Comparable Rates of Gene Loss and Functional Divergence after Genome Duplications Early in Vertebrate Evolution

Duplicated genes are an important source of new protein functions and novel developmental and physiological pathways. Whereas most models for fate of duplicated genes show that they tend to be rapidly lost, models for pathway evolution suggest that many duplicated genes rapidly acquire novel functions. Little empirical evidence is available, however, for the relative rates of gene loss vs. divergence to help resolve these contradictory expectations. Gene families resulting from genome duplications provide an opportunity to address this apparent contradiction. With genome duplication, the number of duplicated genes in a gene family is at most 2(n), where n is the number of duplications. The size of each gene family, e.g., 1, 2, 3, . . . , 2(n), reflects the patterns of gene loss vs. functional divergence after duplication. We focused on gene families in humans and mice that arose from genome duplications in early vertebrate evolution and we analyzed the frequency distribution of gene family size, i.e., the number of families with two, three or four members. All the models that we evaluated showed that duplicated genes are almost as likely to acquire a new and essential function as to be lost through acquisition of mutations that compromise protein function. An explanation for the unexpectedly high rate of functional divergence is that duplication allows genes to accumulate more neutral than disadvantageous mutations, thereby providing more opportunities to acquire diversified functions and pathways.     

On the nature of gene innovation: duplication patterns in microbial genomes

Gene duplication is considered a major force in gene family expansion and gene innovation. As gene copies assume novel functions, they must avoid periods of neutrality or be deleted from the genome. Current opinions state that copies avoid neutrality through gene dosage effects. These copies are therefore selected from an early stage. This study concentrates on the flow of copies from recent duplication to gene innovation. We have studied 21 microbial genomes using amino acid divergence to describe paralog evolution in the long-term perspective. Five of these were studied in closer detail using nucleotide divergence for a shorter perspective. It was found that rates of duplication and deletion are high, with only a small fraction of duplications retained and apparently selected. This leads to a steady accumulation of paralogs, which seems to be of a similar magnitude in most of the genomes. Furthermore, it is found that genes of high expression level, as measured by their codon bias, are strongly underrepresented among the most recent duplications. Based on these and other observations, it is suggested that gene innovation is driven by amplification of weak, ancillary functions rather than strong, established functions.     

Extensive and continuous duplication facilitates rapid evolution and diversification of gene families

The origin of novel gene functions through gene duplication, mutation, and natural selection represents one of the mechanisms by which organisms diversify and one of the possible paths leading to adaptation. Nonetheless, the extent, role, and consequences of duplications in the origins of ecological adaptations, especially in the context of species interactions, remain unclear. To explore the evolution of a gene family that is likely linked to species associations, we investigated the evolutionary history of the A-superfamily of conotoxin genes of predatory marine cone snails (Conus species). Members of this gene family are expressed in the venoms of Conus species and are presumably involved in predator-prey associations because of their utility in prey capture. We recovered sequences of this gene family from genomic DNA of four closely related species of Conus and reconstructed the evolutionary history of these genes. Our study is the first to directly recover conotoxin genes from Conus genomes to investigate the evolution of conotoxin gene families. Our results revealed a phenomenon of rapid and continuous gene turnover that is coupled with heightened rates of evolution. This continuous duplication pattern has not been observed previously, and the rate of gene turnover is at least two times higher than estimates from other multigene families. Conotoxin genes are among the most rapidly evolving protein-coding genes in metazoans, a phenomenon that may be facilitated by extensive gene duplications and have driven changes in conotoxin functions through neofunctionalization. Together these mechanisms led to dramatically divergent arrangements of A-superfamily conotoxin genes among closely related species of Conus. Our findings suggest that extensive and continuous gene duplication facilitates rapid evolution and drastic divergence in venom compositions among species, processes that may be associated with evolutionary responses to predator-prey interactions.     

Genome-level evolution of resistance genes in Arabidopsis thaliana

Pathogen resistance genes represent some of the most abundant and diverse gene families found within plant genomes. However, evolutionary mechanisms generating resistance gene diversity at the genome level are not well understood. We used the complete Arabidopsis thaliana genome sequence to show that most duplication of individual NBS-LRR sequences occurs at close physical proximity to the parent sequence and generates clusters of closely related NBS-LRR sequences. Deploying the statistical strength of phylogeographic approaches and using chromosomal location as a proxy for spatial location, we show that apparent duplication of NBS-LRR genes to ectopic chromosomal locations is largely the consequence of segmental chromosome duplication and rearrangement, rather than the independent duplication of individual sequences. Although accounting for a smaller fraction of NBS-LRR gene duplications, segmental chromosome duplication and rearrangement events have a large impact on the evolution of this multigene family. Intergenic exchange is dramatically lower between NBS-LRR sequences located in different chromosome regions as compared to exchange between sequences within the same chromosome region. Consequently, once translocated to new chromosome locations, NBS-LRR gene copies have a greater likelihood of escaping intergenic exchange and adopting new functions than do gene copies located within the same chromosomal region. We propose an evolutionary model that relates processes of genome evolution to mechanisms of evolution for the large, diverse, NBS-LRR gene family.     

