OrthoPhy: A Program to Construct Ortholog Data Sets Using Taxonomic Information.

Species phylogenetic trees represent the evolutionary processes of organisms, and they are fundamental in evolutionary research. Therefore, new methods have been developed to obtain more reliable species phylogenetic trees. A highly reliable method is the construction of an ortholog data set based on sequence information of genes, which is then used to infer the species phylogenetic tree. However, although methods for constructing an ortholog data set for species phylogenetic analysis have been developed, they cannot remove some paralogs, which is necessary for reliable species phylogenetic inference. To address the limitations of current methods, we developed OrthoPhy, a program that excludes paralogs and constructs highly accurate ortholog data sets using taxonomic information dividing analyzed species into monophyletic groups. OrthoPhy can remove paralogs, detecting inconsistencies between taxonomic information and phylogenetic trees of candidate ortholog groups clustered by sequence similarity. Performance tests using evolutionary simulated sequences and real sequences of 40 bacteria revealed that the precision of ortholog inference by OrthoPhy is higher than that of existing programs. Additionally, the phylogenetic analysis of species was more accurate when performed using ortholog data sets constructed by OrthoPhy than that performed using data sets constructed by existing programs. Furthermore, we performed a benchmark test of the Quest for Orthologs using real sequence data and found that the concordance rate between the phylogenetic trees of orthologs inferred by OrthoPhy and those of species was higher than the rates obtained by other ortholog inference programs. Therefore, ortholog data sets constructed using OrthoPhy enabled a more accurate phylogenetic analysis of species than those constructed using the existing programs, and OrthoPhy can be used for the phylogenetic analysis of species even for distantly related species that have experienced many evolutionary events.

The Evolution of Molybdenum Dependent Nitrogenase in Cyanobacteria.

Nitrogen fixation plays a crucial role in the nitrogen cycle by helping to convert nitrogen into a form usable by other organisms. Bacteria capable of fixing nitrogen are found in six phyla including Cyanobacteria. Molybdenum dependent nitrogenase (nif) genes are thought to share a single origin as they have homologs in various phyla. However, diazotrophic bacteria have a mosaic distribution within the cyanobacterial lineage. Therefore, the aim of this study was to determine the cause of this mosaic distribution. We identified nif gene operon structures in the genomes of 85 of the 179 cyanobacterial strains for which whole genome sequences were available. Four nif operons were conserved in each diazotroph Cyanobacterium, although there were some gene translocations and insertions. Phylogenetic inference of these genes did not reveal horizontal gene transfer from outside the phylum Cyanobacteria. These results support the hypothesis that the mosaic distribution of diazotrophic bacteria in the cyanobacterial lineage is the result of the independent loss of nif genes inherited from common cyanobacterial ancestors in each lineage.

Chronology of emergence of the genus Leptospira and over-representation of gene families enriched by vitamin B2, B12 biosynthesis, cell adhesion and external encapsulating structure in L. interrogans isolates from asymptomatic dogs.

The spirochete species Leptospira interrogans is the most common cause of leptospirosis, producing acute to chronic infections in most mammalian species. This pathogenic bacterium has an ability to evolve in many ways to occupy various environments and hosts. In this study, we performed chronology analysis to look for insight into the emergence of Leptospira species, focusing on L. interrogans, and investigated gene gain and loss related to their adaptation in strains isolated from asymptomatic dogs. Chronology analysis revealed that the emergence of L. interrogans was around 53.7 million years ago (MYA), corresponding to the Paleogene period that coincided with an optimal climatic temperature and the evolution of suitable mammalian hosts. Gene families encoding for vitamin B2, B12 biosynthesis, cell adhesion and external encapsulating structure were found to be enriched in L. interrogans isolated from the urine of asymptomatic dogs. The activity of these gene families may have favored adaptations resulting in chronic infections.

Evolutionary Analysis of the Highly Conserved Insect Odorant Coreceptor (Orco) Revealed a Positive Selection Mode, Implying Functional Flexibility.

