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     

Characterization of a Novel Class of Interspersed LTR Elements in Primate Genomes: Structure, Genomic Distribution, and Evolution

Retrovirus-like sequences and their solitary (solo) long terminal repeats (LTRs) are common repetitive elements in eukaryotic genomes. We reported previously that the tandemly arrayed genes encoding U2 snRNA (the RNU2 locus) in humans and apes contain a solo LTR (U2-LTR) which was presumably generated by homologous recombination between the two LTRs of an ancestral provirus that is retained in the orthologous baboon RNU2 locus. We have now sequenced the orthologous U2-LTRs in human, chimpanzee, gorilla, orangutan, and baboon and examined numerous homologs of the U2-LTR that are dispersed throughout the human genome. Although these U2-LTR homologs have been collectively referred to as LTR13 in the literature, they do not display sequence similarity to any known retroviral LTRs; however, the structure of LTR13 closely resembles that of other retroviral LTRs with a putative promoter, polyadenylation signal, and a tandemly repeated 53-bp enhancer-like element. Genomic blotting indicates that LTR13 is primate-specific; based on sequence analysis, we estimate there are about 2,500 LTR13 elements in the human genome. Comparison of the primate U2-LTR sequences suggests that the homologous recombination event that gave rise to the solo U2-LTR occurred soon after insertion of the ancestral provirus into the ancestral U2 tandem array. Phylogenetic analysis of the LTR13 family confirms that it is diverse, but the orthologous U2-LTRs form a coherent group in which chimpanzee is closest to the humans; orangutan is a clear outgroup of human, chimpanzee, and gorilla; and baboon is a distant relative of human, chimpanzee, gorilla, and orangutan. We compare the LTR13 family with other known LTRs and consider whether these LTRs might play a role in concerted evolution of the primate RNU2 locus. Received: 29 September 1997 / Accepted: 16 January 1998     

Molecular Timing of Primate Divergences as Estimated by Two Nonprimate Calibration Points

The complete mitochondrial DNA (mtDNA) molecule of the hamadryas baboon, Papio hamadryas, was sequenced and included in a molecular analysis of 24 complete mammalian mtDNAs. The particular aim of the study was to time the divergence between Cercopithecoidea and Hominoidea. That divergence, set at 30 million years before present (MYBP) was a fundamental reference for the original proposal of recent hominoid divergences, according to which the split among gorilla, chimpanzee, and Homo took place 5 MYBP. In the present study the validity of the postulated 30 MYBP dating of the Cercopithecoidea/Hominoidea divergence was examined by applying two independent nonprimate molecular references, the divergence between artiodactyls and cetaceans set at 60 MYBP and that between Equidae and Rhinocerotidae set at 50 MYBP. After calibration for differences in evolutionary rates, application of the two references suggested that the Cercopithecoidea/Hominoidea divergence took place >50 MYBP. Consistent with the marked shift in the dating of the Cercopithecoidea/Hominoidea split, all hominoid divergences receive a much earlier dating. Thus the estimated date of the divergence between Pan (chimpanzee) and Homo is 10–13 MYBP and that between Gorilla and the Pan/Homo linage ≈17 MYBP. The same datings were obtained in an analysis of clocklike evolving genes. The findings show that recalculation is necessary of all molecular datings based directly or indirectly on a Cercopithecoidea/Hominoidea split 30 MYBP. Received: 1 April 1998 / Accepted: 1 July 1998     

Interspecific Comparison in the Frequency of Concerted Evolution at the Polyubiquitin Gene Locus

The polyubiquitin gene, encoding tandemly repeated multiple ubiquitins, constitutes a uniquitin gene subfamily. It has been demonstrated that polyubiquitin genes are subject to concerted evolution; namely, the individual ubiquitin coding units contained within a polyubiquitin gene are more similar to one another than they are to the ubiquitin coding units in the orthologous gene from other species. However there has been no comprehensive study on the concerted evolution of polyubiquitin genes in a wide range of species, because the relationships (orthologous or paralogous) among multiple polyubiquitin genes from different species have not been extensively analyzed yet. In this report, we present the results of analyzing the nucleotide sequence of polyubiquitin genes of mammals, available in the DDBJ/EMBL/GenBank nucleotide sequence databases, in which we found that there are two groups of polyubiquitin genes in an orthologous relationship. Based on this result, we analyzed the concerted evolution of the polyubiquitin gene in various species and compared the frequency of concerted evolutionary events interspecifically by taking into consideration that the rate of synonymous substitution at the polyubiquitin gene locus may vary depending on species. We found that the concerted evolutionary events in polyubiquitin genes have been more frequent in rats and Chinese hamsters than those in humans, cows, and sheep. The guinea pig polyubiquitin gene was an intermediate example. The frequency of concerted evolution in the mouse gene was unexpectedly low compared to that of other rodent genes. Received: 18 January 2000 / Accepted: 26 April 2000     

