Institute for Bioscience and Biotechnology Research

A tendency for exons to correspond to discrete units of protein structure in protein-coding genes of ancient origin would provide clear evidence in favor of the exon theory of genes, which proposes that split genes arose not by insertion of introns into unsplit genes, but from combinations of primordial mini-genes (exons) separated by spacers (introns). Although putative examples of such correspondence have strongly influenced previous debate on the origin of introns, a general correspondence has not been rigorously proved. Objective methods for detecting correspondences were developed and applied to four examples that have been cited previously as evidence of the exon theory of genes. No significant correspondence between exons and units of protein structure was detected, suggesting that the putative correspondence does not exist and that the exon theory of genes is untenable.

Alignments of homologous genes typically reveal a great diversity of intron locations, far more than could fit comfortably in a single gene. Thus, a minority of these intron positions could be inherited from a single ancestral gene, but the larger share must be attributed to subsequent events of intron gain or intron ''sliding'' (movement from one position to another within a gene). Intron sliding has been argued from cases of discordant introns and from putative spatial clustering of intron positions. A list of 32 cases of discordant introns is presented here. Most of these cases are found to be artefactual. The spatial and phylogenetic distributions of intron positions from five published compilations of gene data, comprising 205 intron positions, have been examined systematically for evidence of intron sliding. The results suggest that sliding, if it occurs at all, has contributed little to the diversity of intron positions.

Remarkable sequence similarities in the trp region among Escherichia coli strains of diverse natural origins imply the existence of worldwide clones of very recent origin. This in turn implies a low rate of fixation of new universally favorable alleles, which carry adjacent stretches of chromosome to high frequency. These clonal segments begin as entire chromosomes; recombination shortens them progressively by substituting less closely related homologous DNA. The rate of this recombination, comprising the introduction of a homologous chromosomal fragment to a cell and the replacement of part of the original chromosome, is estimated from observations.

PCR fragments, 1500-bp, from 15 previously sequenced regions in the Escherichia coli chromosome have been compared by restriction analysis in a large set of wild (ECOR) strains. Prior published observations of segmental clonality are confirmed: each of several sequence types is shared by a number of strains. The rate of recombinational replacement and the average size of the replacements are estimated in a set of closely related strains in which a clonal frame is dotted with occasional stretches of DNA belonging to other clones. A clonal hierarchy is described. Some new comparative sequencing data are presented.

Background: Evolutionary analysis provides a formal framework for comparative analysis of genomic and other data. In evolutionary analysis, observed data are treated as the terminal states of characters that have evolved (via transitions between states) along the branches of a tree. The NEXUS standard of Syst. Biol. 46: 590-621) provides a portable, expressive and flexible text format for representing character-state data and trees. However, due to its complexity, NEXUS is not well supported by software and is not easily accessible to bioinformatics users and developers.

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.

Theories regarding the evolution of spliceosomal introns differ in the extent to which the distribution of introns reflects either a formative role in the evolution of protein-coding genes or the adventitious gain of genetic elements. Here, systematic methods are used to assess the causes of the present-day distribution of introns in 10 families of eukaryotic protein-coding genes comprising 1,868 introns in 488 distinct alignment positions. The history of intron evolution inferred using a probabilistic model that allows ancestral inheritance of introns, gain of introns, and loss of introns reveals that the vast majority of introns in these eukaryotic gene families were not inherited from the most recent common ancestral genes, but were gained subsequently. Furthermore, among inferred events of intron gain that meet strict criteria of reliability, the distribution of sites of gain with respect to reading-frame phase shows a 5:3:2 ratio of phases 0, 1 and 2, respectively, and exhibits a nucleotide preference for MAG GT (positions 23 to 12 relative to the site of gain). The nucleotide preferences of intron gain may prove to be the ultimate cause for the phase bias. The phase bias of intron gain is sufficient to account quantitatively for the well-known 5:3:2 bias in phase frequencies among extant introns, a conclusion that holds even when taxonomic heterogeneity in phase patterns is considered. Thus, intron gain accounts for the vast majority of extant introns and for the bias toward phase 0 introns that previously was interpreted as evidence for ancient formative introns.

