame, may explain why, despite having lost the E6 open reading frame between 20 and 60 million years ago, evidence of an E6-like protein is conserved.

Constant evolutionary pressure acting on pathogens refines their molecular strategies to attain successful pathogenesis. Recent studies have shown that pathogenicity mechanisms of necrotrophic fungi are far more intricate than earlier evaluated. However, only a few studies have explored necrotrophic fungal pathogens. Ascochyta rabiei is a necrotrophic fungus that causes devastating blight disease of chickpea (Cicer arietinum). Here, we report a 34.6 megabase draft genome assembly of A. rabiei. The genome assembly covered more than 99% of the gene space and 4,259 simple sequence repeats were identified in the assembly. A total of 10,596 high confidence protein-coding genes were predicted which includes a large and diverse inventory of secretory proteins, transporters and primary and secondary metabolism enzymes reflecting the necrotrophic lifestyle of A. rabiei. A wide range of genes encoding carbohydrate-active enzymes capable for degradation of complex polysaccharides were also identified. Comprehensive analysis predicted a set of 758 secretory proteins including both classical and non-classical secreted proteins. Several of these predicted secretory proteins showed high cysteine content and numerous tandem repeats. Together, our analyses would broadly expand our knowledge and offer insights into the pathogenesis and necrotrophic lifestyle of fungal phytopathogens.

Hippophae rhamnoides ssp. sinensis is of ecological and practical importance, PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the rDNA ITS region was conducted to estimate the genetic diversity of H. rhamnoides ssp. sinensis at 19 sites in Northern China. Restriction analysis divided the samples into seven RFLP patterns (I–VII), which implied that H. rhamnoides ssp. sinensis had a degree of genetic diversity. The genetic diversity of H. rhamnoides ssp. sinensis mainly lied within populations other than among populations, and the results also suggested that geographic distances did not have any clear effect on their genetic differentiation. The sequences of rDNA ITS region varied in length from 656 to 658 bp, which exhibited 95–100% sequence identity. Pairwise Kimura 2-parameter distances within these H. rhamnoides ssp. sinensis samples ranged from 0.27% to 5.95%. There were 48 variable sites within the entire ITS region, among them transitions were more frequent than transversions. The ITS1 and ITS2 transcripts of H. rhamnoides ssp. sinensis had conserved secondary structures, implying that the ITS region maybe under evolutionary pressure to maintain the RNA secondary structure involved in the post-transcriptional processing of rRNA.

Conjugative plasmids of Gram-negative bacteria have both vertical and horizontal modes of transmission: they are segregated to daughter cells during division, and transferred between hosts by plasmid-encoded conjugative machinery. Despite maintaining horizontal mobility, many plasmids carry fertility inhibition (fin) systems that repress their own conjugative transfer. To assess the ecological basis of self-transfer repression, we compared the invasion of bacterial populations by fin(+) and fin(-) variants of the plasmid R1 using a computational model and co-culture competitions. We observed that the fin(+) variant had a modest cost to the host (measured by reduction in growth rate), while the fin(-) variant incurred a larger cost. In simulations and empirical competitions the fin(-) plasmid invaded cultures quickly, but was subsequently displaced by the fin(+) plasmid. This indicated a competitive advantage to reducing horizontal transmission and allowing increased host replication. Computational simulations predicted that the advantage associated with reduced cost to the host would be maintained over a wide range of environmental conditions and plasmid costs. We infer that vertical transmission in concert with competitive exclusion favour decreased horizontal mobility of plasmids. Similar dynamics may exert evolutionary pressure on parasites, such as temperate bacteriophages and vertically transmitted animal viruses, to limit their rates of horizontal transfer.

Haptoglobin (Hp) is a hemoglobin-binding plasma protein consisting of two types of chains, called alpha and beta, which originate from a common polypeptide. In humans, but not in other mammals, Hp has been shown to occur in two allelic forms, Hp1 and Hp2, which differ in the length of the alpha-chain. The longer alpha-chain (in Hp2) seems to have arisen by an internal duplication of a gene segment coding for almost the entire alpha-chain of Hp1. In this article we show that Hp of cow (Bos taurus) contains an alpha-chain, the structure of which is similar to that of the human Hp2 alpha-chain. Furthermore, comparison of the structure of bovine Hp and human Hp2 suggests that the bovine gene arose by a duplication of the gene segment homologous to that duplicated in human Hp2. However, a phylogenetic analysis indicates that the two genes were formed independently. The evolutionary pressure that has led to the fixation of the Hps with a longer alpha-chain is not known.