Evidence for mutation by unequal sister chromatid exchange in the Duchenne muscular dystrophy gene.

We have studied three families each containing a male with Duchenne or Becker muscular dystrophy. Southern blot analysis using both genomic and cDNA probes revealed that an exon-containing segment of DNA within the gene is duplicated in the probands, their mothers, and, in two cases, their sisters. The grandpaternal origin of the duplication has been demonstrated in these families by RFLP and duplication analysis. The results suggest that unequal sister-chromatid exchange, which most likely occurred in the germ cell lineage of the proband's grandfather, is responsible for generating these duplications and that this type of intrachromosomal rearrangement, although rarely reported in humans, is not uncommon in the muscular dystrophy gene.     

Extensive Gene Duplication in the Early Evolution of Animals Before the Parazoan–Eumetazoan Split Demonstrated by G Proteins and Protein Tyrosine Kinases from Sponge and Hydra

To know whether genes involved in cell–cell communication typical of multicellular animals dramatically increased in concert with the Cambrian explosion, the rapid evolutionary burst in the major groups of animals, and whether these genes exist in the sponge lacking cell cohesiveness and coordination typical of eumetazoans, we have carried out cloning of the G-protein α subunit (Gα) and the protein tyrosine kinase (PTK) cDNAs from Ephydatia fluviatilis (freshwater sponge) and Hydra magnipapillata strain 105 (hydra). We obtained 13 Gα and 20 PTK cDNAs. Generally animal gene families diverged first by gene duplication (subtype duplication) that gave rise to diverse subtypes with different primary functions, followed by further gene duplication in the same subtype (isoform duplication) that gave rise to isoform genes with virtually identical function. Phylogenetic trees of Gα and PTK families including cDNAs from sponge and hydra revealed that most of the present-day subtypes had been established in the very early evolution of animals before the parazoan–eumetazoan split, the earliest branching among the extant animal phyla, by extensive subtype duplication: for PTK and Gα families, 23 and 9 subtype duplications were observed in the early stage before the parazoan–eumetazoan split, respectively, and after that split, only 2 and 1 subtype duplications were found, respectively. After the separation from arthropods, vertebrates underwent frequent isoform duplications before the fish–tetrapod split. Furthermore, rapid amino acid changes appear to have occurred in concert with the extensive subtype duplication and isoform duplication. Thus the pattern of gene diversification during animal evolution might be characterized by bursts of gene duplication interrupted by considerably long periods of silence, instead of proceeding gradually, and there might be no direct link between the Cambrian explosion and the extensive gene duplication that generated diverse functions (subtypes) of these families. Received: 4 November 1998 / Accepted: 17 November 1998     

Chromosome conformation capture uncovers potential genome-wide interactions between human conserved non-coding sequences

Comparative analyses of various mammalian genomes have identified numerous conserved non-coding (CNC) DNA elements that display striking conservation among species, suggesting that they have maintained specific functions throughout evolution. CNC function remains poorly understood, although recent studies have identified a role in gene regulation. We hypothesized that the identification of genomic loci that interact physically with CNCs would provide information on their functions. We have used circular chromosome conformation capture (4C) to characterize interactions of 10 CNCs from human chromosome 21 in K562 cells. The data provide evidence that CNCs are capable of interacting with loci that are enriched for CNCs. The number of trans interactions varies among CNCs; some show interactions with many loci, while others interact with few. Some of the tested CNCs are capable of driving the expression of a reporter gene in the mouse embryo, and associate with the oligodendrocyte genes OLIG1 and OLIG2. Our results underscore the power of chromosome conformation capture for the identification of targets of functional DNA elements and raise the possibility that CNCs exert their functions by physical association with defined genomic regions enriched in CNCs. These CNC-CNC interactions may in part explain their stringent conservation as a group of regulatory sequences.     