Odorant coreceptor (Orco) represents one of the essential genes in the insect olfactory system, which facilitates signal transduction and heterodimerization with different odorant receptors (Ors) in the insect antennal dendritic membrane. Evolutionary analysis by detecting positive selection is important to examine the functional flexibility of Orco that potentially supports insect survival. The maximum likelihood codon substitution model was applied using CODEML program as implemented in PAML ver 4.9e package across 59 Orco codon sequences available from GenBank. These sequences represented five major insect orders and two reproductive systems (holometabola and nonholometabola). In the site model that identified common ω values for Orco, it was clearly shown that Orco was under strong purifying selection, indicated by the ω value that was far from 1 (ω: 0.03). However, in to the branch model, positive selection was detected to be acting on Dipteran lineages, whereas in the branch-site model, several sites were under significant positive selection occurring in the following four clades: Coleoptera, Diptera, Lepidoptera, and Psocodea. The typical evolutionary mode acting on Orco was consistent with the entropy value [H(x)], confirming that 48.9% of the Orco site was under conservation (H(x) < 0.5), whereas 26.9% of the Orco sites was under high variation (H(x) > 1). These findings confirmed that Orco genes are generally highly conserved and can possibly be used for the manipulation of insect pest control programs. However, positive selection that acts on certain lineages suggested future adaptive evolutionary ability of Orco to anticipate flexible functions for successful olfactory processes.

Genome Sequence of Pectobacterium carotovorum Phage PPWS1, Isolated from Japanese Horseradish [Eutrema japonicum (Miq.) Koidz] Showing Soft-Rot Symptoms.

ITALIC! Pectobacterium carotovorumsubsp. ITALIC! carotovorumand its lytic bacteriophage PPWS1 were isolated from a Japanese horseradish rhizome with soft rot. Sequencing of the phage genomic DNA suggested that PPWS1 is a new species of the family ITALIC! Podoviridaeand has high similarity to the bacteriophage Peat1 infectious to ITALIC! P. atrosepticum.

Ortholog-Finder: A Tool for Constructing an Ortholog Data Set.

Orthologs are widely used for phylogenetic analysis of species; however, identifying genuine orthologs among distantly related species is challenging, because genes obtained through horizontal gene transfer (HGT) and out-paralogs derived from gene duplication before speciation are often present among the predicted orthologs. We developed a program, "Ortholog-Finder," to obtain ortholog data sets for performing phylogenetic analysis by using all open-reading frame data of species. The program includes five processes for minimizing the effects of HGT and out-paralogs in phylogeny construction: 1) HGT filtering: Genes derived from HGT could be detected and deleted from the initial sequence data set by examining their base compositions. 2) Out-paralog filtering: Out-paralogs are detected and deleted from the data set based on sequence similarity. 3) Classification of phylogenetic trees: Phylogenetic trees generated for ortholog candidates are classified as monophyletic or polyphyletic trees. 4) Tree splitting: Polyphyletic trees are bisected to obtain monophyletic trees and remove HGT genes and out-paralogs. 5) Threshold changing: Out-paralogs are further excluded from the data set based on the difference in the similarity scores of genuine orthologs and out-paralogs. We examined how out-paralogs and HGTs affected phylogenetic trees constructed for species based on ortholog data sets obtained by Ortholog-Finder with the use of simulation data, and we determined the effects of confounding factors. We then used Ortholog-Finder in phylogeny construction for 12 Gram-positive bacteria from two phyla and validated each node of the constructed tree by comparison with individually constructed ortholog trees.

HGT-Gen: a tool for generating a phylogenetic tree with horizontal gene transfer.

UNLABELLED: Horizontal gene transfer (HGT) is a common event in prokaryotic evolution. Therefore, it is very important to consider HGT in the study of molecular evolution of prokaryotes. This is true also for conducting computer simulations of their molecular phylogeny because HGT is known to be a serious disturbing factor for estimating their correct phylogeny. To the best of our knowledge, no existing computer program has generated a phylogenetic tree with HGT from an original phylogenetic tree. We developed a program called HGT-Gen that generates a phylogenetic tree with HGT on the basis of an original phylogenetic tree of a protein or gene. HGT-Gen converts an operational taxonomic unit or a clade from one place to another in a given phylogenetic tree. We have also devised an algorithm to compute the average length between any pair of branches in the tree. It defines and computes the relative evolutionary time to normalize evolutionary time for each lineage. The algorithm can generate an HGT between a pair of donor and acceptor lineages at the same evolutionary time. HGT-Gen is used with a sequence-generating program to evaluate the influence of HGT on the molecular phylogeny of prokaryotes in a computer simulation study. AVAILABILITY: The database is available for free at http://www.grl.shizuoka.ac.jp/˜thoriike/HGT-Gen.html.

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip.

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

Phylogenetic construction of 17 bacterial phyla by new method and carefully selected orthologs.

