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.

Dopamine receptor genes and evolutionary differentiation in the domestication of fighting cocks and long-crowing chickens.

The chicken domestication process represents a typical model of artificial selection, and gives significant insight into the general understanding of the influence of artificial selection on recognizable phenotypes. Two Japanese domesticated chicken varieties, the fighting cock (Shamo) and the long-crowing chicken (Naganakidori), have been selectively bred for dramatically different phenotypes. The former has been selected exclusively for aggressiveness and the latter for long crowing with an obedient sitting posture. To understand the particular mechanism behind these genetic changes during domestication, we investigated the degree of genetic differentiation in the aforementioned chickens, focusing on dopamine receptor D2, D3, and D4 genes. We studied other ornamental chickens such as Chabo chickens as a reference for comparison. When genetic differentiation was measured by an index of nucleotide differentiation (NST) newly devised in this study, we found that the NST value of DRD4 for Shamo (0.072) was distinctively larger than those of the other genes among the three populations, suggesting that aggressiveness has been selected for in Shamo by collecting a variety of single nucleotide polymorphisms. In addition, we found that in DRD4 in Naganakidori, there is a deletion variant of one proline at the 24th residue in the repeat of nine prolines of exon 1. We thus conclude that artificial selection has operated on these different kinds of genetic variation in the DRD4 genes of Shamo and Naganakidori so strongly that the two domesticated varieties have differentiated to obtain their present opposite features in a relatively short period of time.

Divergence of East Asians and Europeans estimated using male- and female-specific genetic markers.

To study the male and female lineages of East Asian and European humans, we have sequenced 25 short tandem repeat markers on 453 Y-chromosomes and collected sequences of 72 complete mitochondrial genomes to construct independent phylogenetic trees for male and female lineages. The results indicate that East Asian individuals fall into two clades, one that includes East Asian individuals only and a second that contains East Asian and European individuals. Surprisingly, the European individuals did not form an independent clade, but branched within in the East Asians. We then estimated the divergence time of the root of the European clade as ∼ 41,000 years ago. These data indicate that, contrary to traditional views, Europeans diverged from East Asians around that time. We also address the origin of the Ainu lineage in northern Japan.

Age-dependent changes in the functions and compositions of photosynthetic complexes in the thylakoid membranes of Arabidopsis thaliana.

Photosynthetic complexes in the thylakoid membrane of plant leaves primarily function as energy-harvesting machinery during the growth period. However, leaves undergo developmental and functional transitions along aging and, at the senescence stage, these complexes become major sources for nutrients to be remobilized to other organs such as developing seeds. Here, we investigated age-dependent changes in the functions and compositions of photosynthetic complexes during natural leaf senescence in Arabidopsis thaliana. We found that Chl a/b ratios decreased during the natural leaf senescence along with decrease of the total chlorophyll content. The photosynthetic parameters measured by the chlorophyll fluorescence, photochemical efficiency (F v/F m) of photosystem II, non-photochemical quenching, and the electron transfer rate, showed a differential decline in the senescing part of the leaves. The CO2 assimilation rate and the activity of PSI activity measured from whole senescing leaves remained relatively intact until 28 days of leaf age but declined sharply thereafter. Examination of the behaviors of the individual components in the photosynthetic complex showed that the components on the whole are decreased, but again showed differential decline during leaf senescence. Notably, D1, a PSII reaction center protein, was almost not present but PsaA/B, a PSI reaction center protein is still remained at the senescence stage. Taken together, our results indicate that the compositions and structures of the photosynthetic complexes are differentially utilized at different stages of leaf, but the most dramatic change was observed at the senescence stage, possibly to comply with the physiological states of the senescence process.

Purification and characterization of two phospho-ß-galactosidases, LacG1 and LacG2, from Lactobacillus gasseri ATCC33323(T).

Lactobacillus gasseri ATCC33323(T) expresses four enzymes showing phospho-ß-galactosidase activity (LacG1, LacG2, Pbg1 and Pbg2). We previously reported the purification and characterization of two phospho-ß-galactosidases (Pbg1 and Pbg2) from Lactobacillus gasseri JCM1031 cultured in lactose medium. Here we aimed to characterize LacG1 and LacG2, and classify the four enzymes into 'phospho-ß-galactosidase' or 'phospho-ß-glucosidase.' LacG1 and recombinant LacG2 (rLacG2), from Lb. gasseri ATCC33323(T), were purified to homogeneity using column chromatography. Kinetic experiments were performed using sugar substrates, o-nitrophenyl-ß-D-galactopyranoside 6-phosphate (ONPGal-6P) and o-nitrophenyl-ß-D-glucopyranoside 6-phosphate (ONPGlc-6P), synthesized in our laboratory. LacG1 and rLacG2 exhibited high k(cat)/K(m) values for ONPGal-6P as compared with Pbg1 and Pbg2. The V(max) values for ONPGal-6P were higher than phospho-ß-galactosidases previously purified and characterized from several lactic acid bacteria. A phylogenetic tree analysis showed that LacG1 and LacG2 belong to the phospho-ß-galactosidase cluster and Pbg1 and Pbg2 belong to the phospho-ß-glucosidase cluster. Our data suggest two phospho-ß-galactosidase, LacG1 and LacG2, are the primary enzymes for lactose utilization in Lb. gasseri ATCC33323(T). We propose a reclassification of Pbg1 and Pbg2 as phospho-ß-glucosidase.

