Structural divergence between the human and chimpanzee genomes

The structural microheterogeneity evident between the human and chimpanzee genomes is quite considerable and includes inversions and duplications as well as deletions, ranging in size from a few base-pairs up to several megabases (Mb). Insertions and deletions have together given rise to at least 150 Mb of genomic DNA sequence that is either present or absent in humans as compared to chimpanzees. Such regions often contain paralogous sequences and members of multigene families thereby ensuring that the human and chimpanzee genomes differ by a significant fraction of their gene content. There is as yet no evidence to suggest that the large chromosomal rearrangements which serve to distinguish the human and chimpanzee karyotypes have influenced either speciation or the evolution of lineage-specific traits. However, the myriad submicroscopic rearrangements in both genomes, particularly those involving copy number variation, are unlikely to represent exclusively neutral changes and hence promise to facilitate the identification of genes that have been important for human-specific evolution.     

A comprehensive expression analysis of all members of a gene family encoding cell-wall enzymes allowed us to predict cis-regulatory regions involved in cell-wall construction in specific organs of Arabidopsis.

The Arabidopsis thaliana genome sequencing project has revealed that multigene families, such as those generated by genome duplications, are more abundant among plant genomes than among animal genomes. To gain insight into the evolutionary implications of the multigene families in higher plants, we examined the XTH gene family, a group of genes encoding xyloglucan endotransglucosylase/hydrolase, which are responsible for cell-wall construction in plants. Expression analysis of all members (33 genes) of this family, using quantitative real-time RT-PCR, revealed that most members exhibit distinct expression profiles in terms of tissue specificity and responses to hormonal signals, with some members exhibiting similar expression patterns. By comparing the flanking sequences of individual genes, we identified four sets of large-segment duplications and two sets of solitary gene duplications. In each set of gene duplicates, long nucleotide sequences, ranging from one to two hundred base pairs, are conserved. Furthermore, gene duplicates exhibit similar organ-specific expression profiles. These facts allowed us to predict putative cis-regulatory regions, particularly those responsible for cell-wall construction, and hence for morphogenesis, that are specific for certain organs or tissues in plants.     

Transgenic Arabidopsis expressing osmolyte glycine betaine synthesizing enzymes from halophilic methanogen promote tolerance to drought and salt stress

Gene duplication events exert key functions on gene innovations during the evolution of the eukaryotic genomes. A large portion of the total gene content in plants arose from tandem duplications events, which often result in paralog genes with high sequence identity. Ubiquitin ligases or E3 enzymes are components of the ubiquitin proteasome system that function during the transfer of the ubiquitin molecule to the substrate. In plants, several E3s have expanded in their genomes as multigene families. To gain insight into the consequences of gene duplications on the expansion and diversification of E3s, we examined the evolutionary basis of a cluster of six genes, duplC-ATLs, which arose from segmental and tandem duplication events in Brassicaceae. The assessment of the expression suggested two patterns that are supported by lineage. While retention of expression domains was observed, an apparent absence or reduction of expression was also inferred. We found that two duplC-ATL genes underwent pseudogenization and that, in one case, gene expression is probably regained. Our findings provide insights into the evolution of gene families in plants, defining key events on the expansion of the Arabidopsis Tóxicos en Levadura family of E3 ligases.     

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.     

Pattern of the divergence of olfactory receptor genes during tetrapod evolution

The olfactory receptor (OR) multigene family is responsible for the sense of smell in vertebrate species. OR genes are scattered widely in our chromosomes and constitute one of the largest gene families in eutherian genomes. Some previous studies revealed that eutherian OR genes diverged mainly during early mammalian evolution. However, the exact period when, and the ecological reason why eutherian ORs strongly diverged has remained unclear. In this study, I performed a strict data mining effort for marsupial opossum OR sequences and bootstrap analyses to estimate the periods of chromosomal migrations and gene duplications of OR genes during tetrapod evolution. The results indicate that chromosomal migrations occurred mainly during early vertebrate evolution before the monotreme-placental split, and that gene duplications occurred mainly during early mammalian evolution between the bird-mammal split and marsupial-placental split, coinciding with the reduction of opsin genes in primitive mammals. It could be thought that the previous chromosomal dispersal allowed the OR genes to subsequently expand easily, and the nocturnal adaptation of early mammals might have triggered the OR gene expansion.     