Here, we constructed a phylogenetic tree of 17 bacterial phyla covering eubacteria and archaea by using a new method and 102 carefully selected orthologs from their genomes. One of the serious disturbing factors in phylogeny construction is the existence of out-paralogs that cannot easily be found out and discarded. In our method, out-paralogs are detected and removed by constructing a phylogenetic tree of the genes in question and examining the clustered genes in the tree. We also developed a method for comparing two tree topologies or shapes, ComTree. Applying ComTree to the constructed tree we computed the relative number of orthologs that support a node of the tree. This number is called the Positive Ortholog Ratio (POR), which is conceptually and methodologically different from the frequently used bootstrap value. Our study concretely shows drawbacks of the bootstrap test. Our result of bacterial phylogeny analysis is consistent with previous ones showing that hyperthermophilic bacteria such as Thermotogae and Aquificae diverged earlier than the others in the eubacterial phylogeny studied. It is noted that our results are consistent whether thermophilic archaea or mesophilic archaea is employed for determining the root of the tree. The earliest divergence of hyperthermophilic eubacteria is supported by genes involved in fundamental metabolic processes such as glycolysis, nucleotide and amino acid syntheses.

Extensive analysis of EST sequences reveals that all cichlid species in Lake Victoria share almost identical transcript sets.

Lake Victoria harbors hundreds of cichlid species, which have aggressively developed their morphological features through their evolution. In particular, the shapes of jaws and teeth have diverged markedly, and correlate with feeding ecology. These species are believed to have explosively arisen within the last 15,000 years and are therefore of particular interest to evolutionary biologists. Previously, we reported expressed sequence tags (ESTs) from two cichlid species, Haplochromis chilotes and H. sp. "redtailsheller", and provided a comparative EST analysis. In this study, we further examined the molecular basis for diversification of Lake Victoria cichlids by generating EST from another cichlid species, H. sp. "Matumbi hunter"; 9,219 clones were applied for sequence determination and 16,795 sequences were newly obtained from this EST set. Comparative analysis of the 68 genes common to the three cichlid species revealed that the degrees of genetic distance and the degrees of genetic polymorphism were highly similar for each pair-wise comparison, suggesting that the protein coding regions of the three cichlid genomes are essentially the same. Because the three species used for the comparative analysis were particularly diverged from each other in morphological and also ecological features, we concluded that the uniformity of transcript sequences among cichlid species in Lake Victoria is a global feature.

Comprehensive analysis of the origin of eukaryotic genomes.

There is currently no consensus on the evolutionary origin of eukaryotes. In the search of the ancestors of eukaryotes, we analyzed the phylogeny of 46 genomes, including those of 2 eukaryotes, 8 archaea, and 36 eubacteria. To avoid the effects of gene duplications, we used inparalog pairs of genes with orthologous relationships. First, we grouped these inparalogs into the functional categories of the nucleus, cytoplasm, and mitochondria. Next, we counted the sister groups of eukaryotes in prokaryotic phyla and plotted them on a standard phylogenetic tree. Finally, we used Pearson's chi-square test to estimate the origin of the genomes from specific prokaryotic ancestors. The results suggest the eukaryotic nuclear genome descends from an archaea that was neither euryarchaeota nor crenarchaeota and that the mitochondrial genome descends from alpha-proteobacteria. In contrast, genes related to the cytoplasm do not appear to originate from a specific group of prokaryotes.

Determination of whole prokaryotic phylogeny by the development of a random extraction method.

The construction of accurate prokaryotic phylogeny is important not only in the field of evolutionary biology, but also in microbiology and pathology. However, in constructing a phylogenetic tree to trace prokaryotic evolution, the phylogenetic relationship is often changed by the choice of species. For the estimation of the accurate lineage of prokaryotes, a new method, named the "random extraction method", was developed. In this method, 16S rRNA sequence data were randomly extracted 1000 times from each closely-related taxa such as seven phyla of Eubacteria and one domain of Archaea and phylogenetic trees were constructed by the data to clarify the relationship of those groups. Next, the tree topology was counted and the most supported tree topology was found as the most plausible phylogenetic tree. To evaluate the reliability of each node, we developed the "Branching rate" (BR) and calculated for every tree. And also, computational simulation analysis was carried out to confirm these methods. On the assumption that the root of life is between Archaea and Eubacteria, the obtained phylogenetic relationships of phyla are the following. At first, Archaea (Euryarchaeota, Crenarchaeota and Korarchaeota) diverged, and Thermotogales, Cyanobacteria and Chlamydiales diverged in this order, then Firmicutes (Actinobacteria and Bacillus/Clostridium group cluster) and Proteobacteria (alpha and beta/gamma cluster) diverged. In addition, it was shown by the BR that the position of the node of Firmicutes Actinobacteria and Firmicutes Bacillus/Clostridium was changeable for each extraction. Therefore, it was suggested that the differences among the phylogenetic trees of prokaryotes were caused by the influence of these phyla.

