Functional divergence in the caspase gene family and altered functional constraints: statistical analysis and prediction.

In this article, we explore the pattern of type I functional divergence (i.e., altered functional constraints or site-specific rate difference) in the caspase gene family that is important for apoptosis (programmed cell death) and cytokine maturation. By taking advantage of substantial experimental data from caspases, the functional/structural basis of our posterior predictions from sequence analysis was extensively studied. Our results are as follows: (1) Phylogenetic analysis shows that the evolution of major caspase-mediated pathways has been facilitated by gene duplications, (2) type I functional divergence (altered functional constraints) is statistically significant between two major subfamilies, CED-3 and ICE, (3) 4 of 21 predicted amino acid residues (for site-specific rate difference between CED-3 and ICE) have been verified by experimental evidence, and (4) we found that some CED-3 caspases may inherit more ancestral functions, whereas other members may employ some recently derived functions. Our approach can be cost effective in functional genomics to make statistically sound predictions from amino acid sequences.     

Statistical methods for testing functional divergence after gene duplication

Functional innovations after gene duplication may result in altered functional constraints between member gene clusters of a gene family. This type (type I) of functional divergence is measured by the coefficient of functional divergence (theta lambda), which can be interpreted as the decrease in rate correlation between gene clusters, or the probability that the evolutionary rate at a site is statistically independent between two gene clusters. A simple stochastic model has been developed for estimating theta lambda and testing its statistical significance. The current model includes the model of rate variation among sites as a special case when theta lambda = 0. Moreover, we have developed a site-specific profile based on the hidden Markov model to identify critical amino acid residues that are responsible for these functional differences between two gene clusters, which may have great potential in functional genomics.     

Evolution of Antennapedia-Class Homeobox Genes

Antennapedia (Antp)-class homeobox genes are involved in the determination of pattern formation along the anterior-posterior axis of the animal embryo. A phylogenetic analysis of Antp-class homeodomains of the nematode, Drosophila, amphioxus, mouse, and human indicates that the 13 cognate group genes of this gene family can be divided into two major groups, i.e., groups I and II. Group I genes can further be divided into subgroups A (cognate groups 1-2), B (cognate group 3), and C (cognate groups 4-8), and group II genes can be divided into subgroups D (cognate groups 9-10) and E (cognate groups 11-13), though this classification is somewhat ambiguous. Evolutionary distances among different amino acid sequences suggest that the divergence between group I and group II genes occurred ~1000 million years (MY) ago, and the five different subgroups were formed by ~600 MY ago, probably before the divergence of Pseudocoelomates (e.g., nematodes) and Coelomates (e.g., insects and chordates). Our results show that the genes that are phylogenetically close are also closely located in the chromosome, suggesting that the colinearity between the gene expression and gene arrangement was generated by successive tandem gene duplications and that the gene arrangement has been maintained by some sort of selection.     

Identification of rice TUBBY-like genes and their evolution

The identification of TUBBY-like genes in organisms ranging from single-celled to multicellular eukaryotes has allowed the phylogenetic history of this gene family to be traced back to the early evolutionary stages of eukaryote development. Rice TUBBY-like genes were located on chromosomes 1, 2, 3, 4, 5, 7, 8, 11 and 12 without any obvious clustering. On a genomic scale, it was revealed that the rice TUBBY-like gene family probably evolved mainly through segmental duplication produced by polyploidy. The altered selective constraints (or site-specific rate changes), related to functional divergence during protein evolution between plant and animal TUBBY-like genes, were statistically significant. Based on posterior probability analysis, five amino acid sites (103, 312, 315, 317 and 319) are thought to be responsible for functional divergence.     

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.     

Evolutionary Analysis for Functional Divergence of the Toll-Like Receptor Gene Family and Altered Functional Constraints

The Toll-like receptor (TLR) gene family consists of type 1 transmembrane receptors, which play essential roles in both innate immunity and adaptive immune response by ligand recognition and signal transduction. Using all available vertebrate TLR protein sequences, we inferred the phylogenetic tree and then characterized critical amino acid residues for functional divergence by detecting altered functional constraints after gene duplications. We found that the extracellular domain of TLR genes showed higher functional divergence than that of the cytoplasmic domain, particularly in the region between leucine-rich repeat (LRR) 10 and LRR 15 of TLR 4. Our finding supports the concept that sequence evolution in the extracellular domain may be responsible for the broad diversity of TLR ligand-binding affinity, providing a testable hypothesis for potential targets that could be verified by further experimentation.     