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.

Evolutionary conserved microRNAs are ubiquitously expressed compared to tick-specific miRNAs in the cattle tick Rhipicephalus (Boophilus) microplus.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression in eukaryotes modulating a large diversity of biological processes. The discovery of miRNAs has provided new opportunities to understand the biology of a number of species. The cattle tick, Rhipicephalus (Boophilus) microplus, causes significant economic losses in cattle production worldwide and this drives us to further understand their biology so that effective control measures can be developed. To be able to provide new insights into the biology of cattle ticks and to expand the repertoire of tick miRNAs we utilized Illumina technology to sequence the small RNA transcriptomes derived from various life stages and selected organs of R. microplus. RESULTS: To discover and profile cattle tick miRNAs we employed two complementary approaches, one aiming to find evolutionary conserved miRNAs and another focused on the discovery of novel cattle-tick specific miRNAs. We found 51 evolutionary conserved R. microplus miRNA loci, with 36 of these previously found in the tick Ixodes scapularis. The majority of the R. microplus miRNAs are perfectly conserved throughout evolution with 11, 5 and 15 of these conserved since the Nephrozoan (640 MYA), Protostomian (620MYA) and Arthropoda (540 MYA) ancestor, respectively. We then employed a de novo computational screening for novel tick miRNAs using the draft genome of I. scapularis and genomic contigs of R. microplus as templates. This identified 36 novel R. microplus miRNA loci of which 12 were conserved in I. scapularis. Overall we found 87 R. microplus miRNA loci, of these 15 showed the expression of both miRNA and miRNA* sequences. R. microplus miRNAs showed a variety of expression profiles, with the evolutionary-conserved miRNAs mainly expressed in all life stages at various levels, while the expression of novel tick-specific miRNAs was mostly limited to particular life stages and/or tick organs. CONCLUSIONS: Anciently acquired miRNAs in the R. microplus lineage not only tend to accumulate the least amount of nucleotide substitutions as compared to those recently acquired miRNAs, but also show ubiquitous expression profiles through out tick life stages and organs contrasting with the restricted expression profiles of novel tick-specific miRNAs.

Evolution of protein phosphorylation for distinct functional modules in vertebrate genomes.

Recent publications have revealed that the evolution of phosphosites is influenced by the local protein structures and whether the phosphosites have characterized functions or not. With knowledge of the wide functional range of phosphorylation, we attempted to clarify whether the evolutionary conservation of phosphosites is different among distinct functional modules. We grouped the phosphosites in the human genome into the modules according to the functional categories of KEGG (Kyoto Encyclopedia of Genes and Genomes) and investigated their evolutionary conservation in vertebrate genomes from mouse to zebrafish. We have found that the phosphosites in the vertebrate-specific functional modules (VFMs), such as cellular signaling processes and responses to stimuli, are evolutionarily more conserved than those in the basic functional modules (BFMs), such as metabolic and genetic processes. The phosphosites in the VFMs are also significantly more conserved than their flanking regions, whereas those in the BFMs are not. These results hold for both serine/threonine and tyrosine residues, although the fraction of phosphorylated tyrosine residues is increased in the VFMs. Moreover, the difference in the evolutionary conservation of the phosphosites between the VFMs and BFMs could not be explained by the difference in the local protein structures. There is also a higher fraction of phosphosites with known functions in the VFMs than BFMs. Based on these findings, we have concluded that protein phosphorylation may play more dominant roles for the VFMs than BFMs during the vertebrate evolution. As phosphorylation is a quite rapid biological reaction, the VFMs that quickly respond to outer stimuli and inner signals might heavily depend on this regulatory mechanism. Our results imply that phosphorylation may have an essential role in the evolution of vertebrates.

Biological databases at DNA Data Bank of Japan in the era of next-generation sequencing technologies.