Ascidian and Amphioxus Adh Genes Correlate Functional and Molecular Features of the ADH Family Expansion During Vertebrate Evolution

The alcohol dehydrogenase (ADH) family has evolved into at least eight ADH classes during vertebrate evolution. We have characterized three prevertebrate forms of the parent enzyme of this family, including one from an urochordate (Ciona intestinalis) and two from cephalochordates (Branchiostoma floridae and Branchiostoma lanceolatum). An evolutionary analysis of the family was performed gathering data from protein and gene structures, exon–intron distribution, and functional features through chordate lines. Our data strongly support that the ADH family expansion occurred 500 million years ago, after the cephalochordate/vertebrate split, probably in the gnathostome subphylum line of the vertebrates. Evolutionary rates differ between the ancestral, ADH3 (glutathione-dependent formaldehyde dehydrogenase), and the emerging forms, including the classical alcohol dehydrogenase, ADH1, which has an evolutionary rate 3.6-fold that of the ADH3 form. Phylogenetic analysis and chromosomal mapping of the vertebrate Adh gene cluster suggest that family expansion took place by tandem duplications, probably concurrent with the extensive isoform burst observed before the fish/tetrapode split, rather than through the large-scale genome duplications also postulated in early vertebrate evolution. The absence of multifunctionality in lower chordate ADHs and the structures compared argue in favor of the acquisition of new functions in vertebrate ADH classes. Finally, comparison between B. floridae and B. lanceolatum Adhs provides the first estimate for a cephalochordate speciation, 190 million years ago, probably concomitant with the beginning of the drifting of major land masses from the Pangea. Received: 10 April 2001 / Accepted: 23 May 2001     

Gene Conversions May Obscure Actin Gene Family Relationships

Phylogenetic hypotheses of muscle actin evolution are significantly different when a sea urchin is used as a representative echinoderm than when a sea star is used. While sea urchin muscle actins support an echinoderm–chordate sister relationship, sea star sequences suggest that echinoderm muscle actins are convergent with chordate muscle actins. Our results suggest that gene conversion in the sea star muscle actin may be responsible for these discordant results. Received: 19 July 1999 / Accepted: 1 October 1999     

Characteristic Sequence Pattern in the 5- to 20-bp Upstream Region of Primate Alu Elements

We conducted comprehensive sequence analysis of 5′ flanking regions of primate Alu elements. Information contents were computed and frequencies of 1024 pentanucleotides were measured to approximate the location of a characteristic sequence and to specify its pattern(s), which may be involved in the integration of Alu elements into their host genomes. A large number of samples was used, the wide region of the 5′ end of Alu elements was analyzed, and comparisons were made among different subfamilies. Through our analyses, ``TTTTAAAAA' or ``(T) _m (A) _n ' can be stated as a candidate for the characteristic sequence pattern, which resides around the region 5 to 20 base pairs upstream of the 5′ end of Alu elements. This characteristic sequence pattern was more prominent in the sequences of younger Alus, which is a strong indication that the sequence pattern has a role at the time of Alu integration. Received: 10 May 1999 / Accepted: 1 October 1999     

Unexpected Conservation of the X-Linked Color Vision Gene in Nocturnal Prosimians: Evidence from Two Bush Babies

Bush babies have had a long history of nocturnal life and it would be interesting to know whether their color vision genes have become degenerate. Therefore, we used PCR techniques to sequence the X-linked pigment gene of two of these nocturnal prosimians: Galago senegalensis and Otolemur garnettii. Southern hybridization of genomic DNA of G. senegalensis showed a single X-linked pigment gene. Interestingly, the deduced pigment sequences of the two bush babies are identical. By comparing the X-linked pigments of bush baby, human, squirrel monkey, and marmoset, 38 variable positions were identified. At those positions that may cause a spectral shift, the bush baby pigment has identical or biochemically similar residues to those of the marmoset cone pigment with a spectral peak of 543 nm. This result is consistent with the estimate of 544–545 nm for the spectral peak of the X-linked pigment of Otolemur crassicaudatus, which is closely related to Otolemur garnettii. The neighbor-joining tree of mammalian X-linked pigments showed a significantly shorter branch in the bush baby lineage than in other primate lineages. A relative rate test showed that the nonsynonymous substitution rate of the bush baby X-linked pigment gene is about three times slower than that of the human red pigment gene, though the synonymous substitution rates of the two genes are similar. The slower nonsynonymous rate in the bush baby lineage suggests that the bush baby X-linked pigment gene is under functional constraints, in spite of its nocturnal life. Two radical changes at positions in the intradiskal surface next to the sixth transmembrane domain were observed in the X-linked cone pigment of bush babies but not in other primates. They are changes from Ala to Ser and from Asn to His, which are similar in function to the corresponding residues in rhodopsins. These two changes may be of importance for dim light sensitivity, which is consistent with our proposal that the evolution of the bush baby X-linked pigment gene is under selective pressure. In addition, the 2.5% divergence in introns 2 and 5 of the X-linked pigment gene between the two bush babies supports their classification into two separate genera. Received: 30 November 1996 / Accepted: 17 June 1997     

