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Competing Interests: The author has declared that no competing interests exist.

Gene duplications are believed to facilitate evolutionary innovation. However, the mechanisms shaping the fate of duplicated genes remain heavily debated because the molecular processes and evolutionary forces involved are difficult to reconstruct. Here, we study a large family of fungal glucosidase genes that underwent several duplication events. We reconstruct all key ancestral enzymes and show that the very first preduplication enzyme was primarily active on maltose-like substrates, with trace activity for isomaltose-like sugars. Structural analysis and activity measurements on resurrected and present-day enzymes suggest that both activities cannot be fully optimized in a single enzyme. However, gene duplications repeatedly spawned daughter genes in which mutations optimized either isomaltase or maltase activity. Interestingly, similar shifts in enzyme activity were reached multiple times via different evolutionary routes. Together, our results provide a detailed picture of the molecular mechanisms that drove divergence of these duplicated enzymes and show that whereas the classic models of dosage, sub-, and neofunctionalization are helpful to conceptualize the implications of gene duplication, the three mechanisms co-occur and intertwine., Competing Interests: The authors have declared that no competing interests exist.

Background: Duplicated genes frequently experience asymmetric rates of sequence evolution. Relaxed selective constraints and positive selection have both been invoked to explain the observation that one paralog within a gene-duplicate pair exhibits an accelerated rate of sequence evolution. In the majority of studies where asymmetric divergence has been established, there is no indication as to which gene copy, ancestral or derived, is evolving more rapidly. In this study we investigated the effect of local synteny (gene-neighborhood conservation) and codon usage on the sequence evolution of gene duplicates in the S. cerevisiae genome. We further distinguish the gene duplicates into those that originated from a whole-genome duplication (WGD) event (ohnologs) versus small-scale duplications (SSD) to determine if there exist any differences in their patterns of sequence evolution., Results: For SSD pairs, the derived copy evolves faster than the ancestral copy. However, there is no relationship between rate asymmetry and synteny conservation (ancestral-like versus derived-like) in ohnologs. mRNA abundance and optimal codon usage as measured by the CAI is lower in the derived SSD copies relative to ancestral paralogs. Moreover, in the case of ohnologs, the faster-evolving copy has lower CAI and lowered expression., Conclusions: Together, these results suggest that relaxation of selection for codon usage and gene expression contribute to rate asymmetry in the evolution of duplicated genes and that in SSD pairs, the relaxation of selection stems from the loss of ancestral regulatory information in the derived copy.

Gene and genome duplications create novel genetic material on which evolution can work and have therefore been recognized as a major source of innovation for many eukaryotic lineages. Following duplication, the most likely fate is gene loss; however, a considerable fraction of duplicated genes survive. Not all genes have the same probability of survival, but it is not fully understood what evolutionary forces determine the pattern of gene retention. Here, we use genome sequence data as well as large-scale phosphoproteomics data from the baker's yeast Saccharomyces cerevisiae, which underwent a whole-genome duplication approximately 100 mya, and show that the number of phosphorylation sites on the proteins they encode is a major determinant of gene retention. Protein phosphorylation motifs are short amino acid sequences that are usually embedded within unstructured and rapidly evolving protein regions. Reciprocal loss of those ancestral sites and the gain of new ones are major drivers in the retention of the two surviving duplicates and in their acquisition of distinct functions. This way, small changes in the sequences of unstructured regions in proteins can contribute to the rapid rewiring and adaptation of regulatory networks.

Population genetic theory of gene duplication suggests that the preservation of duplicate copies requires functional divergence upon duplication. Genes that can be readily modified to produce new gene expression patterns may thus be duplicated often. In yeast, genes exhibit dichotomous expression patterns based on their promoter architectures. The expression of genes that contain TATA box or occupied proximal nucleosome (OPN) tends to be variable and respond to external signals. On the other hand, genes without TATA box or with depleted proximal nucleosome (DPN) are expressed constitutively. We find that recent duplicates in the yeast genome are heavily biased to be TATA box containing genes and not to be DPN genes. This suggests that variably expressed genes, due to the functional organization in their promoters, have higher duplicability than constitutively expressed genes.

