Molecular Evolutionary Processes Research Articles

Ribosome display and affinity maturation: from antibodies to single V-domains and steps towards cancer therapeutics

Protein affinity maturation using molecular evolution techniques to produce high-affinity binding proteins is an important step in the generation of reagents for cancer diagnosis and treatment. Currently, the most commonly used molecular evolution processes involve mutation of a single gene into complex gene repertoires followed by selection from a display library. Fd-bacteriophage are the most popular display vectors, but are limited in their capacity for library presentation, speed of processing and mutation frequency. Recently, the potential of ribosome display for directed molecular evolution was recognised and developed into a rapid and simple affinity selection strategy using ribosome complexes to display antibody fragments (scFv). Ribosome display and selection has the potential to generate and display large libraries more representative of the theoretical optima for naı̈ve repertoires (10 14). Even more important is the application of ribosome display for the affinity maturation of individual proteins by rapid mutation and selection cycles. These display strategies can apply to other members of the immunoglobulin superfamily; for example single V-domains which have an important application in providing specific targeting to either novel or refractory cancer markers. We discuss the application of ribosome display and selection in conjunction with variable domain (CTLA-4) libraries as the first step towards this objective and review affinity maturation strategies for in vitro ribosome display systems.

B-chromosome evolution.

B chromosomes are extra chromosomes to the standard complement that occur in many organisms. They can originate in a number of ways including derivation from autosomes and sex chromosomes in intra- and interspecies crosses. Their subsequent molecular evolution resembles that of univalent sex chromosomes, which involves gene silencing, heterochromatinization and the accumulation of repetitive DNA and transposons. B-chromosome frequencies in populations result from a balance between their transmission rates and their effects on host fitness. Their long-term evolution is considered to be the outcome of selection on the host genome to eliminate B chromosomes or suppress their effects and on the B chromosome's ability to escape through the generation of new variants. Because B chromosomes interact with the standard chromosomes, they can play an important role in genome evolution and may be useful for studying molecular evolutionary processes.

Using protein structural information in evolutionary inference: transmembrane proteins.

We present a model of amino acid sequence evolution based on a hidden Markov model that extends to transmembrane proteins previous methods that incorporate protein structural information into phylogenetics. Our model aims to give a better understanding of processes of molecular evolution and to extract structural information from multiple alignments of transmembrane sequences and use such information to improve phylogenetic analyses. This should be of value in phylogenetic studies of transmembrane proteins: for example, mitochondrial proteins have acquired a special importance in phylogenetics and are mostly transmembrane proteins. The improvement in fit to example data sets of our new model relative to less complex models of amino acid sequence evolution is statistically tested. To further illustrate the potential utility of our method, phylogeny estimation is performed on primate CCR5 receptor sequences, sequences of l and m subunits of the light reaction center in purple bacteria, guinea pig sequences with respect to lagomorph and rodent sequences of calcitonin receptor and K-substance receptor, and cetacean sequences of cytochrome b.

Null Model Selection, Compositional Bias, Character State Bias, and the Limits of Phylogenetic Information

Evolutionary trends and processes can distort phylogenetic information in sequences such that they do not reliably reflect the evolutionary processes that generate them. This fact of molecular evolution has a ubiquitous influence on the ability of researchers to adequately reconstruct genealogical relationships and histories of the processes of molecular evolution. This feature of phylogenetic inference can limit the capacity of researchers to adequately specify a relevant null hypothesis for testing hypothesis of relationships, data informativeness, and processes of molecular evolution. We show how this feature of historical inference also influences the exactness of the relative apparent synapomorphy analysis (RASA) test for phylogenetic signal and demonstrate how a permutation modification of the null hypothesis can improve the robustness of the underlying distributional assumption of the test. The RASA test (using either null model) was found not only to appropriately reject the combinability of independent lines of evidence for the relationships among the Physalaemus pustulosusfrog species group, but also to be more appropriately sensitive to individual uninformative data sets than commonly used tree-based measures of signal, including the consistency index, the retention index, and the permutation tail probability test statistic.