Conserved synteny--the sharing of at least one orthologous gene by a pair of chromosomes from two species--can, in the strictest sense, be viewed as sequence conservation between chromosomes of two related species, irrespective of whether coding or non-coding sequence is examined. The recent sequencing of multiple vertebrate genomes indicates that certain chromosomal segments of considerable size are conserved in gene order as well as underlying non-coding sequence across all vertebrates. Some of these segments lost genes or non-coding sequence and/or underwent breakage only in teleost genomes, presumably because evolutionary pressure acting on these regions to remain intact were relaxed after an additional round of whole genome duplication. Random reporter insertions into zebrafish chromosomes combined with computational genome-wide analysis indicate that large chromosomal areas of multiple genes contain long-range regulatory elements, which act on their target genes from several gene distances away. In addition, computational breakpoint analyses suggest that recurrent evolutionary breaks are found in "fragile regions" or "hotspots", outside of the conserved blocks of synteny. These findings cannot be accommodated by the random breakage model and suggest that this view of genome and chromosomal evolution requires substantial reassessment.

p53 is a fundamental determinant of oncogenesis, aging physiology and neurodegenerative pathologies. The complexity of the p53 regulatory network can hinder attempts to fully understand how this oncogenic protein operates within/ between cells to constrain growth potential. Orthologs of p53 in non-mammalian models, such as zebrafish, Drosophila and Caenorhabditis elegans, afford simpler models that illuminate core properties of this ancient network. The existence of p53 in short-lived organisms, where cancers do not occur, argues that tumor suppression per se was not the evolutionary pressure shaping p53. Instead, p53 as a constraint against tumor growth was probably co-opted from more ancestral, nonautonomous functions that are entirely unknown. Describing these functional properties will be essential for a comprehensive picture of the p53 regulatory network in normal and disease states. The tractable systems described here offer sophisticated genetic tools and fundamental insights that will continue to be indispensable models for achieving this goal.

Functionally linked genes generally evolve at similar rates and the knowledge of this particular feature of genomic evolution has been used as the basis for the phylogenetic profiling method. We illustrate here an exception to this rule in the evolution of the cytochrome bd complex. This is a two-component oxidase complex, with the subunits I and II known to be widely present in bacteria. The subunits within the cytochrome bd complex are under the same evolutionary pressure and most likely behave in the same evolutionary manner. However, the sequence similarity of genes encoding subunit II varies considerably across species. Genes encoding subunit II evolve 1.2 times faster on most of the branches of their phylogeny than subunit I genes. Furthermore, the genes encoding subunit II in Oceanobacillus iheyensis, Bacillus halodurans, and Staphylococcus species do not have detectable homologues within E. coli due to their large divergence. Together, the two subunits of cytochrome bd reveal an interesting example of an asymmetric pattern of evolutionary change.

Genes on the mammalian X chromosome may be under unique evolutionary pressure due to their hemizygous expression in males. Since any recessive deleterious mutation would immediately be expressed in males and, therefore, efficiently removed from the population, selective constraint could be more pronounced in X-linked genes. Conversely, if a recessive mutation were beneficial, its immediate exposure to selection would be advantageous, and would facilitate adaptive evolution. We tested for positive selection in a total of 86 genes using a maximum likelihood approach, including 40 sperm-expressed and 46 non-sperm, tissue-specific genes. We find evidence to suggest that X-linkage enhances the effects of positive selection in sperm-expressed genes in terms of the number of codons affected, and report a general trend for positively selected genes to reside on the X chromosome rather than on the autosomes. Our data suggest that hemizygous expression in males makes the X chromosome a preferred location for positively selected sperm genes that do not require postmeiotic transcription.

As more and more protein structures are determined, it has become clear that there is only a limited number of protein folds in nature. To explore whether the protein folds found in nature are the only solutions to the protein folding problem, or that a lack of evolutionary pressure causes the paucity of different protein folds found, we set out to construct protein libraries without any restriction on topology. We generated different libraries (all alpha-helix, all beta-strand and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand and beta-turn) in various proportions, based primarily on the patterning of polar and non-polar residues. From the analysis of proteins chosen randomly from the libraries, we found that a substantial portion of pure alpha-helical proteins show properties similar to native proteins. Using these libraries as a starting point, we aim to establish a selection system which allows us to enrich proteins with favorable folding properties (non-aggregating, compactly folded) from the libraries. We have developed such a method based on ribosome display. This selection is based on two concepts: (1) misfolded proteins are more sensitive to proteolysis, (2) misfolded and/or aggregated proteins are more hydrophobic. We show that by applying each of these selection criteria proteins that are compactly folded and soluble can be enriched over insoluble and random coil proteins.