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.     

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.     

Accepted mutations in a gene family: Evolutionary diversification of duplicated DNA

Summary We report and compare the DNA sequences of 14 silkmoth (Antheraea polyphemus) chorion genes, derived from either cDNA or chromosomal DNA clones. Seven of these genes are members of the A multigene family, and seven are members of the B family. Where available, the previously reported (Jones and Kafatos 1980) intronic and extragenic flanking DNA sequences are also considered. Closely related sequences are compared, revealing the types of spontaneous mutations that were fixed during paralogous evolution. Segmental mutations (i.e. mutations other than substitutions) are nearly always interpretable as small duplications or deletions. related to small direct repeats. Segmental mutations are strongly constrained in the coding regions, although they do occur. Nucleotide substitutions also appear to be under selective constraints: relatively few substitutions leading to amino acid replacements are accepted, silent substitutions leading to some codons (especially purine-terminated ones) are disfavored, and different compositional biases are maintained in different parts of the sequences. Other sequence differences can be interpreted as indicative of neutral drift, including most differences in non-coding regions and most T/C transitions in third-base positions. In the non-coding regions, which are thought to be only loosely constrained by selection, transitions are observed more frequently than might be expected: they account for 52% of all substitutions, and they appear to be favored two to threefold over transversions when allowance is made for the skewed base composition of these regions.     

A novel gene family NBPF: intricate structure generated by gene duplications during primate evolution

Partial and complete genome duplications occurred during evolution and resulted in the creation of new genes and gene families. We identified a novel and intricate human gene family located primarily in regions of segmental duplications on human chromosome 1. We named it NBPF, for neuroblastoma breakpoint family, because one of its members is disrupted by a chromosomal translocation in a neuroblastoma patient. The NBPF genes have a repetitive structure with high intragenic and intergenic sequence similarity in both coding and noncoding regions. These similarities might expose these genomic regions to illegitimate recombination, resulting in structural variation in the NBPF genes. The encoded proteins contain a highly conserved domain of unknown function, which we have named the NBPF repeat. In silico analysis combined with the isolation of multiple full-length cDNA clones showed that several members of this gene family are abundantly expressed in a large variety of tissues and cell lines. Strikingly, no discernable orthologues could be identified in the completed genomes of fruit fly, nematode, mouse, or rat, but sequences with low homology could be isolated from the draft canine and bovine genomes. Interestingly, this gene family shows primate-specific duplications that result in species-specific arrays of NBPF homologous sequences. Overall, this novel NBPF family reflects the continuous evolution of primate genomes that resulted in large physiological differences, and its potential role in this process is discussed.     

Ectopic gene conversions in bacterial genomes.

We characterized the gene conversions found between the duplicated genes of 75 bacterial genomes from five species groups (archaea, nonpathogenic and pathogenic firmicutes, and nonpathogenic and pathogenic proteobacteria). The number of gene conversions is positively correlated with the size of multigene families and the size of multigene families is not significantly different between pathogenic and nonpathogenic taxa. However, gene conversions occur twice as frequently in pathogenic species as in nonpathogenic species. Comparisons between closely related species also indicate a trend towards increased gene conversion in pathogenic species. Whereas the length of the conversions is positively correlated with flanking sequence similarity in all five groups, these correlations are smaller for pathogenic firmicutes and proteobacteria than for nonpathogenic firmicutes and proteobacteria. These results are consistent with our previous work on E. coli genomes and suggest that pathogenic bacteria allow recombination between more divergent gene sequences. This higher permissiveness is likely adaptive because it allows them to generate more genetic variability.     