The Rates of Molecular Evolution in Rodent and Primate Mitochondrial DNA

A higher rate of molecular evolution in rodents than in primates at synonymous sites and, to a lesser extent, at amino acid replacement sites has been reported previously for most nuclear genes examined. Thus in these genes the average ratio of amino acid replacement to synonymous substitution rates in rodents is lower than in primates, an observation at odds with the neutral model of molecular evolution. Under Ohta's mildly deleterious model of molecular evolution, these observations are seen as the consequence of the combined effects of a shorter generation time (driving a higher mutation rate) and a larger effective population size (resulting in more effective selection against mildly deleterious mutations) in rodents. The present study reports the results of a maximum-likelihood analysis of the ratio of amino acid replacements to synonymous substitutions for genes encoded in mitochondrial DNA (mtDNA) in these two lineages. A similar pattern is observed: in rodents this ratio is significantly lower than in primates, again consistent only with the mildly deleterious model. Interestingly the lineage-specific difference is much more pronounced in mtDNA-encoded than in nuclear-encoded proteins, an observation which is shown to run counter to expectation under Ohta's model. Finally, accepting certain fossil divergence dates, the lineage-specific difference in amino acid replacement-to-synonymous substitution ratio in mtDNA can be partitioned and is found to be entirely the consequence of a higher mutation rate in rodents. This conclusion is consistent with a replication-dependent model of mutation in mtDNA. Received: 24 September 1999 / Accepted: 18 September 2000     

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     

Rapid Concerted Evolution via Gene Conversion at the <Emphasis Type="BoldItalic">Drosophila hsp70</Emphasis> Genes

We analyzed nucleotide variation in the hsp70 genes of Drosophila melanogaster (five genes) and D. simulans (four genes) to characterize the homogenizing and diversifying roles of gene conversion in their evolution. Gene conversion within and between the 87A7 and 87C1 gene clusters homogenize the hsp70 coding regions; in both D. melanogaster and D. simulans, same-cluster paralogues are virtually identical, and large intercluster conversion tracts diminish 87A7/87C1 divergence. Same-cluster paralogues share many polymorphisms, consistent with frequent intracluster conversion. Shared polymorphism is highly biased toward silent variation; homogenizing conversion interacts with purifying selection. In contrast to the coding regions, some hsp70 flanking regions show conversion-mediated diversification. Strong reductions of nucleotide variability and linkage disequilibria among conversion-mediated sites in hsp70Ab and hsp70Bb alleles sampled from a single natural population are consistent with a selective sweep. Comparison of the D. melanogaster and D. simulans hsp70 genes reveals whole-family fixed differences, consistent with rapid propagation of novel mutations among duplicate genes. These results suggest that the homogenizing and diversifying roles of conversion interact to drive dynamic concerted evolution of the hsp70 genes. Received: 25 June 2001 / Accepted: 10 October 2001     

SINE Retrotransposition During the Evolution of the Pecoran Ruminants

SINE retrotransposition events have proven their value as phylogenetic markers in several eukaryotic taxa at different taxonomic levels. The genomes of ruminants contain three related SINE elements, Bov-tA, Bov-A2, and Bov-B. To estimate the time points of retrotransposition of individual copies of these SINEs, we designed PCR primers on database sequences containing SINE insertions in cattle, sheep, or goat genomes and tested for the presence of these copies in the genomes of other ruminants. It was checked by sequencing whether length variation of the PCR products reflected a SINE retrotransposition. One Bov-B and nine Bov-tA insertions were shared by cattle, sheep, goat, and giraffe, indicating an early retrotransposition event before the radiation of the Pecora, while three other Bov-tA and two Bov-B elements were apparently inserted later. The ruminant α-lactalbumine gene contains a hotspot of early and more recent Bov-tA insertions, a Bov-tA replacement as well as a recent Bov-B insertion. Three Bov-A2 insertions were found to be shared only by the Bovidae, the Bovini, and the Bos and Bison species, respectively, indicating that most Bov-A2 insertions are relatively recent. The time elapsed since the retrotransposition was also reflected in the degeneration of the direct repeats that flank SINE inserts. We suggest that retrotransposition of SINEs may serve as phylogenetic markers in the ruminant families, subfamilies, and even tribes. In addition, sequencing of SINE insertions revealed several other unique deletions/insertions that also may be informative for phylogenetic reconstructions of ruminants. Received: 19 January 2001 / Accepted: 6 June 2001     