Fasciola spp., commonly known as liver fluke, are significant trematode parasites of livestock and humans. They secrete several cathepsin L-like cysteine proteases, some of which differ in enzymatic properties and timing of expression in the parasite's life cycle. A detailed sequence and evolutionary analysis is presented, based on 18 cathepsin L-like enzymes isolated from Fasciola spp. (including a novel clone identified in this study). The enzymes form a monophyletic group which has experienced several gene duplication events over the last approximately 135 million years, giving rise to the present-day enzymatic repertoire of the parasite. This timing of these duplications appears to correlate with important points in the evolution of the mammalian hosts. Furthermore, the dates suggest that Fasciola hepatica and Fasciola gigantica diverged around 19 million years ago. A novel analysis, based on the pattern of amino acid diversity, was used to identify sites in the enzyme that are predicted to be subject to positive adaptive evolution. Many of these sites occur within the active site cleft of the enzymes, and hence would be expected to lead to differences in substrate specificity. Using homology modeling, with reference to previously obtained biochemical data, we are able to predict S2 subsite specificity for these enzymes: specifically those that can accommodate bulky hydrophobic residues in the P2 position and those that cannot. A number of other positions subject to evolutionary pressure and potentially significant for enzyme function are also identified, including sites anticipated to diminish cystatin binding affinity.

The common assumption of operons as composed of genes that cooperate in a biological process is confirmed here by showing that Escherichia coli operons tend to be composed of genes that belong to the same general class of cellular function. Furthermore, the comparison between the genomic organization of E. coli and that of Bacillus subtilis shows that the genes that are homologous to genes that belong to experimentally characterized E. coli operons tend to cluster in neighboring regions of the genome. This tendency is greater for the subset of E. coli operons whose genes belong to a single functional class. These observations indicate strong evolutionary pressure that, translated into functional constraints, leads to the inclusion of many essential functions in conserved operons and clusters in these two distant species.

Malaria programs aimed at eliminating Plasmodium falciparum (Pf) incorporate active infection detection strategies to target the subclinical transmission reservoir. Currently available immunochromatographic lateral flow tests lack the sensitivity required for Pf malaria elimination; the limit of detection (LOD) of these rapid diagnostic tests (RDTs) is above that which is required to identify all transmissible infections. Malaria RDTs with a significantly improved LOD would enable more effective elimination interventions while retaining the critical advantages of low cost, ease of use, and rural deployment capability. Histidinerich protein 2 (HRP2) is a high-priority target analyte for identifying individuals at risk of transmitting Pf. HRP2 protein is secreted by Pf parasites in relatively high concentrations, is widely conserved among Pf strains, and persists in human plasma for up to four weeks. These characteristics allow HRP2 antigen detection even in the absence of circulating parasites during cyclical sequestration. However, the highly polymorphic nature of HRP2 makes optimization of RDTs challenging. Current HRP2-detecting RDT reagents primarily target Type 2 (AHHAHHAAD) and Type 7 (AHHAAD) HRP2 motifs, and independent antibody discovery confirms the most common selection of a C1-13/PTL-3 antibody conjugate pair yields the best RDT performance for the majority of parasite strains. In scenarios where malaria transmission rapidly decreases, exclusive use of a diagnostic whose sensitivity varies with the number of HRP2 epitope repeats can result in single axis evolutionary pressure, increasing selection for less-detectable parasite strains. To evaluate the feasibility of developing an HRP2-based RDT that is highly effective in mass testing and treatment programs, we have assessed the technical characteristics of HRP2 reagents. Our aim is to facilitate the development of improved malaria HRP2 RDTs by: (1) incorporating an understanding of HRP2 structure-function relationships and their impact on epitope-antibody interactions and adaptation, (2) correlating HRP2 concentration levels (in pg/mL) with traditional malaria diagnostic metrics (parasites/mL) for comparative evaluation of tests, and (3) predicting optimal capture reagents to improve and sustain HRP2 RDT performance. Based on our ongoing analysis, we present a product development strategy that combines capture reagent and platform innovations that can be used to make highly sensitive Pf malaria RDTs. In addition, we present criteria for the rational selection of HRP2 protein and parasite culture controls to support the product lifecycle from development through commercialization and scale-up.

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5' end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5' region of S. franciscanus bindin is a result of neutral evolution.

This paper is a contribution to the discussion of whether the general architecture of electron transfer sites in blue copper proteins is mainly a result of the structural preferences of the metal ion or is induced by the protein. Although the site is probably stable only when protected by the protein, there appears to be no strain from the latter on the structure in the vicinity of the copper atom. For an operative redox site it is further required that the geometry of the site is acceptable for both oxidation states, to avoid high reorganization energy. The site must also be connected to the outer world by suitable tunneling pathways. The blue copper sites appear to fulfill these requirements, but it is difficult to assess the role of evolutionary pressure to form electron transfer proteins in general.

