I. CENTER FOR INFORMATION BIOLOGY AND DNA DATA BANK OF JAPAN
I-a. Laboratory for DNA Data Analysis - Takashi Gojobori Group

RESEARCH ACTIVITIES

(1) Comparative analysis of gene expression for convergent evolution of camera eye between octopus and human

Atsushi Ogura, Kazuho Ikeo and Takashi Gojobori

--Although the camera eye of the octopus is very similar to that of humans, phylogenetic and embryological analyses have suggested that their camera eyes have been acquired independently. It has been known as a typical example of convergent evolution. To study the molecular basis of convergent evolution of camera eyes, we conducted a comparative analysis of gene expression in octopus and human camera eyes. We sequenced 16,432 ESTs of the octopus eye, leading to 1052 nonredundant genes that have matches in the protein database. Comparing these 1052 genes with 13,303 already-known ESTs of the human eye, 729 (69.3%) genes were commonly expressed between the human and octopus eyes. On the contrary, when we compared octopus eye ESTs with human connective tissue ESTs, the expression similarity was quite low. To trace the evolutionary changes that are potentially responsible for camera eye formation, we also compared octopus-eye ESTs with the completed genome sequences of other organisms. We found that 1019 out of the 1052 genes had already existed at the common ancestor of bilateria, and 875 genes were conserved between humans and octopuses. It suggests that a larger number of conserved genes and their similar gene expression may be responsible for the convergent evolution of the camera eye.

(2) Molecular hierarchy in neurons differentiated from mouse ES cells containing a single human chromosome 21

Chi Chiu Wang, Mitsutaka Kadota, Ryuichi Nishigaki, Yasuhiro Kazuki, Yasuaki Shirayoshi, Michael Scott Rogers, Takashi Gojobori, Kazuho Ikeo and Mitsuo Oshimura

--Defects in neurogenesis and neuronal differentiation in the fetal brain of Down syndrome (DS) patients lead to the apparent neuropathological abnormalities and contribute to the phenotypic characters of mental retardation, and premature development of Alzheimer's disease, those being the most common phenotype in DS. In order to understand the molecular mechanism underlying the cause of phenotypic abnormalities in the DS brain, we have utilized an in vitro model of TT2F mouse embryonic stem cells containing a single human chromosome 21 (hChr21) to study neuron development and neuronal differentiation by microarray containing 15K developmentally expressed cDNAs. Defective neuronal differentiation in the presence of extra hChr21 manifested primarily the post-transcriptional and translational modification, such as Mrpl10, SNAPC3, Srprb, and SF3a60 in the early neuronal stem cell stage, and Mrps18a, Eef1g, and Ubce8 in the late differentiated stage. Hierarchical clustering patterned specific expression of hChr21 gene dosage effects on neuron outgrowth, migration, and differentiation, such as Syngr2, Dncic2, Eif3sf, and Peg3.

(3) Strong positive selection and recombination drive the antigenic variation of the PilE protein of the human pathogen Neisseria meningitidis

Daniel T. Andrews and Takashi Gojobori

--The PilE protein is the major component of the Neisseria meningitidis pilus, which is encoded by the pilE/pilS locus that includes an expressed gene and eight homologous silent fragments. The silent gene fragments have been shown to recombine through gene conversion with the expressed gene and thereby provide a means by which novel antigenic variants of the PilE protein can be generated. We have analyzed the evolutionary rate of the pilE gene using the nucleotide sequences of two complete pilE/pilS loci. The very high rate of evolution displayed by the PilE protein appears driven by both recombination and positive selection. Within the semivariable region of the pilE and pilS genes, recombination appears to occur within multiple small sequence blocks that lie between conserved sequence elements. Within the hypervariable region, positive selection was identified from comparison of the silent and expressed genes. The unusual gene conversion mechanism that operates at the pilE/pilS locus is a strategy employed by N. meningitidis to enhance mutation of certain regions of the PilE protein. The silent copies of the gene effectively allow “parallelized" evolution of pilE, thus enabling the encoded protein to rapidly explore a large area of sequence space in an effort to find novel antigenic variants.