Origin and Evolution of Paralogous rRNA Gene Clusters Within the Flatworm Family Dugesiidae (Platyhelminthes, Tricladida)

Analysis of the 18S rDNA sequences of five species of the family Dugesiidae (phylum Platyhelminthes, suborder Tricladida, infraorder Paludicola) and eight species belonging to families Dendrocoelidae and Planaridae and to the infraorder Maricola showed that members of the family Dugesiidae have two types of 18S rDNA genes, while the rest of the species have only one. The duplication event also affected the ITS-1, 5.8S, ITS-2 region and probably the 28S gene. The mean divergence value between the type I and the type II sequences is 9% and type II 18S rDNA genes are evolving 2.3 times more rapidly than type I. The evolutionary rates of type I and type II genes were calibrated from biogeographical data, and an approximate date for the duplication event of 80–120 million years ago was calculated. The type II gene was shown, by RT-PCR, to be transcribed in adult individuals of Schmidtea polychroa, though at very low levels. This result, together with the fact that most of the functionally important positions for small-subunit rRNA in prokaryotes have been conserved, indicates that the type II gene is probably functional. Received: 24 March 1998 / Accepted: 17 March 1999     

Molecular Evolution of the Photolyase–Blue-Light Photoreceptor Family

The photolyase–blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors. CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively. CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity. Blue-light photoreceptors are essential light detectors for the early development of plants. The amino acid sequence of the receptor is similar to those of the photolyases, although the receptor does not show the activity of photoreactivation. To investigate the functional divergence of the family, the amino acid sequences of the proteins were aligned. The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases (class I photolyases) are different from those of class II CPD photolyases (class II photolyases). We reconstructed the phylogenetic trees based on the alignment by the NJ method and the ML method. The phylogenetic analysis suggested that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes. Received: 23 October 1996 / Accepted: 1 April 1997     

Molecular phylogeny,long-term evolution,and functional divergence of flavin-containing monooxygenases

Flavin-containing monooxygenases (FMOs) metabolize xenobiotic compounds, many of which are clinically important, as well as endogenous substrates as part of a discrete physiological process. The FMO gene family is conserved and ancient with representatives present in all phyla so far examined. However, there is a lack of information regarding the long-term evolution and functional divergence of these proteins. This study represents the first attempt to characterize the long-term evolution followed by the members of this family. Our analysis shows that there is extensive silent divergence at the nucleotide level suggesting that this family has been subject to strong purifying selection at the protein level. Invertebrate FMOs have a polyphyletic origin. The functional divergence of FMOs 1–5 started before the split between amphibians and mammals. The vertebrate FMO5 is more ancestral than other four FMOs. Moreover, the existence of higher levels of codon bias was detected at the N-terminal ends, which can be ascribed to the critical role played by the FAD binding motif in this region. Finally, critical amino acid residues for FMO functional divergence (type I & II) after gene duplication were detected and characterized.     

Pattern of Nucleotide Substitutions in Growth Hormone-Prolactin Gene Family: A Paradigm for Evolution by Gene Duplication

The growth hormone-prolactin gene family in mammals is an interesting example of evolution by gene duplication. Divergence among members of duplicated gene families and among species was examined by using reported gene sequences of growth hormone, prolactin and their receptors. Sequence divergence among species was found to show a general tendency in which a generation-time effect is pronounced for synonymous substitutions but not so for nonsynonymous substitutions. Divergence among duplicated genes is characterized by the relatively high rate of nonsynonymous substitutions, i.e., the rate is close to that of synonymous ones. In view of the stage- and tissue-specific expression of duplicated genes, some of the amino acid substitutions among duplicated genes is likely to be caused by positive Darwinian selection.     

Functional divergence outlines the evolution of novel protein function in NifH/BchL protein family

Biological nitrogen fixation is accomplished by prokaryotes through the catalytic action of complex metalloenzyme, nitrogenase. Nitrogenase is a two-protein component system comprising MoFe protein (NifD&K) and Fe protein (NifH). NifH shares structural and mechanistic similarities as well as evolutionary relationships with light-independent protochlorophyllide reductase (BchL), a photosynthesis-related metalloenzyme belonging to the same protein family. We performed a comprehensive bioinformatics analysis of the NifH/BchL family in order to elucidate the intrinsic functional diversity and the underlying evolutionary mechanism among the members. To analyse functional divergence in the NifH/BchL family, we have conducted pair-wise estimation in altered evolutionary rates between the member proteins. We identified a number of vital amino acid sites which contribute to predicted functional diversity. We have also made use of the maximum likelihood tests for detection of positive selection at the amino acid level followed by the structure-based phylogenetic approach to draw conclusion on the ancient lineage and novel characterization of the NifH/BchL protein family. Our investigation provides ample support to the fact that NifH protein and BchL share robust structural similarities and have probably deviated from a common ancestor followed by divergence in functional properties possibly due to gene duplication     

Analysis of the evolution of angiotensin II type 1 receptor gene in mammals (mouse, rat, bovine and human).