The paper contributes to the literature on learning dynamics with heterogeneous agents, demonstrating that heterogeneity as such may be conducive to stability. The point is made in the framework of the cobweb model, where two different forecast procedures are considered. Either one destabilizes the price dynamics when it is uniformly adopted by all firms, or the price equilibrium becomes locally stable if firms are heterogenous and the two rules are suitably mixed within the population. It is also indicated that such a stabilizing composition is endogenously brought about by an adjustment process in which the population shares evolve under evolutionary pressure.

The paper is concerned with genetic algorithm learning in a cobweb economy. Besides discussing several specification details in the genetic operators, the model includes four different types of firm forecasting rules and subjects the demand side to serially correlated random shocks. The main finding of the simulation experiments is that the genetic algorithm is a reasonably good approximation of the moving Walrasian equilibria, and that this process is characterized by the coevolution of different strategies. Accordingly, it is just the persistent heterogeneity of firms, and the persistently changing composition of this heterogeneity, that achieves stability. In this world, convergence is improved by weak, rather than strong, evolutionary pressure.

Any living or non-living surface immersed in seawaterrapidly acquires a bacterial biofilm. For living marineorganisms, biofilm formation can result in the death ofthe host, and thus there is strong evolutionary pressure formarine eukaryotes to evolve mechanisms which inhibit orcontrol the development of biofilms on their surfaces.Some marine eukaryotes are indeed successful incontrolling biofilms on their surfaces, and in manyinstances this control is achieved by the production ofinhibitory chemicals which act at or near the surface ofthe organism. In some cases these natural inhibitors aresimply toxic to bacteria. However, increasingly it appearsthat at least some of these compounds act by interferingspecifically with bacterial characteristics which effect theability of bacteria to colonize their hosts, such asattachment, surface spreading, or the production ofextracellular macromolecules. As an example, theAustralian seaweed Delisea pulchra appears tocontrol bacterial colonization by interfering with abacterial regulatory system (the acylated homoserinelactone system) that regulates several colonizationrelevant bacterial traits. Understanding how marineorganisms control specific bacterial colonization traitsshould provide us with insights into new technologies forthe control of biofilms on artificial surfaces.

The sequence database of HLA class I genes is mainly derived from mRNA analysis. Little is known about the non-coding sequences of the different class I alleles. In this study we have determined the sequence of the 1st through 3rd introns of the majority of HLA-A and -B alleles. The few published sequences emerged to contain substantial errors. The introns turned out to be highly polymorphic with a variability of 14.6% in the 1st intron decreasing to 6.2% in the 3rd intron. Against all expectations, this variability is not characterised by random point mutations but by a highly systematic diversity reflecting the ancestral relationship of the HLA alleles. The variability is arrested on the level of the serological diversity. The striking conservation within each ancestral lineage suggests that point mutations have been negatively selected. This finding could be explained by the evolutionary pressure on base order, promoting the potential to extrude single-strand stem-loops from supercoiled duplex DNA, which is believed to be important for combination. Moreover, the GC content was found to be as high as 78% in the 1st and 2nd introns and 55% in the 3rd intron. These CpG islands are directly involved in the exchange of short stretches of DNA in unequal crossing-over events. Additionally, conversion between different class I sequences is facilitated by regions of strong homology, stabilizing the pairing of variable regions. All these observations indicate the potential of a substantial contribution of introns to the recombinational activity of class I genes. The exclusive clustering of CpG islands in the 1st and 2nd introns restricts the gene conversion events to the regions of the 2nd and 3rd exons and therefore protects the conservation of the 5 flanking region and the 3 part of the gene. Since there are less diversification forces acting on introns they may be more conserved in a trans-species manner than exons. Therefore, they could provide the answer for the controversy regarding intra- or trans-species evolution.

Background In contrast to the majority of mammalian genes, imprinted genes are monoallelically expressed with the choice of the active allele depending on its parental origin. Due to their special inheritance patterns, maternally and paternally expressed genes might be under different evolutionary pressure. Here, we aimed at assessing the evolutionary history of imprinted genes. Results In this study, we investigated the conservation of imprinted genes in vertebrate genomes and their exposition to natural selection. In a genome-wide comparison, orthologs of imprinted genes show a stronger divergence on cDNA and protein level in mammals. This pattern is most pronounced for maternally expressed genes in rodents in comparison to their non-rodent orthologs. The divergence is not attributable to increased mutation of CpG positions. It is contrasted by strong conservation of paternally expressed genes in mouse and rat. Interestingly, we found that the early divergence of imprinted genes was accompanied by an unusually strict conservation of their paralogs. Conclusions The apparent degeneration of maternally expressed genes may reflect a relaxation of selective pressure due to counteracting effects on maternal and embryonic fitness. Functional redundancy provided by the presence of highly conserved (non-imprinted) paralogs may have facilitated the divergence. Moreover, intensification of imprinting in modern rodents seems to have shifted the evolutionary fate of imprinted genes towards strong purifying selection.