Gene Conversion and Evolution of Daphniid Hemoglobins (Crustacea, Cladocera)

The extracellular hemoglobins of cladocerans derive from the aggregation of 12 two-domain globin subunits that are apparently encoded by four genes. This study establishes that at least some of these genes occur as a tandem array in both Daphnia magna and Daphnia exilis. The genes share a uniform structure; a bridge intron separates two globin domains which each include three exons and two introns. Introns are small, averaging just 77 bp, but a longer sequence (2.2–3.2 kb) separates adjacent globin genes. A survey of structural diversity in globin genes from other daphniids revealed three independent cases of intron loss, but exon lengths were identical, excepting a 3-bp insertion in exon 5 of Simocephalus. Heterogeneity in the extent of nucleotide divergence was marked among exons, largely as a result of the pronounced diversification of the terminal exon. This variation reflected, in part, varying exposure to concerted evolution. Conversion events were frequent in exons 1–4 but were absent from exons 5 and 6. Because of this difference, the results of phylogenetic analyses were strongly affected by the sequences employed in this construction. Phylogenies based on total nucleotide divergence in exons 1–4 revealed affinities among all genes isolated from a single species, reflecting the impact of gene conversion events. In contrast, phylogenies based on total nucleotide divergence in exons 5 and 6 revealed affinities among orthologous genes from different taxa. Received: 8 March 1999 / Accepted: 14 July 1999     

Evolution of the nad3-rps12 Gene Cluster in Angiosperm Mitochondria: Comparison of Edited and Unedited Sequences

We have analyzed the nad3-rps12 locus for eight angiosperms in order to compare the utility of mitochondrial DNA and edited mRNA sequences in phylogenetic reconstruction. The two coding regions, containing from 25 to 35 editing sites in the various plants, have been concatenated in order to increase the significance of the analysis. Differing from the corresponding chloroplast sequences, unedited mitochondrial DNA sequences seem to evolve under a quasi-neutral substitution process which undifferentiates the nucleotide substitution rates for the three codon positions. By using complete gene sequences (all codon positions) we found that genomic sequences provide a classical angiosperm phylogenetic tree with a clear-cut grouping of monocotyledons and dicotyledons with Magnoliidae at the basal branch of the tree. Conversely, owing to their low nucleotide substitution rates, edited mRNA sequences were found not to be suitable for studying phylogenetic relationships among angiosperms. Received: 24 January 1996 / Accepted: 5 June 1996     

Evolution of Orthologous Intronless and Intron-Bearing Globin Genes in Two Insect Species

While globin genes ctt-2β and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2β and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species. Received: 31 December 1996 / Accepted: 16 May 1997     

Gene Conversion Drives Within Genic Sequences: Concerted Evolution of Ribosomal RNA Genes in Bacteria and Archaea

Multiple copies of a given ribosomal RNA gene family undergo concerted evolution such that sequences of all gene copies are virtually identical within a species although they diverge normally between species. In eukaryotes, gene conversion and unequal crossing over are the proposed mechanisms for concerted evolution of tandemly repeated sequences, whereas dispersed genes are homogenized by gene conversion. However, the homogenization mechanisms for multiple-copy, normally dispersed, prokaryotic rRNA genes are not well understood. Here we compared the sequences of multiple paralogous rRNA genes within a genome in 12 prokaryotic organisms that have multiple copies of the rRNA genes. Within a genome, putative sequence conversion tracts were found throughout the entire length of each individual rRNA genes and their immediate flanks. Individual conversion events convert only a short sequence tract, and the conversion partners can be any paralogous genes within the genome. Interestingly, the genic sequences undergo much slower divergence than their flanking sequences. Moreover, genomic context and operon organization do not affect rRNA gene homogenization. Thus, gene conversion underlies concerted evolution of bacterial rRNA genes, which normally occurs within genic sequences, and homogenization of flanking regions may result from co-conversion with the genic sequence. Received: 31 March 2000 / Accepted: 15 June 2000