The nucleotide and amino acid sequences for mouse angiotensin II (AII) type 1A and 1B receptors were deduced from their complementary and genomic DNAs. Evolutionary analyses based on the nucleotide sequences of the coding region of AII type 1 receptor genes indicated that the duplication event of the type 1 gene occurred 24 +/- 2 million years ago before the divergence between the rat and mouse but after the divergence between rodents and the human/artiodactyls couple. This conclusion was consistent with the results of genomic Southern blot analyses, which revealed that the mouse and rat possess 2 similar but separate genes, whereas the bovine and human have only a single class gene.     

Rapid expansion of killer cell immunoglobulin-like receptor genes in primates and their coevolution with MHC Class I genes

The gene family of killer cell immunoglobulin-like receptors (KIRs) in primates provides the first line of defense against virus infection and tumor transformation. Interacting with MHC class I molecules, KIRs can regulate the cytotoxic activity of natural killer (NK) cells and distinguish the tumor and virus infected cells from normal body cells. Phylogenetic analysis and comparison of domain structures identified three major groups of KIR genes (group I, II, and III genes). These groups of KIR genes, generated by a series of gene duplications, have acquired different MHC-binding specificity. Inference of ancestral KIR sequences suggested that the functional divergence of group I genes from group II genes occurred by positive selection at the MHC-binding sites after duplication. Our evolutionary study has shown that group I genes diverged from group II genes about 17 million years ago (Mya) apparently after separation of hominoids from Old World (OW) monkeys. Around the same time, gene duplication generating the class I MHC-C locus appears to have occurred. These findings suggest that KIR and MHC class I genes have coevolved as an interacting system. The KIR gene family has experienced a rapid expansion in primate species. The rate of expansion of this gene family seems to be one of the highest among all hominoid gene families. The KIR gene family is also subject to birth-and-death evolution.     

Evolution and functional divergence of monocarboxylate transporter genes in vertebrates

Monocarboxylate transporters (MCTs) form a gene family with an ancient past. The identification of MCTs (MCHs) from bacteria, protozoa, fungi, invertebrates, as well as vertebrates, but not from plants and virus, allowed illuminating the phylogenetic and evolutionary history of this gene family. The significant expansion of vertebrate MCT genes should have primarily occurred after the divergence of vertebrates and invertebrates, but before the divergence time between ray-finned fish and mammals. The divergence of insect MCTs should have at least occurred in the common ancestor of fruit fly, beetle, and honeybee. Fungi monocarboxylate transporter homologues (MCHs) might evolve independently from an ancient ancestor. The results of functional divergence analysis provided statistical evidences for shifted evolutionary rate and/or changes of amino acid property after gene duplication. The sliding window analysis of the d(N)/d(S) ratio values showed that strong functional constraints must impose on the N- and C-terminal domains of vertebrate MCTs. These corresponding regions may play crucial roles for functionality of MCT proteins.     

Mathematical modeling for functional divergence after gene duplication.

In this paper, I present a statistical framework for modeling the functional divergence after gene duplication. A rate-component model to describe the rate covariation among homologous genes of a gene family is implemented when a phylogenetic tree is known. The Markov chain model is rigorous but may require a huge amount of computational time when the number of sequences is large. On the other hand, the Poisson-based model is mathematically analytical so that computation is very fast even for a large dataset. Moreover, under the posterior framework, we have developed a site-specific profile for predicting important amino acid residues responsible for these functional differences between member genes of a gene family. Our study may have great potential for functional genomics because it is cost-effective, and these predictions can be further tested by biological experimentation.     

Opsin gene duplication and divergence in ray-finned fish

Opsin gene sequences were first reported in the 1980s. The goal of that research was to test the hypothesis that human opsins were members of a single gene family and that variation in human color vision was mediated by mutations in these genes. While the new data supported both hypotheses, the greatest contribution of this work was, arguably, that it provided the data necessary for PCR-based surveys in a diversity of other species. Such studies, and recent whole genome sequencing projects, have uncovered exceptionally large opsin gene repertoires in ray-finned fishes (taxon, Actinopterygii). Guppies and zebrafish, for example, have 10 visual opsin genes each. Here we review the duplication and divergence events that have generated these gene collections. Phylogenetic analyses revealed that large opsin gene repertories in fish have been generated by gene duplication and divergence events that span the age of the ray-finned fishes. Data from whole genome sequencing projects and from large-insert clones show that tandem duplication is the primary mode of opsin gene family expansion in fishes. In some instances gene conversion between tandem duplicates has obscured evolutionary relationships among genes and generated unique key-site haplotypes. We mapped amino acid substitutions at so-called key-sites onto phylogenies and this exposed many examples of convergence. We found that dN/dS values were higher on the branches of our trees that followed gene duplication than on branches that followed speciation events, suggesting that duplication relaxes constraints on opsin sequence evolution. Though the focus of the review is opsin sequence evolution, we also note that there are few clear connections between opsin gene repertoires and variation in spectral environment, morphological traits, or life history traits.     