Nexplorer is a web-based program for interactive browsing and manipulation of character data in NEXUS format, well suited for use with alignments and trees representing families of homologous genes or proteins. Users may upload a sequence family data set, or choose from one of several thousand already available. Nexplorer provides a flexible means to develop customized views that combine a tree and a data matrix or alignment, to create subsets of data, and to output data files or publication-quality graphics.

The comparative analysis of protein sequences depends crucially on measures of amino acid similarity or distance. Many such measures exist, yet it is not known how well these measures reflect the operational exchangeability of amino acids in proteins, since most are derived by methods that confound a variety of effects, including effects of mutation. In pursuit of a pure measure of exchangeability, we present (1) a compilation of data on the effects of 9671 amino acid exchanges engineered and assayed in a set of 12 proteins; (2) a statistical procedure to combine results from diverse assays of exchange effects; (3) a matrix of "experimental exchangeability" values EX ij derived from applying this procedure to the compiled data; and (4) a set of three tests designed to evaluate the power of an exchangeability measure to (i) predict the effects of amino acid exchanges in the laboratory, (ii) account for the disease-causing potential of missense mutations in the human population, and (iii) model the probability of fixation of missense mutations in evolution. EX not only captures useful information on exchangeability while remaining free of other effects, but also outperforms all measures tested except for the best-performing alignment scoring matrix, which is comparable in performance.

ince the genetic code first was determined, many have claimed that it is organized adaptively, so as to assign similar codons to similar amino acids. This claim has proved difficult to establish due to the absence of relevant comparative data on alternative primordial codes and of objective measures of amino acid exchangeability. Here we use a recently developed measure of exchangeability to evaluate a null hypothesis and two alternative hypotheses about the adaptiveness of the genetic code. The null hypothesis that there is no tendency for exchangeable amino acids to be assigned to similar codons can be excluded here as expected from earlier work. The first alternative hypothesis is that any such correlation between codon distance and amino acid distance is due to incremental mechanisms of code evolution, and not to adaptation to reduce deleterious effects of future mutations. More specifically, new codon assignments that occur by ambiguity reduction or by codon capture will tend to give rise to correlations, whether due to the condition of amino acid ambiguity, or to the condition of similarity between a new tRNA synthetase (or tRNA) and its parent. The second alternative hypothesis, the adaptive hypothesis, then may be defined as an excess relative to what may be expected given the incremental nature of evolution, reflecting true adaptation for robustness rather than an incidental effect. The results reported here indicate that most of the non-randomness in the amino acids to codon assignments can be explained by incremental code evolution, with a small residue of orderliness that may reflect code adaptation.

Determining the relative contributions of mutation and selection to evolutionary change is a matter of great practical and theoretical significance. In this paper, we examine relative contributions of codon mutation rates and amino acid exchangeability on the frequencies of each type of amino acid difference in alignments of distantly related proteins, alignments of closely related proteins, and among human SNPs, using a model that incorporates prior estimates of mutation and exchangeability parameters. For the operational exchangeability of amino acids in proteins, we use EX, a measure of proteinlevel effects from a recent statistical meta-analysis of nearly 10,000 experimental amino acid exchanges. EX is both free of mutational effects and more powerful than commonly used "biochemical distance" measures (1). For distant protein relationships, mutational effects (genetic code, transition/transversion bias) and operational exchangeability (EX) account for roughly equal portions of variance in off-diagonal values, the complete model accounting for R 2 = 0.35 of the variance. For human/chimpanzee alignments representing closely related proteins relationships, mutational effects (including CpG bias) account for 0.52 of the variance; adding EX to the model increases this to 0.67. For natural variation in human proteins, the variance explained by mutational effects alone, and by mutational effects and operational exchangeability are, respectively, 0.66 and 0.70 for SNPs in HGVBase, and 0.56 and 0.60 for disease-causing missense variants in HGMD. Thus, exchangeability has a stronger relative effect for distant protein evolution than for the cases of closely related proteins or of population variation. A more detailed model for the hominid data suggests that 1) there is a threshold in EX below which substitutions are highly unlikely to be accepted, corresponding to roughly 30 % relative protein activity; 2) selection against missense mutants is a slightly convex function of protein activity, not changing much as long as protein activity is low; and 3) the probability of disease-causing effects decreases nearly linearly with EX.

