The Molecular Evolution of the Vertebrate Trypsinogens

We expand the already large number of known trypsinogen nucleotide and amino acid sequences by presenting additional trypsinogen sequences from the tunicate (Boltenia villosa), the lamprey (Petromyzon marinus), the pufferfish (Fugu rubripes), and the frog (Xenopus laevis). The current array of known trypsinogen sequences now spans the entire vertebrate phylogeny. Phylogenetic analysis is made difficult by the presence of multiple isozymes within species and rates of evolution that vary highly between both species and isozymes. We nevertheless present a Fitch-Margoliash phylogeny constructed from pairwise distances. We employ this phylogeny as a vehicle for speculation on the evolution of the trypsinogen gene family as well as the general modes of evolution of multigene families. Unique attributes of the lamprey and tunicate trypsinogens are noted. Received: 12 July 1997     

Characterization of Two Alcohol Dehydrogenase (Adh) Loci from the Olive Fruit Fly, Bactrocera (Dacus) oleae and Implications for Adh Duplication in Dipteran Insects

We report the cloning and structural characterization of two Adh loci of the olive fruit fly, Bactrocera oleae. Each of the two genes, named Adh1 and Adh2, consists of three exons and two introns for a total length of 1981 and 988 nucleotides, respectively. Their deduced amino acid sequences of 257 and 258 residues exhibit a 77% identity and display the characteristics of the insect ADH enzymes, which belong to the short-chain dehydrogenases/reductases family. The Adh genes of B. oleae are compared to the two genes of the Mediterranean fly, Ceratitis capitata, the only other species of the Tephritidae family in which the Adh genes have been studied. On the basis of amino acid divergence the four genes form two clusters each containing one gene from each species, as expected if there was one duplication event before speciation. On the basis of nucleotide sequence the four sequences form two clusters each containing the two sequences from the same species, as expected if there was a separate duplication event in each species. To help decide between the two alternatives, we compared at both the amino acid and DNA level the Adh genes of five Drosophila species that are known to carry two such genes and observed that, with only one exception at the amino acid level, conspecific loci cluster together. We conclude that the information we have at present does not allow a firm choice between the hypothesis of a single duplication event that occurred before the split of Bactrocera and Ceratitis from their common ancestor and the hypothesis of two independent duplication events, one in each of the two genera. Received: 30 May 2000 / Accepted: 17 August 2000     

Molecular Evolution of Calmodulin and Calmodulin-Like Genes in the Cephalochordate Branchiostoma

Calmodulin is a calcium-binding EF-hand protein that is an activator of many enzymes as well as ion pumps and channels. Due to its multiple targets and its central role in the cell, understanding the evolutionary history of calmodulin genes should provide insights into the origin of genetic complexity in eukaryotes. We have previously isolated and characterized a calmodulin gene from the early-diverging chordate Branchiostoma lanceolatum (CaM1). In this paper, we report the existence of a second calmodulin gene (CaM2) as well as two CaM-like genomic fragments (CaML-2, CaML-3) in B. lanceolatum and a CaM2 and three CaM-like genes (CaML-1, CaML-2, CaML-3) in B. floridae. The CaM-like genes were isolated using low-stringency PCR. Surprisingly, the nucleotide sequences of the B. lanceolatum CaM1 and CaM2 cDNAs differ by 19.3%. Moreover, the CaM2 protein differs at two positions from the amino acid sequence of CaM1; the latter is identical to calmodulins in Drosophila melanogaster, the mollusc Aplysia californica, and the tunicate Halocynthia roretzi. The two B. lanceolatum CaM-like genes are more closely related to the CaM2 than to the CaM1 gene. This relationship is supported by the phylogenetic analyses and the identical exon/intron organization of these three genes, a relationship unique among animal CaM sequences. These data demonstrate the existence of a CaM multigene family in the cephalochordate Branchiostoma, which may have evolved independently from the multigene family in vertebrates. Received: 2 November 1999 / Accepted: 25 April 2000     

Intron Dynamics and the Evolution of Integrin β-Subunit Genes: Maintenance of an Ancestral Gene Structure in the Coral, Acropora millepora