Extensive analysis of ORF sequences from two different cichlid species in Lake Victoria provides molecular evidence for a recent radiation event of the Victoria species flock: identity of EST sequences between Haplochromis chilotes and Haplochromis sp. "Redtailsheller".

The Lake Victoria Cichlid fishes have diverged very rapidly. The estimated 500 species inhabiting the lake are believed to have arisen within the last 14,000 years. The fishes' jaws and teeth have diverged markedly to adapt to different feeding behaviors and environments. To examine how the genomes of these fishes differentiated during speciation, we performed comparative analysis of expressed sequenced tag (EST) sequences. We constructed cDNA libraries derived only from the jaw portions of two cichlid species endemic to Lake Victoria. We sequenced 17,280 cDNA clones from Haplochromis chilotes and 9600 cDNA clones from Haplochromis sp. "Redtailsheller" and obtained 543 different genes common to both species. Of these genes, 441 were essentially identical between species and 102 contained base replacements in their open reading frame (ORF) or untranslated (UTR) regions. Comparative analysis of 71 selected sequences has revealed that while the degree of polymorphism is 0.0054/site for H. chilotes and 0.0047/site for H. sp. "Redtailsheller", genetic distance between the two species is 0.0031/site. The genetic distance particularly indicates that the two species diverged about 890,000 years ago.

The origin of eukaryotes is suggested as the symbiosis of pyrococcus into gamma-proteobacteria by phylogenetic tree based on gene content.

Attempts were made to define the relationship among the three domains (eukaryotes, archaea, and eubacteria) using phylogenetic tree analyses of 16S rRNA sequences as well as of other protein sequences. Since the results are inconsistent, it is implied that the eukaryotic genome has a chimeric structure. In our previous studies, the origin of eukaryotes to be the symbiosis of archaea into eubacteria using the whole open reading frames (ORF) of many genomes was suggested. In these studies, the species participating in the symbiosis were not clarified, and the effect of gene duplication after speciation (in-paralog) was not addressed. To avoid the influence of the in-paralog, we developed a new method to calculate orthologous ORFs. Furthermore, we separated eukaryotic in-paralogs into three groups by sequence similarity to archaea, eubacteria (other than alpha-proteobacteria), and alpha-proteobacteria and treated them as individual organisms. The relationship between the three ORF groups and the functional classification was clarified by this analysis. The introduction of this new method into the phylogenetic tree analysis of 66 organisms (4 eukaryotes, 13 archaea, and 49 eubacteria) based on gene content suggests the symbiosis of pyrococcus into gamma-proteobacteria as the origin of eukaryotes.

Molecular chaperones: proposal of a systematic computer-oriented nomenclature and construction of a centralized database.

Molecular chaperones are a wide group of unrelated protein families whose role is to assist others proteins. Comparably, under environmental stress, stress proteins behave as biocatalysts of protein stabilization. Stress proteins include a large class of proteins that were originally termed heat shock proteins (HSPs) due to their initial discovery in tissues exposed to elevated temperatures. Many, but not all, stress proteins and HSPs are molecular chaperones. Moreover, not all HSPs are derivable from stress. HSPs are structurally diversified by the contribution of various domains having specific roles. HSPs have been grouped, mainly on the basis of their molecular masses, into specific families that include small HSPs (sHSPs)/alpha-crystallins, HSP10s, HSP40s, HSP60s, HSP70s, HSP90s, HSP100s and HSP110s. The names of these major families are historical artefacts with limited information content. Using the current databases, names and proteic domains of many molecular chaperones in different species were analyzed. Although traditional names of HSPs are trivial, it is unrealistic to suggest replacing them, because they are preferred and widely used. Here we suggest that these traditional names be chaperoned, in silico, by a systematic nomenclature. Thus, for example, with the same intent of use of [trioxygen: O3] for ozone, we propose here C7HSP70[Ehsa]ER-P11021 for GRP78 (78 kDa endoplasmic Human molecular chaperone in HSP70 superfamily with P11021 as its accession number in the database of the National Center for Biotechnology Information (NCBI)). The proposed systematic computer-oriented naming and classification method is designed for HSPs and also their partners based on the number of amino acids, domain structure, phylogenetic domain, localization in the cell and accession number as stated in the NCBI. Arabidopsis thaliana was analyzed as a model, because it contains a large number of various HSPs localized in several organelles. Overall, this naming system helps in building, optimizing and managing a novel online database entirely devoted to HSPs. The purported taxonomy, coupled with the newly constructed database, can contribute to studies involving large amounts of stored data on HSPs.