Rates and patterns of gene duplication and loss in the human genome

Gene duplication has certainly played a major role in structuring vertebrate genomes but the extent and nature of the duplication events involved remains controversial. A recent study identified two major episodes of gene duplication: one episode of putative genome duplication ca. 500 Myr ago and a more recent gene-family expansion attributed to segmental or tandem duplications. We confirm this pattern using methods not reliant on molecular clocks for individual gene families. However, analysis of a simple model of the birth-death process suggests that the apparent recent episode of duplication is an artefact of the birth-death process. We show that a constant-rate birth-death model is appropriate for gene duplication data, allowing us to estimate the rate of gene duplication and loss in the vertebrate genome over the last 200 Myr (0.00115 and 0.00740 Myr(-1) lineage(-1), respectively). Finally, we show that increasing rates of gene loss reduce the impact of a genome-wide duplication event on the distribution of gene duplications through time.     

Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens

Gene duplication is thought to be the main potential source of material for the evolution of new gene functions. Several models have been proposed for the evolution of new functions through duplication, most based on ancient events (Myr). We provide molecular evidence for the occurrence of several (at least 3) independent duplications of the ace-1 locus in the mosquito Culex pipiens, selected in response to insecticide pressure that probably occurred very recently (<40 years ago). This locus encodes the main target of several insecticides, the acetylcholinesterase. The duplications described consist of 2 alleles of ace-1, 1 susceptible and 1 resistant to insecticide, located on the same chromosome. These events were detected in different parts of the world and probably resulted from distinct mechanisms. We propose that duplications were selected because they reduce the fitness cost associated with the resistant ace-1 allele through the generation of persistent, advantageous heterozygosis. The rate of duplication of ace-1 in C. pipiens is probably underestimated, but seems to be rather high.     

The fate of duplicated genes: loss or new function?

Gene duplication events are important sources of novel gene functions. However, more often than not, a duplicate gene may lose its function and become a pseudogene. What is the relative frequency of these two scenarios: functional divergence versus gene loss? Given that most non-neutral mutations are deleterious, gene loss should be far more frequent than divergence. However, a recent empirical study suggests that about 50% of all gene duplications will lead to functional divergence. The study infers the frequency of functional divergence from the size distribution of gene families produced by two successive genome duplications early in vertebrate evolution. Reasons for this unexpectedly high frequency of functional divergence are discussed.     

Analysis of lamprey and hagfish genes reveals a complex history of gene duplications during early vertebrate evolution

It has been proposed that two events of duplication of the entire genome occurred early in vertebrate history (2R hypothesis). Several phylogenetic studies with a few gene families (mostly Hox genes and proteins from the MHC) have tried to confirm these polyploidization events. However, data from a single locus cannot explain the evolutionary history of a complete genome. To study this 2R hypothesis, we have taken advantage of the phylogenetic position of the lamprey to study the history of gene duplications in vertebrates. We selected most gene families that contain several paralogous genes in vertebrates and for which lamprey genes and an out-group are known in databases. In addition, we isolated members of the nuclear receptor superfamily in lamprey. Hagfish genes were also analyzed and found to confirm the lamprey gene analysis. Consistent with the 2R hypothesis, the phylogenetic analysis of 33 selected gene families, dispersed through the whole genome, revealed that one period of gene duplication arose before the lamprey-gnathostome split and this was followed by a second period of gene duplication after the lamprey-gnathostome split. Nevertheless, our analysis suggests that numerous gene losses and other gene-genome duplications occurred during the evolution of the vertebrate genomes. Thus, the complexity of all the paralogy groups present in vertebrates should be explained by the contribution of genome duplications (2R hypothesis), extra gene duplications, and gene losses.     

Adaptive evolution of conserved noncoding elements in mammals

Conserved noncoding elements (CNCs) are an abundant feature of vertebrate genomes. Some CNCs have been shown to act as cis-regulatory modules, but the function of most CNCs remains unclear. To study the evolution of CNCs, we have developed a statistical method called the “shared rates test” to identify CNCs that show significant variation in substitution rates across branches of a phylogenetic tree. We report an application of this method to alignments of 98,910 CNCs from the human, chimpanzee, dog, mouse, and rat genomes. We find that ~68% of CNCs evolve according to a null model where, for each CNC, a single parameter models the level of constraint acting throughout the phylogeny linking these five species. The remaining ~32% of CNCs show departures from the basic model including speed-ups and slow-downs on particular branches and occasionally multiple rate changes on different branches. We find that a subset of the significant CNCs have evolved significantly faster than the local neutral rate on a particular branch, providing strong evidence for adaptive evolution in these CNCs. The distribution of these signals on the phylogeny suggests that adaptive evolution of CNCs occurs in occasional short bursts of evolution. Our analyses suggest a large set of promising targets for future functional studies of adaptation.     