(4) Difference of organelles involved in membrane traffic

Hajime Ohyanagi and Takashi Gojobori

--Contrary to prokaryotes, eukaryotes have a number of membranous organelles involved in membrane traffic in cells. Therefore, the gain of membrane systems could be one of the most epochal events in the evolution of eukaryotes. However, details about the evolution of a membrane system are still unclear. Each protein involved in a membrane system must have been evolved with its own characteristics, so that proteins localized in the same position in cells may show the same characteristics in the context of molecular evolution. With the aim of giving insight to the evolutionary studies of membrane systems, we are conducting the following analyses. First, we obtained subcellular localization data of yeast proteins that have already published (http://yeastgfp.ucsf.edu/). Second, in order to classify these yeast proteins into groups according to their evolutionary emergence times, we performed the BLAST reciprocal best hit analysis of yeast proteome against the proteomes of other species. From these analyses, we observed that protein sets in endoplasmic reticulum and Golgi apparatus, major components of the membrane traffic system, clearly showed evolutionary emergence times different from each other. Thus, it suggests that endoplasmic reticulum and Golgi apparatus appeared independently in the evolution of eukaryotes.

(5) Highly conserved upstream sequences for transcription factor genes and implications for the regulatory network

Hisakazu Iwama and Takashi Gojobori

--Identifying evolutionarily conserved blocks in orthologous genomic sequences is an effective way to detect regulatory elements. In this study, with the aim of elucidating the architecture of the regulatory network, we systematically estimated the degree of conservation of the upstream sequences of 3,750 human-mouse orthologue pairs along 8-kb stretches. We found that the genes with high upstream conservation are predominantly transcription factor (TF) genes. In particular, developmental process-related TF genes showed significantly higher conservation of the upstream sequences than other TF genes. Such extreme upstream conservation of the developmental process-related TF genes suggests that the regulatory networks involved with developmental processes have been evolutionarily well conserved in both human and mouse lineages.

(6) Computational prediction of microRNA (miRNA) target gene in human and its experimental varidation

Hitomi Sakurai, Roberto Barrero, Shiho Hayakawa, Takuro Tamura, Yoshio Tateno, Ikeo Kazuho, Imanishi Tadashi and Takashi Gojobori

--MicroRNAs (miRNAs) form a novel class of small RNA genes of 21-25 nucleotides derived from highly conserved hairpin precursors (pre-miRNAs) present from mollusc to mammals. miRNAs act as post-transcriptional repressors of target transcripts via mRNA degradation or translation inhibition. We present a novel large scale RNA-affinity screening tool combined with the evaluation of RNA secondary structures to identify putative microRNA recognition elements (MREs) on target mRNAs. We identified 1,476 MREs for 115 known mammalian miRNAs. Eleven of the top predicted MREs for Let-7a, miR-20, miR-97 and miR-182a were analyzed using a reporter assay. As a result, we found that seven MREs mediated inhibition of reporter expression. It suggests that our target prediction algorithm, RNAFFY, identifies highly reliable miRNA targets.

(7) Detection of apoptosis during planarian regeneration by the expression of apoptosis-related genes and TUNEL assay

Jung Shan Hwang, Chiyoko Kobayashi, Kiyokazu Agata, Kazuho Ikeo and Takashi Gojobori

--Apoptosis is a tightly organized cell death process that plays a crucial role in metazoan development, but it has not yet been revealed whether apoptotic events are involved in the process of regeneration. Here, we tried to detect apoptotic cells during planarian regeneration using the TdT-mediated dUTP nick-end labeling (TUNEL) assay as well as the expression of apoptosis-related genes. Three novel cDNAs were isolated from a planarian cDNA library and shown to be closely related to other metazoan caspases at the amino acid sequence level. One of these cDNAs, Caspase-like gene 3 (DjClg3), was expressed primarily in apoptotic cells by double detections with the TUNEL assay. Whole mount in situ studies indicated that DjClg3 was expressed in the cells of the mesenchymal space and also around the pharynx of the intact body. Its expression in the regenerating head piece was seen in the blastema and less significantly in the brain, while in the regenerating tail piece, DjClg3 expression was detected uniformly throughout the entire region. In parallel experiments, we performed in situ TUNEL assays to localize the regions where cell death occurred during regeneration and comparable results to the DjClg3 expression patterns were obtained. This is the first report to show that planarians have apoptosis-related genes, and the results suggest that the apoptotic mechanism probably takes place to a large extent in normal intact worms as well as during their regeneration. We hypothesize that the presence of apoptosis in planarians may have a role in controlling cell numbers, eliminating unnecessary tissues or cells and remodeling the old tissues of regenerating body parts.