Several studies have suggested the existence of backup machinery of transcriptional regulatory networks (TRNs). Here, we have quantified the backup machinery of yeast gene's TRNs under five different conditions in terms of alternate paths and have revealed that a statistically significant (p

Background Shannon entropy applied to columns of multiple sequence alignments as a score of residue conservation has proven one of the most fruitful ideas in bioinformatics. This straightforward and intuitively appealing measure clearly shows the regions of a protein under increased evolutionary pressure, highlighting their functional importance. The inability of the column entropy to differentiate between residue types, however, limits its resolution power. Results In this work we suggest generalizing Shannon's expression to a function with similar mathematical properties, that, at the same time, includes observed propensities of residue types to mutate to each other. To do that, we revisit the original construction of BLOSUM matrices, and re-interpret them as mutation probability matrices. These probabilities are then used as background frequencies in the revised residue conservation measure. Conclusion We show that joint entropy with BLOSUM-proportional probabilities as a reference distribution enables detection of protein functional sites comparable in quality to a time-costly maximum-likelihood evolution simulation method (rate4site), and offers greater resolution than the Shannon entropy alone, in particular in the cases when the available sequences are of narrow evolutionary scope.

GATA genes are an evolutionarily conserved family, which encode a group of important transcription factors involved in the regulation of diverse processes including the development of the heart, haematopoietic system and sex gonads. However, the evolutionary history of the GATA family has not been completely understood. We constructed a complete phylogenetic tree with functional domain information of the GATA genes of both vertebrates and several invertebrates, and mapped the GATA genes onto relevant chromosomes. Conserved synteny was observed around the GATA loci on the chromosomes. GATAs have a tendency to segregate onto different chromosomes during evolution. The phylogenetic tree is consistent with the relevant functions of GATA members. Analysis of the zinc finger domain showed that the domain tends to be duplicated during evolution from invertebrates to vertebrates. We propose that the balance between duplications of zinc finger domains and GATA members should be maintained to exert their physiological roles in each evolutionary stage. Therefore, evolutionary pressure on the GATAs must exist to maintain the balance during evolution from invertebrates to vertebrates. These results reveal the evolutionary characteristics of the GATA family and contribute to a better understanding of the relationship between evolution and biological functions of the gene family, which will help to uncover the GATAs’ biological roles, evolution and their relationship with associated diseases.

Background The elastin gene (ELN) is implicated as a factor in both supravalvular aortic stenosis (SVAS) and Williams Beuren Syndrome (WBS), two diseases involving pronounced complications in mental or physical development. Although the complete spectrum of functional roles of the processed gene product remains to be established, these roles are inferred to be analogous in human and mouse. This view is supported by genomic sequence comparison, in which there are no large-scale differences in the ~1.8 Mb sequence block encompassing the common region deleted in WBS, with the exception of an overall reversed physical orientation between human and mouse. Results Conserved synteny around ELN does not translate to a high level of conservation in the gene itself. In fact, ELN orthologs in mammals show more sequence divergence than expected for a gene with a critical role in development. The pattern of divergence is non-conventional due to an unusually high ratio of gaps to substitutions. Specifically, multi-sequence alignments of eight mammalian sequences reveal numerous non-aligning regions caused by species-specific insertions and deletions, in spite of the fact that the vast majority of aligning sites appear to be conserved and undergoing purifying selection. Conclusions The pattern of lineage-specific, in-frame insertions/deletions in the coding exons of ELN orthologous genes is unusual and has led to unique features of the gene in each lineage. These differences may indicate that the gene has a slightly different functional mechanism in mammalian lineages, or that the corresponding regions are functionally inert. Identified regions that undergo purifying selection reflect a functional importance associated with evolutionary pressure to retain those features.