Retroposition of autosomal mRNA yielded testis-specific gene family on human Y chromosome.

Most genes in the human NRY (non-recombining portion of the Y chromosome) can be assigned to one of two groups: X-homologous genes or testis-specific gene families with no obvious X-chromosomal homologues. The CDY genes have been localized to the human Y chromosome, and we report here that they are derivatives of a conventional single-copy gene, CDYL (CDY-like), located on human chromosome 13 and mouse chromosome 6. CDY genes retain CDYL exonic sequences but lack its introns. In mice, whose evolutionary lineage diverged before the appearance of the Y-linked derivatives, the autosomal Cdyl gene produces two transcripts; one is expressed ubiquitously and the other is expressed in testes only. In humans, autosomal CDYL produces only the ubiquitous transcript; the testis-specific transcript is the province of the Y-borne CDY genes. Our data indicate that CDY genes arose during primate evolution by retroposition of a CDYL mRNA and amplification of the retroposed gene. Retroposition contributed to the gene content of the human Y chromosome, together with two other molecular evolutionary processes: persistence of a subset of genes shared with the X chromosome and transposition of genomic DNA harbouring intact transcription units.

Mutual catalysis in sets of prebiotic organic molecules: Evolution through computer simulated chemical kinetics

A thorough outlook on the origin of life needs to delineate a chemically rigorous, self-consistent path from highly heterogeneous, random ensembles of relatively simple organic molecules, to an entity that has rudimentary life-like characteristics. Such entity should be endowed with a capacity to express variation, undergo mutation-like changes and manifest a simple evolutionary process. For simulating such system we developed the Graded Autocatalysis Replication Domain (GARD) model for explicit kinetic analysis of mutual catalysis in sets of random oligomers derived from energized precursor monomers. The kinetic properties of the GARD model are based on vesicle enclosure and expansion. With the additional assumption of spontaneous vesicle splitting, a GARD evolution scenario is envisaged as a consequence of pure chemical kinetics. Here we show how the GARD model can serve as a platform for investigating the dynamics of self-organization mechanisms in molecular evolutionary processes.

Comparisons of the molecular evolutionary process at rbcL and ndhF in the grass family (Poaceae).

We examine rate heterogeneity among evolutionary lineages of the grass family at two plasmid loci, ndhF and rbcL, and we introduce a method to determine whether patterns of rate heterogeneity are correlated between loci. We show both that rates of synonymous evolution are heterogeneous among grass lineages and that are heterogeneity is correlated between loci at synonymous sites. At nonsynonymous sites, the pattern of rate heterogeneity is not correlated between loci, primarily due to an aberrant pattern of rate heterogeneity at nonsynonymous sites of rbcL. We compare patterns of synonymous rate heterogeneity to predictors based on the generation time effect and the speciation rate hypotheses. Although there is some evidence for generation time effects, neither generation time effects nor speciation rates appear to be sufficient to explain patterns of rate heterogeneity in the grass plastid sequences.

Annexin gene structures and molecular evolutionary genetics.

Annexins provide an exemplary model for studying the pattern and process of molecular evolution in multigene families. Their related gene structures, broad dispersal in eukaryotic genomes and abundant coding sequences permit a phylogenetic reconstruction of their genetic history. The emerging picture is one of prolific expansion by gene duplication to more than 27 paralogous subfamilies that have undergone steady sequence divergence, speciation and differential selection. Homologous recombination via the common tetrad of internal repeats has, nevertheless, strictly preserved this core structure for over 1200 million years, implying a basic functional role. The existence of multiple annexins with unique 5' coding and regulatory regions has facilitated their adaptation to the varying ontogenetic and cell-specific needs of diverse organisms. Computational and cladistic sequence analyses have permitted the determination of original gene duplication dates and mutation rates for the ten known vertebrate annexins. Molecular genetic and evolutionary studies of annexins can help to define their structure-function relationships elucidate their individual physiological roles and ultimately link them to hereditary phenotypes.

Clustering Molecular Sequences with Their Components.