We have determined the genomic structure of an integrin β-subunit gene from the coral, Acropora millepora. The coding region of the gene contains 26 introns, spaced relatively uniformly, and this is significantly more than have been found in any integrin β-subunit genes from higher animals. Twenty-five of the 26 coral introns are also found in a β-subunit gene from at least one other phylum, indicating that the coral introns are ancestral. While there are some suggestions of intron gain or sliding, the predominant theme seen in the homologues from higher animals is extensive intron loss. The coral baseline allows one to infer that a number of introns found in only one phylum of higher animals result from frequent intron loss, as opposed to the seemingly more parsimonious alternative of isolated intron gain. The patterns of intron loss confirm results from protein sequences that most of the vertebrate genes, with the exception of β4, belong to one of two β subunit families. The similarity of the patterns within each of the β1,2,7 and β3,5,6,8 groups indicates that these gene structures have been very stable since early vertebrate evolution. Intron loss has been more extensive in the invertebrate genes, and obvious patterns have yet to emerge in this more limited data set. Received: 5 March 2001 / Accepted: 17 May 2001     

Molecular Characterization and Evolution of the Amylase Multigene Family of Drosophila ananassae

Drosophila ananassae is known to produce numerous alpha-amylase variants. We have cloned seven different Amy genes in an African strain homozygous for the AMY1,2,3,4 electrophoretic pattern. These genes are organized as two main clusters: the first one contains three intronless copies on the 2L chromosome arm, two of which are tandemly arranged. The other cluster, on the 3L arm, contains two intron-bearing copies. The amylase variants AMY1 and AMY2 have been assigned to the intronless cluster, and AMY3 and AMY4 to the second one. The divergence of coding sequences between clusters is moderate (6.1% in amino acids), but the flanking regions are very different, which could explain their differential regulation. Within each cluster, coding and noncoding regions are conserved. Two very divergent genes were also cloned, both on chromosome 3L, but very distant from each other and from the other genes. One is the Amyrel homologous (41% divergent), the second one, Amyc1 (21.6% divergent) is unknown outside the D. ananassae subgroup. These two genes have unknown functions. Received: 30 May 2000 / Accepted: 17 July 2000     

Phylogenetic Analysis of Vertebrate Lactate Dehydrogenase (LDH) Multigene Families

In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human–killifish pair than a human–lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60–70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B. Received: 5 October 2001 / Accepted: 24 October 2001     

Evolutionary Patterns of Gene Families Generated in the Early Stage of Vertebrates

In this paper we have analyzed 49 vertebrate gene families that were generated in the early stage of vertebrates and/or shortly before the origin of vertebrates, each of which consists of three or four member genes. We have dated the first (T₁) and second (T₂) gene duplications of 26 gene families with 3 member genes. The means of T₁ (594 mya) and T₂ (488 mya) are largely consistent to a well-cited version of two-round (2R) genome duplication theory. Moreover, in most cases, the time interval between two successive gene duplications is large enough that the fate of duplicate genes generated by the first gene duplication was likely to be determined before the second one took place. However, the phylogenetic pattern of 23 gene families with 4 members is complicated; only 5 of them are predicted by 2R model, but 11 families require an additional gene (or genome) duplication. For the rest (7 families), at least one gene duplication event had occurred before the divergence between vertebrate and Drosophila, indicating a possible misleading of the 4:1 rule (member gene ratio between vertebrates and invertebrates). Our results show that Ohno's 2R conjecture is valid as a working hypothesis for providing a most parsimonious explanation. Although for some gene families, additional gene duplication is needed, the credibility of the third genome duplication (3R) remains to be investigated. Received: 13 December 1999 / Accepted: 7 April 2000     

The Evolution of the Calpain Family as Reflected in Paralogous Chromosome Regions

Calpains, the Ca²⁺-dependent intracellular proteinases, are involved in the regulation of distinct cellular pathways including signal transduction and processing, cytoskeleton dynamics, and muscle homeostasis. To investigate the evolutionary origin of diverse calpain subfamilies, a phylogenetic study was carried out. The topology of the calpain phylogenetic tree has shown that some of the gene duplications occurred before the divergence of the protostome and deuterostome lineages. Other gene doublings, leading to vertebrate-specific calpain forms, took place during early chordate evolution and coincided with genome duplications as disclosed by the localization of calpain genes to paralogous chromosome regions in the human genome. On the basis of the phylogenetic tree, the time of gene duplications, and the localization of calpain genes, we propose a model of tandem and chromosome duplications for the evolution of vertebrate-specific calpain forms. The data presented here are consistent with scenarios proposed for the evolution of other multigene families. Received: 17 November 1998 / Accepted: 30 April 1999     