We have investigated the ability of different regions of the left arm of Saccharomyces cerevisiae chromosome V to participate in the formation of gross chromosomal rearrangements (GCRs). We found that the 4.2-kilobase HXT13-DSF1 region sharing divergent homology with chromosomes IV, X and XIV, similar to mammalian segmental duplications, was 'at risk' for participating in duplication-mediated GCRs generated by homologous recombination. Numerous genes and pathways, including SGS1, TOP3, RMI1, SRS2, RAD6, SLX1, SLX4, SLX5, MSH2, MSH6, RAD10 and the DNA replication stress checkpoint requiring MRC1 and TOF1, were highly specific for suppressing these GCRs compared to GCRs mediated by single-copy sequences. These results indicate that the mechanisms for formation and suppression of rearrangements occurring in regions containing at-risk sequences differ from those occurring in regions of single-copy sequence. This explains how extensive genome instability is prevented in eukaryotic cells whose genomes contain numerous divergent repeated sequences.

Background: The direct examination of large, unbiased samples of young gene duplicates in their early stages of evolution is crucial to understanding the origin, divergence and preservation of new genes. Furthermore, comparative analysis of multiple genomes is necessary to determine whether patterns of gene duplication can be generalized across diverse lineages or are species-specific. Here we present results from an analysis comprising 68 duplication events in the Saccharomyces cerevisiae genome. We partition the yeast duplicates into ohnologs (generated by a whole-genome duplication) and non-ohnologs (from small-scale duplication events) to determine whether their disparate origins commit them to divergent evolutionary trajectories and genomic attributes., Results: We conclude that, for the most part, ohnologs tend to appear remarkably similar to non-ohnologs in their structural attributes (specifically the relative composition frequencies of complete, partial and chimeric duplicates), the discernible length of the duplicated region (duplication span) as well as genomic location. Furthermore, we find notable differences in the features of S. cerevisiae gene duplicates relative to those of another eukaryote, Caenorhabditis elegans, with respect to chromosomal location, extent of duplication and the relative frequencies of complete, partial and chimeric duplications., Conclusions: We conclude that the variation between yeast and worm duplicates can be attributed to differing mechanisms of duplication in conjunction with the varying efficacy of natural selection in these two genomes as dictated by their disparate effective population sizes.

Since complete redundancy between extant duplicates (paralogs) is evolutionarily unfavorable, some degree of functional congruency is eventually lost. However, in budding yeast, experimental evidence collected for duplicated metabolic enzymes and in global physical interaction surveys had suggested widespread functional overlap between paralogs. While maintained functional overlap is thought to confer robustness against genetic mutation and facilitate environmental adaptability, it has yet to be determined what properties define paralogs that can compensate for the phenotypic consequence of deleting a sister gene, how extensive this epistasis is, and how adaptable it is toward alternate environmental states. To this end, we have performed a comprehensive experimental analysis of epistasis as indicated by aggravating genetic interactions between paralogs resulting from an ancient whole-genome duplication (WGD) event occurring in the budding yeast Saccharomyces cerevisiae, and thus were able to compare properties of large numbers of epistatic and non-epistatic paralogs with identical evolutionary times since divergence. We found that more than one-third (140) of the 399 examinable WGD paralog pairs were epistatic under standard laboratory conditions and that additional cases of epistasis became obvious only under media conditions designed to induce cellular stress. Despite a significant increase in within-species sequence co-conservation, analysis of protein interactions revealed that paralogs epistatic under standard laboratory conditions were not more functionally overlapping than those non-epistatic. As experimental conditions had an impact on the functional categorization of paralogs deemed epistatic and only a fraction of potential stress conditions have been interrogated here, we hypothesize that many epistatic relationships remain unresolved.