Functional divergence after gene duplication and sequence-structure relationship: a case study of G-protein alpha subunits

In this article, we use animal G-protein alpha subunit family as an example to illustrate a comprehensive analytical pipeline for detecting different types of functional divergence of protein families, which is phylogeny-dependent, combined with ancestral sequence inference and available protein structure information. In particular, we focus on (i) Type-I functional divergence, or site-specific rate shift, as typically exemplified by amino acid residue highly conserved in a subset of homologous genes but highly variable in a different subset of homologous genes, and (ii) Type-II functional divergence, or the shift of cluster-specific amino acid property, as exemplified by a radical shift of amino acid property between duplicate genes, which is otherwise evolutionally conserved. We utilized the software DIVERGE2 to carry out these analyses. In the case of G-protein alpha subunit gene family, we have predicted amino acid residues that are related to either Type-I or Type-II functional divergence. The inferred ancestral sequences for these sites are helpful to explore the trends of functional divergence. Finally, these predicted residues are mapped to the protein structures to test whether these residues may have 3D structure or solvent accessibility preference.     

Sequence and structural aspects of the functional diversification of plant alcohol dehydrogenases

The glycolytic proteins in plants are coded by small multigene families, which provide an interesting contrast to the high copy number of gene families studied to date. The alcohol dehydrogenase (Adh) genes encode glycolytic enzymes that have been characterized in some plant families. Although the amino acid sequences of zinc-containing long-chain ADHs are highly conserved, the metabolic function of this enzyme is variable. They also have different patterns of expression and are submitted to differences in nonsynonymous substitution rates between gene copies. It is possible that the Adh copies have been retained as a consequence of adaptative amino acid replacements which have conferred subtle changes in function. Phylogenetic analysis indicates that there have been a number of separate duplication events within angiosperms, and that genes labeled Adh1, Adh2 and Adh3 in different groups may not be homologous. Nonsynonymous/synonymous ratios yielded no signs of positive selection. However, the coefficients of functional divergence (theta) estimated between the Adh1 and Adh2 gene groups indicate statistically significant site-specific shift of evolutionary rates between them, as well as between those of different botanical families, suggesting that altered functional constraints may have taken place at some amino acid residues after their diversification. The theoretical three-dimensional structure of the alcohol dehydrogenase from Arabis blepharophylla was constructed and verified to be stereochemically valid.     

Multiple tandem gene duplications in a neutral lipase gene cluster in Drosophila

We have examined a highly dynamic section of the Drosophila melanogaster genome which contains neutral lipase family genes that have undergone multiple tandem duplication events. We have identified the orthologous clusters, encoding between five and eight apparently functional lipases, in other Drosophila genomes: yakuba, ananassae, pseudoobscura, virilis, mojavensis, persimilis, grimshawi and willistoni. We examined their gene structure, duplication and pseudogene formation, and the presence of transposable elements. Based on phylogenetic comparisons, the lipase genes contained in each of the clusters fall into four distinct clades. Clades I and II have distinct evolutionary constraints to clades III and IV. Multiple gene duplications have occurred in different lineages of clades I and II while clades III and IV contain a single lipase gene from each species. Compared with lipases from other clades, clade IV genes contain an additional 3' domain of tandemly repeated sequence of varying length and composition, and a substitution in the residue adjacent to the key catalytic serine in the encoded proteins. A comparison of non-synonymous to synonymous nucleotide substitution (dN/dS) rates within each clade showed the highest rate of divergence was between paralogous lipase gene pairs suggesting selection pressure on duplicated genes. Analysis of the encoded lipase protein sequences within each species using PAML identified positively selected sites; structure homology modeling based on human pancreatic lipase indicated many of these residues formed part of the active site of the enzyme. As some of the cluster lipase genes are known to be expressed in the insect midgut and respond to changes in dietary components, we propose that the lipase cluster has undergone dynamic evolutionary changes to maximize absorption of lipid nutrients from the diet.