The Ghost of Selection Past: Rates of Evolution and Functional Divergence of Anciently Duplicated Genes

The duplication of genes and even complete genomes may be a prerequisite for major evolutionary transitions and the origin of evolutionary novelties. However, the evolutionary mechanisms of gene evolution and the origin of novel gene functions after gene duplication have been a subject of many debates. Recently, we compiled 26 groups of orthologous genes, which included one gene from human, mouse, and chicken, one or two genes from the tetraploid Xenopus and two genes from zebrafish. Comparative analysis and mapping data showed that these pairs of zebrafish genes were probably produced during a fish-specific genome duplication that occurred between 300 and 450 Mya, before the teleost radiation (Taylor et al. 2001). As discussed here, many of these retained duplicated genes code for DNA binding proteins. Different models have been developed to explain the retention of duplicated genes and in particular the subfunctionalization model of Force et al. (1999) could explain why so many developmental control genes have been retained. Other models are harder to reconcile with this particular set of duplicated genes. Most genes seem to have been subjected to strong purifying selection, keeping properties such as charge and polarity the same in both duplicates, although some evidence was found for positive Darwinian selection, in particular for Hox genes. However, since only the cumulative pattern of nucleotide substitutions can be studied, clear indications of positive Darwinian selection or neutrality may be hard to find for such anciently duplicated genes. Nevertheless, an increase in evolutionary rate in about half of the duplicated genes seems to suggest that either positive Darwinian selection has occurred or that functional constraints have been relaxed at one point in time during functional divergence. Received: 4 January 2001 / Accepted: 29 March 2001     

Circular Permutations in the Molecular Evolution of DNA Methyltransferases

Circular permutations of genes during molecular evolution often are regarded as elusive, although a simple model can explain these rearrangements. The model assumes that first a gene duplication of the precursor gene occurs in such a way that both genes become fused in frame, leading to a tandem protein. After generation of a new start codon within the 5′ part of the tandem gene and a stop at an equivalent position in the 3′ part of the gene, a protein is encoded that represents a perfect circular permutation of the precursor gene product. The model is illustrated here by the molecular evolution of adenine-N⁶ DNA methyltransferases. β- and γ-type enzymes of this family can be interconverted by a single circular permutation event. Interestingly, tandem proteins, proposed as evolutionary intermediates during circular permutation, can be directly observed in the case of adenine methyltransferases, because some enzymes belonging to type IIS, like the FokI methyltransferase, are built up by two fused enzymes, both of which are active independently of each other. The mechanism for circular permutation illustrated here is very easy and applicable to every protein. Thus, circular permutation can be regarded as a normal process in molecular evolution and a changed order of conserved amino acid motifs should not be interpreted to argue against divergent evolution. Received: 17 November 1998 / Accepted: 19 February 1999     

Molecular Evolution of the Myeloperoxidase Family

Animal myeloperoxidase and its relatives constitute a diverse protein family, which includes myeloperoxidase, eosinophil peroxidase, thyroid peroxidase, salivary peroxidase, lactoperoxidase, ovoperoxidase, peroxidasin, peroxinectin, cyclooxygenase, and others. The members of this protein family share a catalytic domain of about 500 amino acid residues in length, although some members have distinctive mosaic structures. To investigate the evolution of the protein family, we performed a comparative analysis of its members, using the amino acid sequences and the coordinate data available today. The results obtained in this study are as follows: (1) 60 amino acid sequences belonging to this family were collected by database searching. We found a new member of the myeloperoxidase family derived from a bacterium. This is the first report of a bacterial member of this family. (2) An unrooted phylogenetic tree of the family was constructed according to the alignment. Considering the branching pattern in the obtained phylogenetic tree, together with the mosaic features in the primary structures, 60 members of the myeloperoxidase family were classified into 16 subfamilies. (3) We found two molecular features that distinguish cyclooxygenase from the other members of the protein family. (4) Several structurally deviated segments were identified by a structural comparison between cyclooxygenase and myeloperoxidase. Some of the segments seemed to be associated with the functional and/or structural differences between the enzymes. Received: 25 January 2000 / Accepted: 19 July 2000     