Motivation: Several methods in genetic information have recently been developed to estimate classification of protein sequences through their sequence similarity. These methods are essential for understanding the function of predicted open reading frames (ORFs) and their molecular evolutionary processes. However, since many protein sequences consist of a number of independently evolved structural units (we refer to these units as components), the combinatorial nature of the components makes it difficult to classify the sequences. Results: This paper presents a new method for classifying uncharacterized protein sequences. As the measure of sequence similarity, we use similarity score computed by a method based on the Smith-Waterman local alignment algorithm. Here we introduce how this method cope when sequences have multi-component structure. This method was applied to predicted ORFs on the Escherichia coli genome and we discuss the algorithm and experimental results.

Emergence of template-and-sequence-directed (TSD) syntheses: II. A computer simulation model.

The initiation of the bio-geochemical scenario described in Part I serves in the present work as the basis for computer modeling, where the central process of the simulation algorithm. i.e., peptide-catalyzed oligomeric growth, is based on mass action equations. The computer model starts with a minimal system in which catalyzed growth processes of proto-RNA templates and small peptides take place, starting from their building blocks. The emerging populations of random oligomers also include a very small fraction of proto-tRNAs and a small fraction of catalytic peptides. Using simplifying assumptions regarding catalyzed proto-RNA template-replication, as well as selectivity of certain molecules and processes, the proportion of proto-tRNA in the proto-RNA molecular population increases rapidly; it is followed by TSD peptide synthesis, based on an ad hoc genetic code and specific peptide catalysts allocated for this synthesis. Consequently, a feedback system is initiated in which TSD peptides involved in the relevant catalytic reactions of the TSD syntheses also start to accumulate. The initial sporadic formation of TSD peptides is thus replaced gradually by cycles of positive feedback and autocatalysis characterized by accumulation of catalytic peptides and Proto-tRNAs and TSD-Reaction-Takeover. The model system which can be considered a 'toy model' can synthesize its templates and catalysts under a wide range of reaction parameters and initial concentrations, thus demonstrating a robustness which is essential for molecular evolution processes. The critical stage of the buildup of a molecular mechanism for the initiation of a minimal TSD reaction cycle has thus been described; because of the centrality of TSD reaction cycles in biology, it is assumed to be central also in the origin of life processes.

Molecular Ecology and Evolution: Approaches and Applications.

I: DNA fingerprinting and behavioral ecology.- Arbitrary primer mediated fingerprinting in plants: Case studies in plant breeding, taxonomy and phylogeny.- DNA amplification fingerprinting: A general tool with applications in breeding, identification and phylogenetic analysis of plants.- The analysis of simple repeat loci as applied in evolutionary and behavioral sciences.- Multilocus DNA fingerprinting and genetic relatedness in plants: A case study with banana and tomato.- Measuring reproductive success in insects.- Unravelling the components that underlie insect reproductive traits using a simple molecular approach.- Molecular analysis of kinship in birds: Interesting questions and useful techniques.- II: Population biology.- Molecular techniques in population genetics: A brief history.- Organization of genetic variation at the molecular level: Lessons from Drosophila.- The use of microsatellite analysis in population biology: Background, methods and potential applications.- The use of microsatellites for genetic analysis of natural populations.- PCR assays of variable nucleotide sites for identification of conservation units.- Concerted evolution and RAPping in mitochondrial VNTRs and the molecular geography of cricket populations.- Molecular markers and evolutionary processes in hermaphrodite freshwater snails.- Extinction and the formation of phylogenetic lineages: Diagnosing units of conservation management in the tiger beetle Cicindela dorsalis.- Perspective on conservation genetics.- III: Molecular systematics.- Advances in the theory and practice of DNA-hybridization as a systematic method.- Allozymes in mammalian population genetics and systematics: Indicative function of a marker system reconsidered.- Analysis of DNA from natural history museum collections.- Sources of ambiguity in nucleic acid sequence alignment.- Computational problems in molecular systematics.- The comparison of morphological and molecular data in phylogenetic systematics.- Non-coding chloroplast DNA for plant molecular systematics at the infrageneric level.- Developing model systems for molecular biogeography: Vicariance and interchange in marine invertebrates.- Bridging phylogenetics and population genetics with gene tree models.- IV: Speciation, development and genome organization.- The role of molecular genetics in speciation studies.- The origin and evolution of species differences in Escherichia coli and Salmonella typhimurium.- The evolutionary ecology of Daphnia.- Diversity within diversity: Molecular approaches to studying microbial interactions with insects.- Evolutionary analysis of genes involved in early embryonic pattern formation in Drosophila.- Developmental genes and the origin and evolution of Metazoa.- To what extent does genetic information determine structural characteristics and document homologies?.- Evolution and multi-functionality of the chitin system.- Genome evolution: Between the nucleosome and the chromosome.- Individual genes underlying quantitative traits: Molecular and analytical methods.- Perspectives on future applications of experimental biology to evolution.

