Tokumasa Horiike

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.

The Origin of Eukaryotes Is Suggested as the Symbiosis of Pyrococcus into γ-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 α-proteobacteria), and α-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 γ-proteobacteria as the origin of eukaryotes.

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.

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.

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

ABSTRACT Pectobacterium carotovorum subsp. carotovorum and 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 Podoviridae and has high similarity to the bacteriophage Peat1 infectious to P. atrosepticum.

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

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

Does endo-symbiosis explain the origin of the nucleus? - Reply

To the editor — Horiike et al. give an excellent bioinformatic analysis showing relationships between yeast genes that function in the nucleus and archaeal genes, and between yeast genes that function in the cytoplasm and bacterial genes. However, their conclusion that the nucleus originated as an archaeal endosymbiont fails to explain the following features of the nucleus: the structure of the nuclear envelope; the nuclear pore complex; linear chromosomes; absence of phagocytic bacteria; the preservation of RNA-world relics in eukaryotes, and reduction of these in prokaryotes. Furthermore, their explanation contradicts the general trend of gene loss reported in parasitic, endosymbiotic and organellar genomes. Clear parallels exist between bacterial, mitochondrial, hydrogenosomal and chloroplast membranes. No such parallel exists for the nuclear envelope where the inner and outer membranes are continuous. Likewise, the nuclear pore complex bears no resemblance to prokaryotic transmembrane pores. Hence, unlike for other organelles, ultrastructure does not favour endosymbiotic origins. The nucleus contains linear chromosomes with telomeres, which have not been found in archaea and arguably predate circular chromosomes. Forterre’s thermoreduction hypothesis, that prokaryotes arose through reductive evolution at high temperature, argues for circularization being derived; circular DNA is more thermostable than linear. Maintenance of telomeres by telomerase probably originated in the RNA world, before modern cells; telomerase has an RNA core and is highly conserved among eukaryotes. Using RNA relics to root the tree of life argues that some eukaryote nuclear traits are ancestral, having been lost through reductive evolution in prokaryotes; thermoreduction explains this pattern because RNA is thermolabile. If some eukaryote nuclear traits predate archaeal traits, these cannot be explained by an archaeal endosymbiont. The conclusion of Horiike and colleagues requires that the endosymbiont gained genes from its host, which is counter to known examples of endosymbiosis (including eukaryotic organelles) and intracellular parasitism, where the unifying feature is gene loss. Intracellular existence makes primary synthetic pathways redundant. Furthermore, the yeast cytoplasmic–bacterial gene relationship described can be explained by Muller’s ratchet — the irreversible accumulation of mutations in small asexual populations. Relocation of organellar genes to the nucleus results in escape of the effects of the ratchet but extensive transfer from host to endosymbiont would place genes under greater mutational pressure. Neither reductive evolution nor endosymbiosis explains nuclear origins. The former, however, explains RNA-world relics and linear chromosomes in eukaryotes, is consistent with Horiike and colleagues’ results and argues against an archaeal origin for the nucleus.