Molecular Evolution Within the L-Malate and L-Lactate Dehydrogenase Super-Family

The NAD(P)-dependent malate (L-MalDH) and NAD-dependent lactate (L-LDH) form a large super-family that has been characterized in organisms belonging to the three domains of life. In the first part of this study, the group of [LDH-like] L-MalDH, which are malate dehydrogenases resembling lactate dehydrogenase, were analyzed and clearly defined with respect to the other enzymes. In the second part, the phylogenetic relationships of the whole super-family were presented by taking into account the [LDH-like] L-MalDH. The inferred tree unambiguously shows that two ancestral genes duplications, and not one as generally thought, are needed to explain both the distribution into two enzymatic functions and the observation of three main groups within the super-family: L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH. In addition, various cases of functional changes within each group were observed and analyzed. The direction of evolution was found to always be polarized: from enzymes with a high stringency of substrate recognition to enzymes with a broad substrate specificity. A specific phyletic distribution of the L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH over the Archaeal, Bacterial, and Eukaryal domains was observed. This was analyzed in the light of biochemical, structural, and genomic data available for the L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH. This analysis led to the elaboration of a refined evolutionary scenario of the super-family, in which the selection of L-LDH and the fate of L-MalDH during mitochrondrial genesis are presented.     

Origin and Evolution of Viral Interleukin-10 and Other DNA Virus Genes with Vertebrate Homologues

Phylogenies of gene families including members in both vertebrates and DNA viruses of the poxvirus and/or herpesvirus families showed that the viral genes originated at widely different times over the history of life. Certain of these viral genes (for example, the genes encoding the large and small subunits of ribonucleoside–diphosphate reductase) originated before animals diverged from fungi, while others originated much more recently. The most striking examples of recent origin involved viral genes encoding the cytokine interleukin-10 (IL-10), which originated independently in viruses at least three times since the divergence of the orders of eutherian mammals, presumably by viral capture of host genes. In certain domains, viral IL-10 genes showed significantly higher rates of nonsynonymous substitution than their nearest mammalian homologues. Though the mutation rate in these viral genes is up to 20 times that of the corresponding mammalian genes, a high mutation rate alone did not account for these differences because they were not seen in all domains. Rather, in certain domains it appears that functional constraints present in the case of mammalian IL-10 are relaxed in the case of the viral homologues. Furthermore, a nonrandom pattern of change with respect to amino acid residue charge in the N-terminal portion of the mature protein has occurred repeatedly in independently derived viral IL-10 genes, strongly suggesting that positive selection has led to divergence of this functionally important domain in viral IL-10. Received: 11 January 2001 / Accepted: 23 May 2001     

The Family of Major Royal Jelly Proteins and Its Evolution

A cDNA encoding a new member of the gene family of major royal jelly proteins (MRJPs) from the honeybee, Apis mellifera, was isolated and sequenced. Royal jelly (RJ) is a secretion of the cephalic glands of nurse bees. The origin and biological function of the protein component (12.5%, w/w) of RJ is unknown. We show that the MRJP gene family encodes a group of closely related proteins that share a common evolutionary origin with the yellow protein of Drosophila melanogaster. Yellow protein functions in cuticle pigmentation in D. melanogaster. The MRJPs appear to have evolved a novel nutritional function in the honeybee. Received: 26 September 1998 / Accepted: 28 February 1999     

Molecular Evolution of the Aldo-keto Reductase Gene Superfamily

The aldo-keto reductase enzymes comprise a functionally diverse gene family which catalyze the NADPH-dependant reduction of a variety of carbonyl compounds. The protein sequences of 45 members of this family were aligned and phylogenetic trees were deduced from this alignment using the neighbor-joining and Fitch algorithms. The branching order of these trees indicates that the vertebrate enzymes cluster in three groups, which have a monophyletic origin distinct from the bacterial, plant, and invertebrate enzymes. A high level of conservation was observed between the vertebrate hydroxysteroid dehydrogenase enzymes, prostaglandin F synthase, and ρ-crystallin of Xenopus laevis. We infer from the phylogenetic analysis that prostaglandin F synthase may represent a recent recruit to the eicosanoid biosynthetic pathway from the hydroxysteroid dehydrogenase pathway and furthermore that, in the context of gene recruitment, Xenopus laevisρ-crystallin may represent a shared gene. Received: 26 August 1996 / Accepted: 5 June 1997     