A Likelihood Ratio Test to Detect Conflicting Phylogenetic Signal

Molecular data are commonly used to reconstruct the evolutionary histories of organ- isms. However, evolutionary reconstructions from different molecular data sets sometimes conflict. It is generally unknown whether these different estimates of history result from random variation in the processes of nucleotide substitution or from fundamentally different evolutionary mecha- nisms underlying the histories of the genes analyzed. We describe a novel likelihood ratio test that compares different topologies (each estimated from a different data partition for the same taxa) to determine if they are significantly different. The results of this test indicate that different genes provide significantly different phylogenies for amniotes, supporting earlier suggestions based on less direct tests. These results suggest that some molecular data can give misleading information about evolutionary history. (Likelihood ratio test; maximum likelihood; phylogenetic methods; phylogenetic heterogeneity.) Debate about which kinds of data pro- vide the most accurate estimates of phy- logeny is fueled by observations that dif- ferent data sets for the same taxa sometimes yield discordant estimates. In some cases there is a good explanation for the different phylogenetic estimates. For example, horizontal gene transfer and re- combination in bacteria cause different portions of the genome to have different histories (Dykhuizen and Green, 1991; Maynard Smith et al., 1991; Medigue et al., 1991; Souza et al., 1992; Valdez and Pinero, 1992). Also, in many organisms, polymor- phisms that predate speciation may fail to reflect the phylogenetic history of the rest of the genome (the gene-tree vs. species- tree problem; Wilson et al., 1977; Pesole et al., 1991; Doyle, 1992). It is nonetheless generally accepted that most genes in higher taxa are free from these problems and that any differences in phylogenies produced with different data sets can be attributed to sampling. Statis- tical procedures such as bootstrapping re- veal that different estimates of phylogeny may arise purely as a consequence of sam- pling error. However, conflicting phyloge- nies may also arise when the underlying processes of molecular evolution differ among genes, even though the genes have

MOLECULAR EVIDENCE FOR NATURAL SELECTION

neutral theory, nearly neutral theory, population genetics ABSTRACT Our understandi ng of the causes of molec ular evolution is not as certain as it was a decade ago when Kimura's neutral theory appeared to explain major features of DNA conservation and change. The last ten years have seen the development of empirical approaches and statistical tests for detecting selec­ tion in DNA and a proliferation of data that challenge our current understanding of the molecular evolutionary process. We begin this review with a discussion of protein polymorphism and divergence: two major areas of research where the strictly neutral model cannot explain general patterns in the data. We then present a survey of statistical methods for detecting positive selection, which includes tests for balancing selection, for sequence convergence, and for un­ usually high rates of evolution that cannot be accounted for by neutral models. Finally, we present findings of a number of groups working on within- and between-species variation in Drosophila: These highlight the importance of adaptive evolution, purifying selection, and recombination in understanding levels and patterns of nucleotide variation.

Substitution processes in molecular evolution. III. Deleterious alleles.