Some species evolve to resemble another species so as to protect themselves from predation, but this mimicry is often imprecise. An analysis of hoverflies suggests why imperfect imitation persists in the face of natural selection. See Letter p.461 Batesian mimics are potential prey species that are harmless to predators but gain protection through a resemblance to unpalatable prey species. Surprisingly, many Batesian species seem to be fairly mediocre mimics, despite presumably strong evolutionary pressure to improve the resemblance. This paper presents a morphological and phylogenetic analysis of harmless hoverfly species that mimic — with various degrees of success — stinging hymenopteran species. The authors rule out several hypotheses, such as that the imperfect mimicry is an artefact of human perception and that the imperfect mimics are actually hedging their bets by resembling several hymenopteran species at the same time. Instead, the authors find a link between imperfect mimicry and small body size, which suggests that the imperfect mimics are simply not subject to particularly intense selection.

Chemical Ecology focuses on how chemicals mediate interactions among organisms. This role of chemistry in organismal interactions generates an evolutionary pressure of pests and hosts to enter into a repetitive cycle of defense novelization and counter-response. This cycle provides the central evolutionary hypothesis about the pressure to derive novel chemicals and thus generate a massive chemical diversification among and within plant and fungal lineages. In the past decades, significant efforts from a large research community have made great progress in cloning numerous enzymes from diverse biochemical pathways leading to a general genomic thesis for this chemical diversification. This thesis can be stated as follows: genes duplicate either individually or as whole genomes. These duplicated genes can either “neo-functionalize” (evolve completely new biochemical functions) or “sub-functionalize” (evolve a fraction of the original function). It is the presence of these changed duplicated genes that provides new enzymatic capacity that can somehow be coordinated into a single pathway to produce a single compound. However, this gene evolution model and the data supporting it focus almost entirely on how the enzymatic capacity to make a new compound arises, while saying little about the regulatory processes that may have accompanied this evolution. This is despite the fact that such regulatory processes are essential to coordinate the pathway and allow it to function. It is possible that part of this lack of enquiry into regulatory evolution comes from the common observation that most secondary metabolite pathways show similar inductive regimes in response to pathogens or herbivores, generating the illusion that they may involve conserved regulators that trap the biochemical pathway promoters. However, recent work is suggesting that there is as much regulatory evolution at play in chemical diversification as there is enzyme evolution. A common observation in secondary metabolism is that biosynthetic pathways frequently are controlled by bHLH andMYB transcription factors. A recent multi-species genomic phylogenetic analysis of the bHLH and MYB families showed that like secondary metabolite enzymes, these families have undergone a massive expansion within the vascular plants (Feller et al., 2011). There were similar levels of gene numbers as for key secondary metabolite enzymes like CYP450s and 2ODDs, and there was even a similar propensity for lineagespecific families of both bHLH and MYB genes. One of these lineage-specific MYB families includes thoseMYBs that control the expression of glucosinolates, amino acid derived defense compounds present in the Capparales. A recent investigation of how the glucosinolate enzymes and their affiliated MYBs evolved in the Capparales showed that early in their evolutionary history, a full biosynthetic pathway along with a specific MYB evolved to create a functioning glucosinolate system (Bekaert et al., 2012). During Capparales evolution, there were whole genome duplication events that duplicated the entire glucosinolate pathway and the MYBs. Following one of these duplications, the genes partitioned into largely two pathways, one for making glucosinolates from Tryptophan and the other using Methionine. Central to the issue of regulatory evolution, the MYBs also split along downstream pathway lines. Thus, as suggested by the phylogenetic analysis, chemical diversification also is likely driving regulatory factor diversification within plants. Intriguingly, recent work suggests that secondary metabolite transcription factors are not limited to a role in secondary metabolism. Analysis of volatile production and regulation within Petunia identified the missing link in a regulatory cascade of MYBs. In this system, three MYBs (EOBII, EOBI, andODO1) form a hierarchical regulatory cascade leading to the production of volatiles in Petunia (Spitzer-Rimon et al., 2013). These MYBs also directly regulated primary metabolite genes within the shikimate pathway, presumably controling precursor availability for the production of benzenoids. Importantly, these genes are not broadly evolutionarily conserved and appear to have arisen specifically in this lineage for secondary volatile production. This function in regulating secondary metabolism, however, did not prevent them from accruing regulatory control over a primary metabolite pathway like the shikimate pathway. Thus, secondary metabolite regulatory factors may also evolve roles in mediating primary metabolic or physiological processes as a part of coordinating secondary metabolite production, in this case regulating precursor availability. Together, these works suggest that the chemical diversification so central to the expansion of biochemical capacity is also driving an unrecognized expansion of regulatory capacity within vascular plants. More dramatically, if lineagespecific secondary metabolite transcription factors frequently acquire the capacity to control central physiological processes that enable the optimization of chemical defenses, then the chemical diversification central to Chemical Ecology may play a fundamental role in shaping areas outside the field’s traditional boundaries, such as core plant physiology and metabolism. In either case, studying how chemical diversification and regulatory evolution have proceeded in coordination will provide unique insight into how organisms and their networks evolve.