The Center for Information Biology and DNA Data Bank of Japan (CIB-DDBJ) has operated biological databases since 1987 in collaboration with NCBI and EBI. As one of the three major public databases, CIB-DDBJ has run four primary databases DDBJ, CIBEX, DDBJ Trace Archive (DTA), and DDBJ Read Archive (DRA) to collect, archive, and provide various kinds of biological data. As the massively parallel new sequencing platforms are increasingly in use, huge amounts of the raw data have been produced. To archive these raw data, we at CIB-DDBJ began operating a new repository, the DDBJ Read Archive (DRA). To accommodate efficiently the processed data as well, we have developed a new pipeline, the DDBJ Read Annotation Pipeline that deals with both data submission and analysis. For data produced by the next generation platforms, the three archives DRA, DDBJ, and CIBEX, which are interconnected by the pipeline, collect the raw, processed sequence, and quantitative data, respectively. The public biological databases at CIB-DDBJ, EBI, and NCBI will together construct world-wide archives for biological data by data sharing to accelerate research in life sciences in the era of next generation sequencing technologies.

The GTOP database in 2009: updated content and novel features to expand and deepen insights into protein structures and functions.

The Genomes TO Protein Structures and Functions (GTOP) database (http://spock.genes.nig.ac.jp/~genome/gtop.html) freely provides an extensive collection of information on protein structures and functions obtained by application of various computational tools to the amino acid sequences of entirely sequenced genomes. GTOP contains annotations of 3D structures, protein families, functions, and other useful data of a protein of interest in user-friendly ways to give a deep insight into the protein structure. From the initial 1999 version, GTOP has been continually updated to reap the fruits of genome projects and augmented to supply novel information, in particular intrinsically disordered regions. As intrinsically disordered regions constitute a considerable fraction of proteins and often play crucial roles especially in eukaryotes, their assignments give important additional clues to the functionality of proteins. Additionally, we have incorporated the following features into GTOP: a platform independent structural viewer, results of HMM searches against SCOP and Pfam, secondary structure predictions, color display of exon boundaries in eukaryotic proteins, assignments of gene ontology terms, search tools, and master files.

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.

An evolutionary origin and selection process of goldfish.

Many different physical characteristics of goldfish (Carassius auratus auratus), such as celestial and telescopic eyes, fancy but uncontrollable shapes of tail fin, an unfittingly fat body, and loss of dorsal fins, provide us with a unique opportunity of studying artificial selection on phenotypic changes on the basis of molecular evolution. The aim of the present study is to elucidate the evolutionary origin and history of goldfish, taking into account the different characteristics of goldfish and human culture. Collecting 44 samples of a variety of goldfish from Japan and China as well as common and Crucian carps, we determined the nucleotide sequences for a substantial portion of mitochondrial genome including eight gene regions (D-loop, 12SrRNA, 16SrRNA, ND1, ND2, COI, ND5 and Cyt b) of approximately 11,180 bps. We, then, constructed phylogenetic trees for a total of 78 fishes, adding the 19 sequence data available in the international DNA database DDBJ/EMBL/GenBank to our 59 sequence data determined. From the phylogenetic trees obtained, we found that Japanese goldfish are not relative to Japanese Crucian carp (Carassius auratus langsdorfi) and that all the goldfish examined were originated from one of the two groups of the Chinese Crucian carp "Gibelio" (Carassius auratus gibelio). Moreover, we found that the process of artificial selection began from losing the dorsal fin followed by diversification of other characters such as eyes. This is supported by our further observations that the improvement of celestial and telescope eyes took place independently at different times, implying that goldfish was imposed by strong artificial selection only to meet diversified needs of human preferences in a unsystematic way.

DDBJ dealing with mass data produced by the second generation sequencer.

DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp) collected and released 2 368 110 entries or 1 415 106 598 bases in the period from July 2007 to June 2008. The releases in this period include genome scale data of Bombyx mori, Oryzas latipes, Drosophila and Lotus japonicus. In addition, from this year we collected and released trace archive data in collaboration with National Center for Biotechnology Information (NCBI). The first release contains those of O. latipes and bacterial meta genomes in human gut. To cope with the current progress of sequencing technology, we also accepted and released more than 100 million of short reads of parasitic protozoa and their hosts that were produced by using a Solexa sequencer.

BioCaster: detecting public health rumors with a Web-based text mining system.

SUMMARY: BioCaster is an ontology-based text mining system for detecting and tracking the distribution of infectious disease outbreaks from linguistic signals on the Web. The system continuously analyzes documents reported from over 1700 RSS feeds, classifies them for topical relevance and plots them onto a Google map using geocoded information. The background knowledge for bridging the gap between Layman's terms and formal-coding systems is contained in the freely available BioCaster ontology which includes information in eight languages focused on the epidemiological role of pathogens as well as geographical locations with their latitudes/longitudes. The system consists of four main stages: topic classification, named entity recognition (NER), disease/location detection and event recognition. Higher order event analysis is used to detect more precisely specified warning signals that can then be notified to registered users via email alerts. Evaluation of the system for topic recognition and entity identification is conducted on a gold standard corpus of annotated news articles. AVAILABILITY: The BioCaster map and ontology are freely available via a web portal at http://www.biocaster.org.