How gene duplication and divergence contribute to genetic novelty and adaptation has been of intense interest, but experimental evidence has been limited. The genetic switch controlling the yeast galactose use pathway includes two paralogous genes in Saccharomyces cerevisiae that encode a co-inducer (GAL3) and a galactokinase (GAL1). These paralogues arose from a single bifunctional ancestral gene as is still present in Kluyveromyces lactis. To determine which evolutionary processes shaped the evolution of the two paralogues, here we assess the effects of precise replacement of coding and non-coding sequences on organismal fitness. We suggest that duplication of the ancestral bifunctional gene allowed for the resolution of an adaptive conflict between the transcriptional regulation of the two gene functions. After duplication, previously disfavoured binding site configurations evolved that divided the regulation of the ancestral gene into two specialized genes, one of which ultimately became one of the most tightly regulated genes in the genome.

To investigate functional divergence of gene duplicates, we examined the protein-protein interactions and coexistence in complexes of paralogs resulting from an ancient whole-genome duplication in yeast. Strikingly, half the surveyed paralog pairs were found to be co-clustered in protein complexes, and were more conserved and highly expressed than non-co-clustered paralogs; however, their discordant expression patterns and conservation rates indicate differential regulation of subfunctionalized paralogs. These results highlight the value of protein complex membership in studying functional divergence among gene duplicates.

In a set of 190 duplicate gene pairs in yeast Saccharomyces cerevisiae, the sharing of transcription factors tended to decrease with increased divergence in coding sequence, at both synonymous and nonsynonymous sites. Our results showed a significantly higher sharing of transcription factors by duplicated gene pairs falling within duplicated genomic blocks than in other duplicated gene pairs; and genes in duplicated blocks also showed significantly greater conservation at the coding sequence level. In spite of the overall trends, there were certain gene pairs, both in duplicated blocks and in other genomic regions, which were highly divergent in coding sequence and yet had identical patterns of transcription factor binding. These results suggest that functional differentiation of genes after duplication is a multi-dimensional process, with different duplicate pairs differentiating in different ways.

Much recent work has explored molecular and population-genetic constraints on the rate of protein sequence evolution. The best predictor of evolutionary rate is expression level, for reasons that have remained unexplained. Here, we hypothesize that selection to reduce the burden of protein misfolding will favor protein sequences with increased robustness to translational missense errors. Pressure for translational robustness increases with expression level and constrains sequence evolution. Using several sequenced yeast genomes, global expression and protein abundance data, and sets of paralogs traceable to an ancient whole-genome duplication in yeast, we rule out several confounding effects and show that expression level explains roughly half the variation in Saccharomyces cerevisiae protein evolutionary rates. We examine causes for expression's dominant role and find that genome-wide tests favor the translational robustness explanation over existing hypotheses that invoke constraints on function or translational efficiency. Our results suggest that proteins evolve at rates largely unrelated to their functions and can explain why highly expressed proteins evolve slowly across the tree of life.

Gene duplication is the fundamental source of new genes. Biases in duplication have profound implications for the dynamics of gene content during evolution. In this article, we compare genes arising from whole gene duplication (WGD), smaller scale duplication (SSD) and singletons in Saccharomyces cerevisiae. Our results demonstrate that genes duplicated by WGD and SSD are similarly biased with respect to codon bias and evolutionary rate, although differing significantly in their functional constituency.

Background: Quantification of intracellular metabolite fluxes by 13C-tracer experiments is maturing into a routine higher-throughput analysis. The question now arises as to which mutants should be analyzed. Here we identify key experiments in a systems biology approach with a genome-scale model of Saccharomyces cerevisiae metabolism, thereby reducing the workload for experimental network analyses and functional genomics., Results: Genome-scale 13C flux analysis revealed that about half of the 745 biochemical reactions were active during growth on glucose, but that alternative pathways exist for only 51 gene-encoded reactions with significant flux. These flexible reactions identified in silico are key targets for experimental flux analysis, and we present the first large-scale metabolic flux data for yeast, covering half of these mutants during growth on glucose. The metabolic lesions were often counteracted by flux rerouting, but knockout of cofactor-dependent reactions, as in the adh1, ald6, cox5A, fum1, mdh1, pda1, and zwf1 mutations, caused flux responses in more distant parts of the network. By integrating computational analyses, flux data, and physiological phenotypes of all mutants in active reactions, we quantified the relative importance of 'genetic buffering' through alternative pathways and network redundancy through duplicate genes for genetic robustness of the network., Conclusions: The apparent dispensability of knockout mutants with metabolic function is explained by gene inactivity under a particular condition in about half of the cases. For the remaining 207 viable mutants of active reactions, network redundancy through duplicate genes was the major (75%) and alternative pathways the minor (25%) molecular mechanism of genetic network robustness in S. cerevisiae.