Background Dentin sialophosphoprotein (DSPP) is the largest member of the SIBLING family and is the most abundant noncollagenous protein in dentin. DSPP is also expressed in non-mineralized tissues including metabolically active ductal epithelia and some cancers. Its function, however, is poorly defined. The carboxy-terminal fragment, dentin phosphoprotein (DPP) is encoded predominantly by a large repetitive domain that requires separate cloning/sequencing reactions and is, therefore, often incomplete in genomic databases. Comparison of DPP sequences from at least one member of each major branch in the mammalian evolutionary tree (including some "toothless" mammals) as well as one reptile and bird may help delineate its possible functions in both dentin and ductal epithelia. Results The BMP1-cleavage and translation-termination domains were sufficiently conserved to permit amplification/cloning/sequencing of most species' DPP. While the integrin-binding domain, RGD, was present in about half of species, only vestigial remnants of this tripeptide were identified in the others. The number of tandem repeats of the nominal SerSerAsp phosphorylation motif in toothed mammals (including baleen whale and platypus which lack teeth as adults), ranged from ~75 (elephant) to >230 (human). These repeats were not perfect, however, and patterns of intervening sequences highlight the rapidity of changes among even closely related species. Two toothless anteater species have evolved different sets of nonsense mutations shortly after their BMP1 motifs suggesting that while cleavage may be important for DSPP processing in other tissues, the DPP domain itself may be required only in dentin. The lizard DSPP had an intact BMP1 site, a remnant RGD motif, as well as a distinctly different Ser/Asp-rich domain compared to mammals. Conclusions The DPP domain of DSPP was found to change dramatically within mammals and was lost in two truly toothless animals. The defining aspect of DPP, the long repeating phosphorylation domain, apparently undergoes frequent slip replication and recombination events that rapidly change specific patterns but not its overall biochemical character in toothed animals. Species may have to co-evolve protein processing mechanisms, however, to handle increased lengths of DSP repeats. While the RGD domain is lost in many species, some evolutionary pressure to maintain integrin binding can be observed.

Background Different regions in a genome evolve at different rates depending on structural and functional constraints. Some genomic regions are highly conserved during metazoan evolution, while other regions may evolve rapidly, either in all species or in a lineage-specific manner. A strong or even moderate change in constraints in functional regions, for example in coding regions, can have significant evolutionary consequences. Results Here we discuss a novel framework, 'BaseDiver', to classify groups of genes in humans based on the patterns of evolutionary constraints on polymorphic positions in their coding regions. Comparing the nucleotide-level divergence among mammals with the extent of deviation from the ancestral base in the human lineage, we identify patterns of evolutionary pressure on nonsynonymous base-positions in groups of genes belonging to the same functional category. Focussing on groups of genes in functional categories, we find that transcription factors contain a significant excess of nonsynonymous base-positions that are conserved in other mammals but changed in human, while immunity related genes harbour mutations at base-positions that evolve rapidly in all mammals including humans due to strong preference for advantageous alleles. Genes involved in olfaction also evolve rapidly in all mammals, and in humans this appears to be due to weak negative selection. Conclusion While recent studies have identified genes under positive selection in humans, our approach identifies evolutionary constraints on Gene Ontology groups identifying changes in humans relative to some of the other mammals.

Despite a strong evolutionary pressure to reduce genome size, proteins vary in length over a surprisingly wide range also in very compact genomes. Here we investigated the evolutionary forces that act on protein size in the yeast Saccharomyces cerevisiae utilizing a system-wide bioinformatics approach. Data on yeast protein size was compared to global experimental data on protein expression, phenotypic pleiotropy, protein-protein interactions, protein evolutionary rate and biochemical classification. Comparing the experimentally determined abundance of individual proteins, highly expressed proteins were found to be consistently smaller than lowly expressed proteins, in accordance with the biosynthetic cost minimization hypothesis. Yeast proteins able to maintain a high expression level despite a large size tended to belong to a very distinct set of protein families, notably nuclear transport and translation initiation/elongation. Large proteins have significantly more protein-protein interactions than small proteins, suggesting that a requirement for multiple interaction domains may constitute a positive selective pressure for large protein size in yeast. The higher frequency of protein-protein interactions in large proteins was not accompanied by a higher phenotypic pleiotropy. Hence, the increase in interactions may not reflect an increase in function differentiation. Proteins of different sizes also evolved at similar rates. Finally, whereas the biological process involved was found to have little influence on protein size the biochemical activity exerted by the protein represented a dominant factor. More than one third of all biochemical activity classes were enriched in one or more size intervals. In yeast, there is an inverse relationship between protein size and protein expression such that highly expressed proteins tend to be of smaller size. Also, protein size is moderately affected by protein connectivity and strongly affected by biochemical activity. Phenotypic pleiotropy does not seem to affect protein size.