The substitution processes for various models of deleterious alleles are examined using computer simulations and mathematical analyses. Most of the work focuses on the house-of-cards model, which is a popular model of deleterious allele evolution. The rate of substitution is shown to be a concave function of the strength of selection as measured by alpha = 2N sigma, where N is the population size and sigma is the standard deviation of fitness. For alpha < 1, the house-of-cards model is essentially a neutral model; for alpha > 4, the model ceases to evolve. The stagnation for large alpha may be understood by appealing to the theory of records. The house-of-cards model evolves to a state where the vast majority of all mutations are deleterious, but precisely one-half of those mutations that fix are deleterious (the other half are advantageous). Thus, the model is not a model of exclusively deleterious evolution as is frequently claimed. It is argued that there are no biologically reasonable models of molecular evolution where the vast majority of all substitutions are deleterious. Other models examined include the exponential and gamma shift models, the Hartl-Dykhuizen-Dean (HDD) model, and the optimum model. Of all those examined, only the optimum and HDD models appear to be reasonable candidates for silent evolution. None of the models are viewed as good candidates for protein evolution, as none are both biologically reasonable and exhibit the variability in substitutions commonly observed in protein sequence data.

Substitution Processes in Molecular Evolution. II. Exchangeable Models from Population Genetics

Substitution Processes in Molecular Evolution. II. Exchangeable Models from Population Genetics

Molecular evolutionary processes and conflicting gene trees: The hominoid case

Molecular evolutionary processes modify DNA over time, creating both newly derived substitutions shared by related descendant lineages (phylogenetic signal) and "false" similarities which confound phylogenetic reconstruction (homoplasy). However, some types of DNA regions, for example those containing tandem duplicate repeats, are preferentially subject to homoplasy-inducing processes such as sporadically occurring concerted evolution and DNA insertion/deletion. This added level of homoplasic "noise" can make DNA regions with repeats less reliable in phylogenetic reconstruction than those without repeats. Most molecular datasets which distinguish among African hominoids support a human-chimpanzee clade; the most notable exception is from the involucrin gene. However, phylogenetic resolution supporting a chimpanzee-gorilla clade is based entirely on involucrin DNA repeat regions. This is problematic because (1) involucrin repeats are difficult to align, and published alignments are contradictory; (2) involucrin repeats are subject to DNA insertion/deletion; (3) gorillas are polymorphic in that some do not have repeats reported to be synapomorphies linking chimpanzees and gorillas. Gene tree/species tree conflicts can occur due to the sorting of ancestrally polymorphic alleles during speciation. Because hominoid females transfer between groups, mitochondrial and nuclear gene flow occur to the same extent, and the probability of conflict between mitochondrial and nuclear gene trees is theoretically low. When hominoid intraspecific mitochondrial variability is taken into account [based on cytochrome oxidase subunit II (COII) gene sequences], humans and chimpanzees are most closely related, showing the same relative degree of separation from gorillas as when single individuals representing species are analyzed. Conflicting molecular phylogenies can be explained in terms of molecular evolutionary processes and sorting of ancient polymorphisms. This perspective can enhance our understanding of hominoid molecular phylogenies.

Substitution processes in molecular evolution. I. Uniform and clustered substitutions in a haploid model.

A computer simulation of the process of nucleotide substitutions in a finite haploid population subject to selection in a randomly fluctuating environment provides a number of unexpected results. For rapidly fluctuating environments, substitutions are more regular than random. A small mutation-rate approximation is used to explain the regularity. The explanation does not depend heavily on the particulars of the haploid model, leading to the conjecture that many symmetrical models of molecular evolution with rapidly changing parameters may exhibit substitutions that are more regular than random. When fitnesses change very slowly, the simulation shows that substitutions are more clumped than random. Here a small-mutation approximation shows that the clustering is due to the increase in fitness that accompanies each successive substitution with a consequent lowering of the effective mutation rate. The two observations taken together suggest that the common observation that amino acid substitutions are clustered in time is due to the presence of parameters that change very slowly.