Under laboratory conditions 80% of yeast genes seem not to be essential for viability. This raises the question of what the mechanistic basis for dispensability is, and whether it is the result of selection for buffering or an incidental side product. Here we analyse these issues using an in silico flux model of the yeast metabolic network. The model correctly predicts the knockout fitness effects in 88% of the genes studied and in vivo fluxes. Dispensable genes might be important, but under conditions not yet examined in the laboratory. Our model indicates that this is the dominant explanation for apparent dispensability, accounting for 37-68% of dispensable genes, whereas 15-28% of them are compensated by a duplicate, and only 4-17% are buffered by metabolic network flux reorganization. For over one-half of those not important under nutrient-rich conditions, we can predict conditions when they will be important. As expected, such condition-specific genes have a more restricted phylogenetic distribution. Gene duplicates catalysing the same reaction are not more common for indispensable reactions, suggesting that the reason for their retention is not to provide compensation. Instead their presence is better explained by selection for high enzymatic flux.

Two new genome sequences confirm that a whole genome duplication occurred in an ancestor of Saccharomyces cerevisiae. This left a legacy of about 500 pairs of duplicated genes, many of which contribute to this yeast's ability to ferment glucose anaerobically; a few have been evolving so quickly they retain almost no sequence similarity to each other.

Gene duplication with subsequent divergence plays a central role in the acquisition of genes with novel function and complexity during the course of evolution. With reduced functional constraints or through positive selection, these duplicated genes may experience accelerated evolution. Under the model of subfunctionalization, loss of subfunctions leads to complementary acceleration at sites with two copies, and the difference in average rate between the sequences may not be obvious. On the other hand, the classical model of neofunctionalization predicts that the evolutionary rate in one of the two duplicates is accelerated. However, the classical model does not tell which of the duplicates experiences the acceleration in evolutionary rate. Here, we present evidence from the Saccharomyces cerevisiae genome that a duplicate located in a genomic region with a low-recombination rate is likely to evolve faster than a duplicate in an area of high recombination. This observation is consistent with population genetics theory that predicts that purifying selection is less effective in genomic regions of low recombination (Hill-Robertson effect). Together with previous studies, our results suggest the genomic background (e.g., local recombination rate) as a potential force to drive the divergence between nontandemly duplicated genes. This implies the importance of structure and complexity of genomes in the diversification of organisms via gene duplications.

Background: Studying the evolution of the function of duplicated genes usually implies an estimation of the extent of functional conservation/divergence between duplicates from comparison of actual sequences. This only reveals the possible molecular function of genes without taking into account their cellular function(s). We took into consideration this latter dimension of gene function to approach the functional evolution of duplicated genes by analyzing the protein-protein interaction network in which their products are involved. For this, we derived a functional classification of the proteins using PRODISTIN, a bioinformatics method allowing comparison of protein function. Our work focused on the duplicated yeast genes, remnants of an ancient whole-genome duplication., Results: Starting from 4,143 interactions, we analyzed 41 duplicated protein pairs with the PRODISTIN method. We showed that duplicated pairs behaved differently in the classification with respect to their interactors. The different observed behaviors allowed us to propose a functional scale of conservation/divergence for the duplicated genes, based on interaction data. By comparing our results to the functional information carried by GO annotations and sequence comparisons, we showed that the interaction network analysis reveals functional subtleties, which are not discernible by other means. Finally, we interpreted our results in terms of evolutionary scenarios., Conclusions: Our analysis might provide a new way to analyse the functional evolution of duplicated genes and constitutes the first attempt of protein function evolutionary comparisons based on protein-protein interactions.