Background: Cellular functions are regulated by complex webs of interactions that might be schematically represented as networks. Two major examples are transcriptional regulatory networks, describing the interactions among transcription factors and their targets, and proteinprotein interaction networks. Some patterns, dubbed motifs, have been found to be statistically over-represented when biological networks are compared to randomized versions thereof. Their function in vitro has been analyzed both experimentally and theoretically, but their functional role in vivo, that is, within the full network, and the resulting evolutionary pressures remain largely to be examined. Results: We investigated an integrated network of the yeast Saccharomyces cerevisiae comprising transcriptional and protein-protein interaction data. A comparative analysis was performed with respect to Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii and Yarrowia lipolytica, which belong to the same class of hemiascomycetes as S. cerevisiae but span a broad evolutionary range. Phylogenetic profiles of genes within different forms of the motifs show that they are not subject to any particular evolutionary pressure to preserve the corresponding interaction patterns. The functional role in vivo of the motifs was examined for those instances where enough biological information is available. In each case, the regulatory processes for the biological function under consideration were found to hinge on post-transcriptional regulatory mechanisms, rather than on the transcriptional regulation by network motifs. Conclusion: The overabundance of the network motifs does not have any immediate functional or evolutionary counterpart. A likely reason is that motifs within the networks are not isolated, that is, they strongly aggregate and have important edge and/or node sharing with the rest of the network.

The collagens represent an interesting example of a structurally related but genetically distinct family of proteins. Type I, the most abundant of the vertebrate collagens, comprises two pro alpha 1(I) chains and one pro alpha 2(I) chain, each containing terminal propeptides and a central domain of 338 (Gly, X, Y) repeats. The structure of the chicken pro alpha 2(I) gene shows an intriguing relationship between exon organization and the arrangement of (Gly, X, Y) repeats (see ref. 2 for review). This has led to the suggestion that the collagens evolved from a common ancestral unit of 54 base pairs (bp). Here we present the structure of the entire human pro alpha 1(I) gene and compare this with the chicken pro alpha 2(I). The exon arrangement of the two genes is remarkably similar, although the human pro alpha 1(I) is more compact because of the shorter length of its introns. The data strongly support the notion that the type I genes have evolved from an ancestral multi-exon unit, and that once the gene was translated, a strong evolutionary pressure caused it to maintain this elaborate structure.

Despite the accumulated evidence, many of the investigators as of quite recently have failed to understand that behavioral strategy or decision rules can be inherited and evolve and that they are subject to certain constraints that cannot be “surmounted” other than by natural selection. For example, in [9, p. 422] we read: “The evolutionary pressure cannot act upon the relative contents of social interactions favoring certain individuals over others.” The present study shows that the following scheme of conflict, which favors those individuals that conform to it with a better accuracy, can be evolutionary stable. On the basis of the information they receive, the partners estimate the role situation and the equilibrium pair of elementary strategies and use their strategies. If the role situations of the partners are coordinated, the partners cooperate in reducing the conflict and define its result according to AIS. If AIS are not coordinated, they cooperate in reducing the conflict if its expected loss for each is greater than the expected payoff; they escalate the against those that have a lesser set of elementary strategies but also against those that have a less accurate mechanism of AIS recognition. A poor accuracy of recognition mechanism would replace a strictly equilibrium ESS with a mixed strategy. From the point of view of behavioral control, the multilevel conflict model shows that generally for controlling the choices at a certain level actions of an equally high level are necessary. It is impossible, for example, to accomplish certain economic choices by using only informational actions.

MOST sequenced RNAs have the potential to form secondary structures by means of local base pairing and loop formation. Arguments have been advanced that these structures are the result of evolutionary pressure, or of requirements for unique functions such as virus packaging1–7. To determine whether secondary structure is an intrinsic or anomalous property of RNA we have investigated random sequences generated by computer. These sequences, which contain no real biological information, can be arranged into thermo-dynamically stable secondary structures with about 50% base pairing. The results imply that one should expect a high degree of base pairing in RNA, consistent with early estimates of helical content in random copolynucleotides8 and the constraints accompanying structure formation in a hypothetical tRNA molecule9,10. Therefore, the mere existence of secondary structure does not necessarily implicate special biological functions, although the potential for functional interactions clearly exists.