Identification of five phospho-beta-glycosidases from Lactobacillus gasseri ATCC33323T cultured in lactose medium.

Lactobacillus gasseri ATCC33323(T) has seven putative phospho-beta-glycosidase genes. Using column chromatography, we found that this strain cultured in lactose medium expresses five phospho-beta-glycosidases (LacG1, LacG2, Pbg1, Pbg2, and Pbg3), where these gene expressions can be suppressed by glucose. To our knowledge, this is the first report indicating that five glycosidases are induced from a single bacterial strain using a single carbon source, lactose.

The Rice Annotation Project Database (RAP-DB): 2008 update.

The Rice Annotation Project Database (RAP-DB) was created to provide the genome sequence assembly of the International Rice Genome Sequencing Project (IRGSP), manually curated annotation of the sequence, and other genomics information that could be useful for comprehensive understanding of the rice biology. Since the last publication of the RAP-DB, the IRGSP genome has been revised and reassembled. In addition, a large number of rice-expressed sequence tags have been released, and functional genomics resources have been produced worldwide. Thus, we have thoroughly updated our genome annotation by manual curation of all the functional descriptions of rice genes. The latest version of the RAP-DB contains a variety of annotation data as follows: clone positions, structures and functions of 31 439 genes validated by cDNAs, RNA genes detected by massively parallel signature sequencing (MPSS) technology and sequence similarity, flanking sequences of mutant lines, transposable elements, etc. Other annotation data such as Gnomon can be displayed along with those of RAP for comparison. We have also developed a new keyword search system to allow the user to access useful information. The RAP-DB is available at: http://rapdb.dna.affrc.go.jp/ and http://rapdb.lab.nig.ac.jp/.

Development and public release of a comprehensive hepatitis virus database.

AIM: Currently, approximately 44 000 hepatitis C virus (HCV), 11 000 hepatitis B virus (HBV), and 1600 hepatitis E virus (HEV) sequences are available at the International Nucleotide Sequence Database Collaboration (INSDC, previously known as DDBJ/EMBL/GenBank), and the number of these virus sequences is growing rapidly. However, since INDSC is not specialized to hepatitis viruses, it is difficult to retrieve information of virological or clinical interests from it. Thus, it is quite worthwhile to construct a specialized database for the hepatitis virus sequences and to make it accessible to researchers worldwide. METHODS: We developed a WWW-based database hepatitis virus database (HVDB), which contains all the HCV, HBV, and HEV sequences available at INSDC. In the HVDB, all piece sequences obtained from INSDC are arranged to the genomesequence of each virus. Also given in the database are the phylogenetic relationships of each locus on the genome among variants for each virus. RESULTS: Users of the database can easily retrieve entries (sequences with annotations) of the specific genotype by referring to the phylogenetic relationships or those of specific loci by referring to the genome map information. HVDB provides users with a tool for phylogenetic analysis that can be used in combination with the data retrieval tools. CONCLUSION: The latest release is publicly accessible at the HVDB website: http://s2as02.genes.nig.ac.jp.

A tree of life based on protein domain organizations.

It is desirable to estimate a tree of life, a species tree including all available species in the 3 superkingdoms, Archaea, Bacteria, and Eukaryota, using not a limited number of genes but full-scale genome information. Here, we report a new method for constructing a tree of life based on protein domain organizations, that is, sequential order of domains in a protein, of all proteins detected in a genome of an organism. The new method is free from the identification of orthologous gene sets and therefore does not require the burdensome and error-prone computation. By pairwise comparisons of the repertoires of protein domain organizations of 17 archaeal, 136 bacterial, and 14 eukaryotic organisms, we computed evolutionary distances among them and constructed a tree of life. Our tree shows monophyly in Archaea, Bacteria, and Eukaryota and then monophyly in each of eukaryotic kingdoms and in most bacterial phyla. In addition, the branching pattern of the bacterial phyla in our tree is consistent with the widely accepted bacterial taxonomy and is very close to other genome-based trees. A couple of inconsistent aspects between the traditional trees and the genome-based trees including ours, however, would perhaps urge to revise the conventional view, particularly on the phylogenetic positions of hyperthermophiles.

DDBJ working on evaluation and classification of bacterial genes in INSDC.

DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp) newly collected and released 12,927,184 entries or 13,787,688,598 bases in the period from July 2005 to June 2006. The released data contain honeybee expressed sequence tags (ESTs), re-examined and re-annotated complete genome data of Escherichia coli K-12 W3110, medaka WGS and human MGA. We also systematically evaluated and classified the genes in the complete bacterial genomes submitted to the International Nucleotide Sequence Database Collaboration (INSDC, http://insdc.org) that is composed of DDBJ, EMBL Bank and GenBank. The examination and classification selected 557,000 genes as reliable ones among all the bacterial genes predicted by us.