The classic view of evolution as ''shifting gene frequencies'' in the Modern Synthesis literally means that evolution is the modulation of existing variation (''standing variation''), as opposed to a ''new mutations'' view of evolution as a 2-step process of mutational origin followed by acceptance-or-rejection (via selection and drift). The latter view has received renewed attention, yet its implications for evolutionary causation still are not widely understood. We review theoretical results showing that this conception of evolution allows for a role of mutation as a cause of nonrandomness, a role that could be important but has been misconceived and associated misleadingly with neutral evolution. Specifically, biases in the introduction of variation, including mutational biases, may impose predictable biases on evolution, with no necessary dependence on neutrality. As an example of how important such effects may be, we present a new analysis partitioning the variance in mean rates of amino acid replacement during human-chimpanzee divergence to components of codon mutation and amino acid exchangeability. The results indicate that mutational effects are not merely important but account for most of the variance explained. The challenge that such results pose for comparative genomics is to address mutational effects as a necessary part of any analysis of causal factors. To meet this challenge requires developing knowledge of mutation as a biological process, understanding how mutation imposes propensities on evolution, and applying methods of analysis that incorporate mutational effects.

Evolutionary trends responsible for systematic differences in genome and proteome composition have been attributed to GC:AT mutation bias in the context of neutral evolution, or to selection acting on genome composition.  A possibility that has been ignored, presumably because it is recognized neither in the Modern Synthesis nor in the Neutral Theory, is that mutation may impose a directional bias on adaptation.  This possibility is explored here with simulations of the effect of a GC:AT bias on amino acid composition during adaptive walks on an abstract protein fitness landscape called an “NK” model.  The results indicate that adaptation does not preclude mutation-biased evolution.  In the complete absence of neutral evolution, a modest GC:AT bias of realistic magnitude can displace the trajectory of adaptation in a mutationally favored direction, to such a degree that amino acid composition is biased substantially and persistently.  Thus, patterns of divergence suggesting mutational explanations do not require the hypothesis of neutral evolution.

The rediscovery of Mendel’s laws a century ago launched the science that William Bateson called “genetics”, and led to a new view of evolution combining selection, particulate inheritance, and the newly characterized phenomenon of “mutation”.  This “mutationist” view clashed with the earlier view of Darwin, and the later “Modern Synthesis”, by allowing discontinuity, and by recognizing mutation (or more properly, mutation-and-altered-development) as a source of creativity, direction, and initiative.  By the mid-20th century, the opposing Modern Synthesis view was a prevailing orthodoxy: under its influence, “evolution” was redefined as “shifting gene frequencies”, i.e., the sorting out of pre-existing variation without new mutations, and the notion that mutation-and-altered-development can exert a predictable influence on the course of evolutionary change was seen as heretical.  Nevertheless, mutationist ideas re-surfaced: the notion of mutational determinants of directionality emerged in molecular evolution by 1962, followed in the 1980’s by an interest among evolutionary developmental biologists in a shaping or creative role of developmental propensities of variation, and more recently, a recognition by theoretical evolutionary geneticists of the importance of discontinuity and of new mutations in adaptive dynamics.  The synthetic challenge presented by these innovations is to integrate mutation-and-altered-development into a new understanding of the dual causation of evolutionary change— a broader and more predictive understanding that already can lay claim to important empirical and theoretical results— and to develop a research program appropriately emphasizing the emergence of variation as a cause of propensities of evolutionary change.