Whether nonessential genes evolve faster than essential genes has been a controversial issue. To resolve this issue, we use the data from a nearly complete set of single-gene deletions in the yeast Saccharomyces cerevisiae to assess protein dispensability. Also, instead of the nematode, which was used previously but is only distantly related to S. cerevisiae, we use another yeast, Candida albicans, as a second species to estimate the evolutionary distances between orthologous genes in two species. Our analysis reveals only a weak correlation between protein dispensability and evolutionary rate. More important, the correlation disappears when duplicate genes are removed from the analysis. And surprisingly, the average rate of nonsynonymous substitution is considerably lower than that for single-copy genes in the yeast genome. This observation suggests that structural constraints are more important in determining the rate of evolution of a protein than dispensability because duplicate genes are on average more dispensable than single-copy genes. For duplicate genes, those with only a weak effect or no effect of deletion on fitness evolve on average faster than those with a moderate or strong effect of deletion on fitness, which in turn evolve on average faster than those with a lethal effect of deletion.

Budding yeast provides a useful resource for studies of gene function. A new analysis of the fitness effects of deletion mutations in budding yeast reveals that genes that have duplicates create lower fitness losses when inactivated than do genes that are singletons.

Gene redundancy has been observed in yeast, plant and human genomes, and is thought to be a consequence of whole-genome duplications. Baker's yeast, Saccharomyces cerevisiae, contains several hundred duplicated genes. Duplication(s) could have occurred before or after a given speciation. To understand the evolution of the yeast genome, we analysed orthologues of some of these genes in several related yeast species. On the basis of the inferred phylogeny of each set of genes, we were able to deduce whether the gene duplicated and/or specialized before or after the divergence of two yeast lineages. Here we show that the gene duplications might have occurred as a single event, and that it probably took place before the Saccharomyces and Kluyveromyces lineages diverged from each other. Further evolution of each duplicated gene pair-such as specialization or differentiation of the two copies, or deletion of a single copy--has taken place independently throughout the evolution of these species.

Deleting a gene in an organism often has little phenotypic effect, owing to two mechanisms of compensation. The first is the existence of duplicate genes: that is, the loss of function in one copy can be compensated by the other copy or copies. The second mechanism of compensation stems from alternative metabolic pathways, regulatory networks, and so on. The relative importance of the two mechanisms has not been investigated except for a limited study, which suggested that the role of duplicate genes in compensation is negligible. The availability of fitness data for a nearly complete set of single-gene-deletion mutants of the Saccharomyces cerevisiae genome has enabled us to carry out a genome-wide evaluation of the role of duplicate genes in genetic robustness against null mutations. Here we show that there is a significantly higher probability of functional compensation for a duplicate gene than for a singleton, a high correlation between the frequency of compensation and the sequence similarity of two duplicates, and a higher probability of a severe fitness effect when the duplicate copy that is more highly expressed is deleted. We estimate that in S. cerevisiae at least a quarter of those gene deletions that have no phenotype are compensated by duplicate genes.

Nhp6Ap and Nhp6Bp, which are 87% identical in sequence, are moderately abundant, chromosome-associated proteins from Saccharomyces cerevisiae. In wild type cells Nhp6Ap is present at three times the level of Nhp6Bp. The effects of altering NHP6A or NHP6B gene number on the expression of its partner has been examined using Northern blots and reporter genes. Deletion of NHP6A led to a three-fold increase in NHP6B synthesis while an extra copy of NHP6A reduced NHP6B expression two-fold. Changes in the NHP6B gene copy number caused more moderate changes in NHP6A synthesis. The regulation of one NHP6 gene by the other uses a mechanism that detects the level of Nhp6 protein (or RNA) rather than gene number, since overexpression of Nhp6B protein from a single gene led to a dramatic decrease in NHP6A synthesis. Deletion analysis showed that the regulatory element involved in gene dosage compensation maps to a 190 bp segment in the NHP6B promoter. The simplest model, that each Nhp6 protein can act as a transcriptional repressor at the other NHP6 gene, is not true since purified Nhp6A protein does not bind specifically to the NHP6B promoter region. Instead, Nhp6p appears to interact with or through another protein in regulating transcription from the NHP6 genes.