Processes of fungal proteome evolution and gain of function: gene duplication and domain rearrangement

During evolution, organisms have gained functional complexity mainly by modifying and improving existing functioning systems rather than creating new ones ab initio. Here we explore the interplay between two processes which during evolution have had major roles in the acquisition of new functions: gene duplication and protein domain rearrangements. We consider four possible evolutionary scenarios: gene families that have undergone none of these event types; only gene duplication; only domain rearrangement, or both events. We characterize each of the four evolutionary scenarios by functional attributes. Our analysis of ten fungal genomes indicates that at least for the fungi clade, species significantly appear to gain complexity by gene duplication accompanied by the expansion of existing domain architectures via rearrangements. We show that paralogs gaining new domain architectures via duplication tend to adopt new functions compared to paralogs that preserve their domain architectures. We conclude that evolution of protein families through gene duplication and domain rearrangement is correlated with their functional properties. We suggest that in general, new functions are acquired via the integration of gene duplication and domain rearrangements rather than each process acting independently.     

The roles of segmental and tandem gene duplication in the evolution of large gene families in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background

Most genes in Arabidopsis thaliana are members of gene families. How do the members of gene families arise, and how are gene family copy numbers maintained? Some gene families may evolve primarily through tandem duplication and high rates of birth and death in clusters, and others through infrequent polyploidy or large-scale segmental duplications and subsequent losses.

Results

Our approach to understanding the mechanisms of gene family evolution was to construct phylogenies for 50 large gene families in Arabidopsis thaliana, identify large internal segmental duplications in Arabidopsis, map gene duplications onto the segmental duplications, and use this information to identify which nodes in each phylogeny arose due to segmental or tandem duplication. Examples of six gene families exemplifying characteristic modes are described. Distributions of gene family sizes and patterns of duplication by genomic distance are also described in order to characterize patterns of local duplication and copy number for large gene families. Both gene family size and duplication by distance closely follow power-law distributions.

Conclusions

Combining information about genomic segmental duplications, gene family phylogenies, and gene positions provides a method to evaluate contributions of tandem duplication and segmental genome duplication in the generation and maintenance of gene families. These differences appear to correspond meaningfully to differences in functional roles of the members of the gene families.

     

High spontaneous rate of gene duplication in Caenorhabditis elegans

Gene and genome duplications are the primary source of new genes and novel functions and have played a pivotal role in the evolution of genomic and organismal complexity. The spontaneous rate of gene duplication is a critical parameter for understanding the evolutionary dynamics of gene duplicates; yet few direct empirical estimates exist and differ widely. The presence of a large population of recently derived gene duplicates in sequenced genomes suggests a high rate of spontaneous origin, also evidenced by population genomic studies reporting rampant copy-number polymorphism at the intraspecific level. An analysis of long-term mutation accumulation lines of Caenorhabditis elegans for gene copy-number changes with array comparative genomic hybridization yields the first direct estimate of the genome-wide rate of gene duplication in a multicellular eukaryote. The gene duplication rate in C. elegans is quite high, on the order of 10(-7) duplications/gene/generation. This rate is two orders of magnitude greater than the spontaneous rate of point mutation per nucleotide site in this species and also greatly exceeds an earlier estimate derived from the frequency distribution of extant gene duplicates in the sequenced C. elegans genome.     

Patterns of Gene Duplication and Their Contribution to Expansion of Gene Families in Grapevine

Grapevine is an important fruit crop that has undergone a long history of evolution. Analysis of the whole genome sequence of grapevine has revealed presence of an early palaeo-hexaploid along with three complements. Thus, gene duplication and genome expansion are common in this genome. In this study, we identified 17,922 duplicated genes in the whole grapevine genome. Among these, 2,039; 628; 1,428; 722; and 2,942 were identified respectively as produced by genome-wide, tandem, proximal, retrotransposed, and DNA-based transposed duplications. Analyses of the evolutionary patterns for different types of duplication using non-synonymous and synonymous substitution rates uncovered a series of underlying rules. Thereafter, all the grapevine genes were classified into families, and the contributions of different types of duplication to the expansion of large families were revealed. No duplication type was solely responsible for the formation of any large gene family, but some families showed enrichment of a special type of duplication. On the basis of this study, we believe that uncovering the underlying rules for gene duplications, expansions of gene families, and their evolutionary styles will contribute significantly to a comprehensive understanding of the features of the grapevine genome.     