Promiscuous mitochondrial group II intron sequences in plant nuclear genomes

Gene translocations from the organelles to the nucleus are postulated by the endosymbiont hypothesis. We here report evidence for sequence insertions in the nuclear genomes of plants that are derived from noncoding regions of the mitochondrial genome. Fragments of mitochondrial group II introns are identified in the nuclear genomes of tobacco and a bean species. The duplicated intron sequences of 75–140 bp are derived from cis- and trans-splicing introns of genes encoding subunits 1 and 5 of the NADH dehydrogenase. The mitochondrial sequences are inserted in the vicinities of a lectin gene, different glucanase genes and a gene encoding a subunit of photosystem II. Sequence similarities between the nuclear and mitochondrial copies are in the range of 80 to 97%, suggesting recent transfer events that occurred in the basic glucanase genes before and in the lectin gene after the gene duplications in the evolution of the nuclear gene families. Overlapping regions of the same introns are in two instances also involved in intramitochondrial sequence duplications. Correspondence to: V. Knoop     

Pattern of divergence of amino acid sequences encoded by paralogous genes in human and pufferfish

We used phylogenetic analyses of protein families containing two or more pairs of orthologues in the genomes of human and pufferfish (Takifugu rubripes) to test the hypothesis that these sequences show a strong signal of polyploidization events hypothesized to have occurred early in vertebrate history. In order to test for evidence of two distinct rounds of polyploidization (the 2R hypothesis), we compared the pattern of amino acid sequence divergence of proteins encoded by genes duplicated just prior to the most recent common ancestor of human and pufferfish with that of proteins encoded genes duplicated earlier. These sequence divergences were statistically indistinguishable, contrary to the prediction of the 2R hypothesis. The variance of amino acid sequence divergences between paralogues was significantly greater than expected from that of orthologues in the same families. Estimation of gene duplication times assuming a molecular clock provided earlier estimates than expected, suggesting that it may not be appropriate to time the duplication of paralogues using rate estimates derived from orthologous comparisons. Overall, the results indicate that amino acid sequences do not provide a strong signal supporting the hypothesis that gene duplications early in vertebrate history occurred by polyploidization. On the other hand, the data are easily explained under an alternative model that gene duplications occurred at different times in different vertebrate gene families.     

Arthropod Hox genes: insights on the evolutionary forces that shape gene functions

Comparative studies suggest that gene duplication, changes in cis-regulatory elements and changes in protein sequence all contribute to the evolution of Hox gene functions, but the evolutionary dynamics of these changes are probably different. It seems likely that gene duplications arise as neutral changes and acquire an adaptive significance later on. By contrast, some changes in regulatory and protein-coding sequences can have immediate consequences in morphological evolution.     

Identification of members of gene families in Arabidopsis thaliana by contig construction from partial cDNA sequences: 106 genes encoding 50 cytoplasmic ribosomal proteins

Partial cDNA sequencing to obtain expressed sequence tags (ESTs) has led to the identification of tags to about 8000 of the estimated 20 000 genes in Arabidopsis thaliana . This figure represents four to five times the number of complete coding sequences from this organism available in international databases. In contrast to mammals, many proteins are encoded by multigene families in A. thaliana . Using ribosomal protein gene families as an example, it is possible to construct relatively long sequences from overlapping ESTs which are of sufficiently high quality to be able to unambiguously identify tags to individual members of multigene families, even when the sequences are highly conserved. A total of 106 genes encoding 50 different cytoplasmic ribosomal protein types have been identified, most proteins being encoded by at least two and up to four genes. Coding sequences of members of individual gene families are almost always very highly conserved and derived amino acid sequences are almost, if not completely, identical in the vast majority of cases. Sequence divergence is observed in untranslated regions which allows the definition of gene-specific probes. The method can be used to construct high-quality tags to any protein.     

On the evolution of multigene families

Multigene families are classified into three groups: small families as exemplified by hemoglobin genes of mammals; middlesize multigene families, by genes of mammalian histocompatibility antigens; and large multigene families, by variable region genes of immunoglobulins. Facts and theories on these evolving multigene families are reviewed, with special reference to the population genetics of their concerted evolution. It is shown that multigene families are evolving under continued occurrence of unequal (but homologous) crossing-over and gene conversion, and that mechanisms for maintaining genetic variability are totally different from the conventional models of population genetics. Thus, in view of widespread occurrence of multigene families in genomes of higher organisms, the evolutionary theory based mainly on change of gene frequency at each locus would appear to need considerable revision.     