Diversification Pattern of the HMG and SOX Family Members During Evolution

From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity. Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used. This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA. The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages. Particular emphasis was placed on the analysis of the SOX subfamily. For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18. The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box. In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family. The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG. The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans. Received: 20 July 1998 / Accepted: 19 October 1998     

Sequential Loss of Two Neighboring Exons of the Tropoelastin Gene During Primate Evolution

Previous evidence has demonstrated the absence of exons 34 and 35 within the 3′ end of the human tropoelastin (ELN) gene. These exons encode conserved polypeptide domains within tropoelastin and are found in the ELN gene in vertebrate species ranging from chickens to rats to cows. We have analyzed the ELN gene in a variety of primate species to determine whether the absence of exons 34 and 35 in humans either is due to allelic variation within the human population or is a general characteristic of the Primates order. An analysis of the 3′ end of the ELN gene in several nonhuman primates and in 546 chromosomes from humans of varying ethnic background demonstrated a sequential loss of exons 34 and 35 during primate evolution. The loss of exon 35 occurred at least 35–45 million years ago, when Catarrhines diverged from Platyrrhines (New World monkeys). Exon 34 loss, in contrast, occurred only about 6–8 million years ago, when Homo separated from the common ancestor shared with chimpanzees and gorillas. Loss of both exons was probably facilitated by Alu-mediated recombination events and possibly conferred a functional evolutionary advantage in elastic tissue. Received: 6 July 1998 / Accepted: 18 February 1999     

Plasticity of Fitness and Diversification Process During an Experimental Molecular Evolution

A simplified experimental evolution encompassing the essence of natural one was designed in an attempt to understand the involved mechanism. In our system, molecular evolution was observed through three serial cycles of consecutive random mutagenesis of the glutamine synthetase gene and chemostat culture of the transformed Escherichia coli cells containing the mutated genes. Selection pressure was imposed solely on the glutamine synthetase gene when varieties of mutant genes compete in an unstructured environment of the chemostat. The molecular phylogeny and population dynamics were deduced from the nucleotide sequences of the genes isolated from each of the chemostat runs. An initial mutant population in each cycle, comprised of diversified closely-related genes, ended up with several varieties of mutants in a state of coexistence. Competition between two mutant genes in the final population of the first cycle ascertained that the observed coexisting state is not an incidental event and that cellular interaction via environmental nutrients is a possible mechanism of coexistence. In addition, the mutant gene once extinct in the previous passage was found to have the capacity to reinvade and constitute the gene pool of the later cycle of molecular evolution. These results, including the kinetic characteristics of the purified wild-type and mutant glutamine synthetases in the phylogenetic tree, revealed that the enzyme activity had diverged, rather than optimized, to a fittest value during the course of evolution. Here, we proposed that the plasticity of gene fitness in consequence of cellular interaction via the environment is an essential mechanism governing molecular evolution. Received: 29 August 2000 / Accepted: 25 January 2001     

Phylogenies of Developmentally Important Proteins Do Not Support the Hypothesis of Two Rounds of Genome Duplication Early in Vertebrate History

It has been proposed that two rounds of duplication of the entire genome (polyploidization) occurred early in vertebrate history (the 2R hypothesis); and the observation that certain gene families important in regulating development have four members in vertebrates, as opposed to one in Drosophila, has been adduced as evidence in support of this hypothesis. However, such a pattern of relationship can be taken as support of the 2R hypothesis only if (1) the four vertebrate genes can be shown to have diverged after the origin of vertebrates, and (2) the phylogeny of the four vertebrate genes (A–D) exhibits a topology of the form (AB) (CD), rather than (A) (BCD). In order to test the 2R hypothesis, I constructed phylogenies for nine protein families important in development. Only one showed a topology of the form (AB) (CD), and that received weak statistical support. In contrast, four phylogenies showed topologies of the form (A) (BCD) with statistically significant support. Furthermore, in two cases there was significant support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes: in one case there was significant support for duplication of the vertebrate genes at least prior to the divergence of vertebrates and urochordates, and in one case there was weak support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes. Taken together with other recently published phylogenies of developmentally important genes, these results provide strong evidence against the 2R hypothesis. Received: 22 December 1997 / Accepted: 5 October 1998