Plant noncoding RNA gene discovery by "single-genome comparative genomics"

Plant genomes have undergone multiple rounds of duplications that contributed massively to the growth of gene families. The structure of resulting families has been studied in depth for protein-coding genes. However, little is known about the impact of duplications on noncoding RNA (ncRNA) genes. Here we perform a systematic analysis of duplicated regions in the rice genome in search of such ncRNA repeats. We observe that, just like their protein counterparts, most ncRNA genes have undergone multiple duplications that left visible sequence conservation footprints. The extent of ncRNA gene duplication in plants is such that these sequence footprints can be exploited for the discovery of novel ncRNA gene families on a large scale. We developed an SVM model that is able to retrieve likely ncRNA candidates among the 100,000+ repeat families in the rice genome, with a reasonably low false-positive discovery rate. Among the nearly 4000 ncRNA families predicted by this means, only 90 correspond to putative snoRNA or miRNA families. About half of the remaining families are classified as structured RNAs. New candidate ncRNAs are particularly enriched in UTR and intronic regions. Interestingly, 89% of the putative ncRNA families do not produce a detectable signal when their sequences are compared to another grass genome such as maize. Our results show that a large fraction of rice ncRNA genes are present in multiple copies and are species-specific or of recent origin. Intragenome comparison is a unique and potent source for the computational annotation of this major class of ncRNA.     

The Roles of Whole-Genome and Small-Scale Duplications in the Functional Specialization of Saccharomyces cerevisiae Genes

Researchers have long been enthralled with the idea that gene duplication can generate novel functions, crediting this process with great evolutionary importance. Empirical data shows that whole-genome duplications (WGDs) are more likely to be retained than small-scale duplications (SSDs), though their relative contribution to the functional fate of duplicates remains unexplored. Using the map of genetic interactions and the re-sequencing of 27 Saccharomyces cerevisiae genomes evolving for 2,200 generations we show that SSD-duplicates lead to neo-functionalization while WGD-duplicates partition ancestral functions. This conclusion is supported by: (a) SSD-duplicates establish more genetic interactions than singletons and WGD-duplicates; (b) SSD-duplicates copies share more interaction-partners than WGD-duplicates copies; (c) WGD-duplicates interaction partners are more functionally related than SSD-duplicates partners; (d) SSD-duplicates gene copies are more functionally divergent from one another, while keeping more overlapping functions, and diverge in their sub-cellular locations more than WGD-duplicates copies; and (e) SSD-duplicates complement their functions to a greater extent than WGD–duplicates. We propose a novel model that uncovers the complexity of evolution after gene duplication.     

Rapid evolution in a pair of recent duplicate segments of rice

Gene duplication has been considered the most important way of generating genetic novelties. The subsequent evolution right after gene duplication is critical for new function to occur. Here we analyzed the evolutionary pattern for a recently duplicated segment between rice chromosomes 11 and 12. This duplication event was estimated to occur about 6 million years ago, during the divergence of the B- and C-genome rice species. The duplicate segment in chromosome 12 has significantly higher frequency of sequence rearrangement rate than non-duplicated regions. The rearrangement rate is approximately 6.5 breakages/Mb per million years, about six times higher than the fastest rate ever reported in eukaryotes. The genes within both segments experienced accelerated nucleotide substitution rates revealed by synonymous (Ks) and non-synonymous divergence (Ka) between Oryza sativa indica and O. sativa japonica. Analysis using EST data also implicates rapid divergence in expression between these segmental duplicate genes. These overall rapid changes from different perspective for the first time provide evidence that relaxation of selection also occurs in large-scale duplications.     

Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution, origin, and phenotypegenotype correlation.

Partial gene deletion is the major cause of mutation leading to Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Partial gene duplication has also been recognized in a few cases. We have conducted a survey for duplication in 72 unrelated nondeletion patients, analyzed by Southern blot hybridization with clones representing the entire DMD cDNA. With careful quantitative analysis of hybridization band intensity, 10 cases were found to carry a duplication of part of the gene, a frequency of 14% for nondeletion cases (10/72), or 6% for all cases (10/181). The extent of these duplications has been characterized according to the published exon-containing HindIII fragment map, and in six of the 10 duplications a novel restriction fragment that spanned the duplication junction was detected. The resulting translational reading frame of mRNA has been predicted for nine duplications. A shift of the reading frame was predicted in four of the six DMD cases and in one of the two intermediate cases, while the reading frame remained uninterrupted in both BMD cases. RFLP and quantitative Southern blot analyses revealed a grandpaternal origin of duplication in four families and grandmaternal origin in one family. In all five families, the duplication was found to originate from a single X chromosome. Unequal sister-chromatid exchange is proposed to be the mechanism for the formation of these duplications.