Presence of isochore structures in reptile genomes suggested by the relationship between GC contents of intron regions and those of coding regions.

Vertebrate genomes are mosaics of isochores. On the assumption that marked differences exist in the isochore structure between warm-blooded and cold-blooded animals, variations among vertebrates were previously attributed to adaptation to homeothermy. However, based on the data of coding regions from representatives of extant vertebrates, including a turtle, a crocodile (Archosauromorpha) and a few kinds of snakes (Lepidosauromorpha), it was recently hypothesized that the common ancestors of mammals, birds and extant reptiles already had the "warm-blooded" isochore structure. To test this hypothesis, the nucleotide sequences of alpha-globin genes including non-coding regions (introns) from two snakes, N. kaouthia and E. climacophora, were determined (accession number: AB104824, AB104825). The correlation between the GC contents in the introns and exons of alpha-globin genes from snakes and those from other vertebrates supports the above hypothesis. Similar analysis using data for exons and introns of other genes obtained from the GenBank (Release 131) also support the above hypothesis.

Origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria supported by the newly clarified origin of functional genes.

In the previous report, we demonstrated the origin of eukaryotic cell nuclei as the symbiosis of Archaea in Bacteria by the newly developed "Homology-Hit Analysis". In that case, we counted yeast Open Reading Frames (ORFs) showing the highest similarity to a bacterial ORF as orthologous ORFs (Orthologous ORFs were produced by speciation from a common ancestor, and have the highest similarity to each other.) by comparing whole ORFs of yeast with those of individual bacteria. However, we could not count all yeast ORFs showing the highest similarity to a bacterial ORF in functional categories of yeast. Therefore, the origin of ORFs in the functional categories of yeast could not be inferred strictly. Here, we have improved the method for detecting orthologous ORFs. In this method, we count the numbers of ORF with the highest similarity between individual yeast functional categories and individual bacteria as orthologous ORFs. By this method, it was possible to detect the correct orthologous ORFs and to infer the origins of the functional categories in eukaryotic cells. As a result, two categories, assembly of protein complexes and DNA repair were newly judged to be of Archaeal origin, while five categories, lipid (fatty-acid and isoprenoid) metabolism, protein folding and stabilization, signal transduction, organization of the plasma membrane and organization of the cytoplasm, were newly judged to be of Bacterial origin. On the other hand, the origins of two categories (meiosis and cellular import, which were determined in the previous analysis) could not be judged. It is considered that functional categories related to the nucleus have origins common to Archaea, while those related to the cytoplasm have origins common to Bacteria. From these data including the origin of plasma membrane, it was further clarified that cell nucleus originated by the symbiosis of Archaea in Bacteria.

Changes in body temperature pattern in vertebrates do not influence the codon usages of alpha-globin genes.

Codon usages are known to vary among vertebrates chiefly due to variations in isochore structure. Under the assumption that marked differences exist in isochore structure between warm-blooded and cold-blooded animals, the variations among vertebrates were previously attributed to an adaptation to homeothermy. However, based on data from a turtle species and a crocodile (Archosauromorpha), it was recently proposed that the common ancestors of mammals, birds and extent reptiles already had the "warm-blooded" isochore structure. We determined the nucleotide sequences of alpha-globin genes from two species of heterotherms, cuckoo (Cuculus canorus) and bat (Pipistrellus abramus), and three species of snakes (Lepidosauromorpha), Naja kaouthia from a tropical terrestrial habitat, Elaphe climacophora from a temperate terrestrial habitat, and Hydrophis melanocephalus from a tropical marine habitat. Our purposes were to assess the influence of differential body temperature patterns on codon usage and GC content at the third position of a codon (GC3), and to test the hypothesis concerning the phylogenetic position at which GC contents had increased in vertebrates. The results of principal component analysis (PCA) using the present data and data for other taxa from GenBank indicate that the primary difference in codon usage in globin genes among amniotes and other vertebrates lies in GC3. The codon usages (and GC3) in alpha-globin genes from two heterotherms and three snakes are similar to those in alpha-globin genes from warm-blooded vertebrates. These results refute the influence of body temperature pattern upon codon usages (and GC3) in alpha-globin genes, and support the hypothesis that the increase in GC content in the genome occurred in the common ancestor of amniotes.