An integrated approach for the comparative analysis of a multigene family: The nicotianamine synthase genes of barley

Recent genomic projects reveal that about half of the gene repertoire in plant genomes is made up by multigene families. In this paper, a set of structural and phylogenetic analyses have been applied to compare the differently sized nicotianamine synthase (NAS) gene families in barley and rice. Nicotianamine acts as a chelator of iron and other heavy metals and plays a key role in uptake, phloem transport and cytoplasmic distribution of iron, challenging efforts for the breeding of iron-efficient crop plants. Nine barley NAS genes have been mapped, and co-linearity of flanking genes in barley and rice was determined. The combined analyses reveal that the NAS multigene family members in barley originated through at least one duplication event that occurred before the divergence of rice and barley. Additional duplications appear to have occurred within each of the species. Although we detected no evidence for positive selection of recently duplicated genes within species, codon-based tests revealed evidence for positive selection having contributed to the divergence of some amino acids. The integrated comparative and phylogenetic analysis improved our current view of NAS gene family evolution, might facilitate the functional characterization of individual members and is applicable to other multigene families. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A comparison of vertebrate interferon gene families detected by hybridization with human interferon DNA

Cloned human interferon complementary DNAs were used as hybridization probes to detect interferon alpha and beta gene families in restriction endonuclease digests of total genomic DNA isolated from a wide range of vertebrates and invertebrates. A complex interferon-alpha multigene family was detected in all mammals examined, whereas there was little or no cross-hybridization of human interferon-alpha complementary DNA to non-mammalian vertebrates or invertebrates. In contrast, human interferon-beta complementary DNA detected one or two interferon-beta genes in all mammals tested, with the exception of the cow and the blackbuck, both of which possessed a complex interferon-beta multigene family which has presumably arisen by a recent series of gene duplications. Interferon-beta sequences could also be detected in non-mammalian vertebrates ranging from birds to bony fish. Detailed restriction endonuclease mapping of DNA sequences neighbouring the interferon-beta gene in a variety of primates indicated a strong evolutionary conservation of flanking sequences, particularly on the 3' side of the gene.     

Evolutionary diversification of multigene families: allelic selection of toxins in predatory cone snails

In order to investigate the evolution of conotoxin multigene families among two closely related vermivorous CONUS: species, we sequenced 104 four-loop conotoxin mRNAs from two individuals of CONUS: ebraeus and compared these with sequences already obtained from CONUS: abbreviatus. In contrast to the diversity of conotoxin sequences obtained from C. abbreviatus, only two common sequence variants were recovered from C. ebraeus. Segregation patterns of the variants in these two individuals and restriction digests of four-loop conotoxin amplification products from nine additional individuals suggest that the common variants are alleles from a single locus. These two putative alleles differ at nine positions that occur nonrandomly in the toxin-coding region of the sequences. Moreover, all substitutions are at nonsynonymous sites and are responsible for seven amino acid differences among the predicted amino acid sequences of the alleles. These results imply that conotoxin diversity is driven by strong diversifying selection and some form of frequency-dependent or overdominant selection at conotoxin loci, and they suggest that diverse conotoxin multigene families can originate from duplications at polymorphic loci. Furthermore, none of the sequences recovered from C. ebraeus appeared to be orthologs of loci from C. abbreviatus, and attempts to amplify orthologous sequences with locus-specific primers were unsuccessful among these species. These patterns suggest that venoms of closely related CONUS: species may differ due to the differential expression of conotoxin loci.     

Genome sequences and evolutionary biology, a two-way interaction

Complete genome sequences are accumulating rapidly, culminating with the announcement of the human genome sequence in February 2001. In addition to cataloguing the diversity of genes and other sequences, genome sequences will provide the first detailed and complete data on gene families and genome organization, including data on evolutionary changes. Reciprocally, evolutionary biology will make important contributions to the efforts to understand functions of genes and other sequences in genomes. Large-scale, detailed and unbiased comparisons between species will illuminate the evolution of genes and genomes, and population genetics methods will enable detection of functionally important genes or sequences, including sequences that have been involved in adaptive changes.