Some Statistical Problems in the Assessment of Inhomogeneities of DNA Sequence Data

Abstract The fields of molecular genetics and medicine are accumulating DNA and protein sequence data at an accelerating rate. Discovering and interpreting sequence patterns can contribute to understanding molecular mechanisms and evolutionary processes. This article considers two types of statistical problems in these contexts: (1) identifying anomalies in the distribution of a specified biochemical marker along a DNA string; in particular, new statistical methods are set forth by which to assess excessive clustering, over dispersion, and too much regularity of the marker along the sequence. Applications are given to the physical map data of the bacterium Escherichia coli. (2) Some results and statistical problems on the assembly of cloned DNA segments are also described. Sections 2 and 3 of the article present helpful background material on DNA organization and inheritance.

Some Statistical Problems in the Assessment of Inhomogeneities of DNA Sequence Data

Abstract The fields of molecular genetics and medicine are accumulating DNA and protein sequence data at an accelerating rate. Discovering and interpreting sequence patterns can contribute to understanding molecular mechanisms and evolutionary processes. This article considers two types of statistical problems in these contexts: (1) identifying anomalies in the distribution of a specified biochemical marker along a DNA string; in particular, new statistical methods are set forth by which to assess excessive clustering, over dispersion, and too much regularity of the marker along the sequence. Applications are given to the physical map data of the bacterium Escherichia coli. (2) Some results and statistical problems on the assembly of cloned DNA segments are also described. Sections 2 and 3 of the article present helpful background material on DNA organization and inheritance.

Accuracy of estimating genetic distance between species from short sequences of mitochondrial DNA.

Recent advances in molecular techniques allow rapid determination of genetic divergence, and in many circumstances the polymerase chain reaction (PCR) and DNA sequencing have become the technologies of choice for estimating genetic distance. For evolutionary studies, considerable effort is being focused on “universal” oligonucleotide primers that amplify short sequences of mitochondrial DNA. Kocher et al. ( 1989, p. 6 199) encapsulate this approach when they state that a “short sequence from a piece of the cytochrome b gene contains phylogenetic information extending from the infraspecific level to the intergeneric level.” If this is a valid generalization, then the advent of universal primers and the ease of direct sequencing of PCR products provides an efficient and reliable means of determining genetic divergence and inferring phylogenetic relationships among taxa. Indeed, DNA sequence derived from the cytochrome b primers (usually amounting to 250 bp) have proved successful for inferring evolutionary relationships and estimating rates and processes of molecular evolution (Kocher et al. 1989; Thomas et al. 1989). However, before evolutionary biologists turn their attention to such short DNA sequences, it is important to understand the accuracy of genetic distances estimated from such sequences. It is well known that the distribution of substitution events is leptokurtic. Thomas and Beckenbach ( 1989) showed that there is spatial heterogeneity in the distribution of substitutions in mitochondrial protein-coding genes from salmonid fishes. Nonrandom distribution of substitutions is a problem in estimating evolutionary relatedness from short, amplified DNA sequences because the sampling scheme is nonrandom, being entirely dependent on the choice of primers used for amplifications. Amplification and sequencing of fragments that are too small to encompass the scope of spatial heterogeneity in the distribution of substitutions will give biased estimates of genetic distance. * We investigated the influence of the number of basepairs on genetic distance estimates based on mtDNA sequence data by randomly subsampling known DNA sequences and then determining the variance of genetic distance estimates among subsamples. This approach allows us to investigate the extent to which substitution differences in different parts of a gene interfere with the robustness of genetic distance estimates based on short sequences. The accuracy of between-taxa genetic divergence estimation based on a small number of basepairs was evaluated by subsampling regions of the mitochondrial genomes containing protein-coding genes for five pairs of vertebrate taxa (fig. 1). The analysis focuses on fourfold-degenerate sites within protein-coding regions because these sites are free from selective constraints and therefore provide the best type of data for inferring evolutionary distance ( Wu and Li 1985 ) . For each of the five paired vertebrate comparisons, continuous, homologous sequences of 250, 500, 750, 1,000, 1,250, and 1,500 bp were selected at random from the complete sequences. For these sequence lengths, the average numbers of fourfold-