(8) Analysis of sexual dimorphism of gene expression in mouse brain

Kazuya Yuge, Kazuho Ikeo and Takashi Gojobori

--There are sexual differences in morphological features of the mammalian brain. The so-called sexual dimorphism of mammalian brains is thought to be determined by the gonadal hormones; this view is called “gonadal origin theory". However, recent studies suggest that differences in gene expression of sex-liked genes in neuronal cells directly generate sexual dimorphism in the brain. To examine this possibility we conducted microarray experiments to screen the genes that were expressed differentially in the brain between male and female mice before gonadal hormone secretion. In this study we have identified 57 female enhanced genes and 24 male enhanced genes in brains at embryonic day 10.5 (E 10.5). These results indicate that sexual differences in gene expression in neuronal cells before gonadal hormone secretion would play an important role in sexual dimorphism in the brain.

(9) Seeking for signes of aging in Hydra, a primitive metazoan

Kengo Yoshida, Hwang Jung Shang, Chimi Fujisawa, Toshitaka Fujisawa, Kazuho Ikeo and Takashi Gojobori

--Aging occurs in a variety of organisms including yeast, nematode, fly, and mammals. However, hydra belonging to phylum Cnidaria has been considered to be immortal (Martinez 1998). This apparent immortality was observed when hydra were maintained so that they underwent asexual reproduction, budding. Do they remain immortal if they undergo sexual reproduction? A previous study using Hydra Oligactis showed that the depression occurred after sexual reproduction (Littlefield 1985). To seek for possible signs of aging in hydra, we studied the process of the depression in detail. As a result, reduction in the capacity of food capture, digestive movements and contractile movements was all observed. Moreover, exponential increase in mortality rate of population was also observed. From these observations, we conclude that aging-like phenomenon occurs in hydra. Aging research on hydra, one of the most primitive metazoans, has potential for providing further insight into the understanding of evolution of aging.

(10) A large variation in the rates of synonymous substitution for RNA viruses and its relationship to a diversity of viral infection and transmission modes

Kousuke Hanada, Yoshiyuki Suzuki and Takashi Gojobori

--RNA viruses successfully adapt to various environments by repeatedly producing new mutants, often through generating a number of nucleotide substitutions. To estimate the degree of variation in mutation rates of RNA viruses and to understand the source of such variation, we studied the synonymous substitution rate because synonymous substitution is exempt from functional constraints at the protein level, and its rate reflects the mutation rate to a great extent. We estimated the synonymous substitution rates for a total of 49 different species of RNA viruses, and we found that the rates had tremendous variation by 5 orders of magnitude (from 1.3 x 10^-7 to 6.2 x 10^-2 /synonymous site/year). Comparing the synonymous substitution rates with the replication frequencies and replication error rates for the RNA viruses, we found that the main source of the rate variation was differences in the replication frequency because the rates of replication error were roughly constant over different RNA viruses. Moreover, we examined a relationship between viral life strategies and synonymous substitution rates to understand which viral life strategies affect replication frequencies. The results show that the variation of synonymous substitution rates has been influenced most by either the difference in the infection modes or the differences in the transmission modes. In conclusion, the variation of mutation rates for RNA viruses is caused by different replication frequencies, which are affected strongly by the infection and transmission modes.

(11) Evolutionary processes of gene splicing and gene silencing

Lihua Jin, Yoshiyuki Suzuki, Kazuho Ikeo and Takashi Gojobori

--Both gene splicing and gene silencing have become known to contribute significantly to functional diversification of genes and gene networks. For gene splicing, we conducted an evolutionary study of the relationship between gene duplication and alternative splicing. For gene silencing, we studied the evolutionary process of small RNA-guided pathways, focusing on the Rnase III family enzymes. For the first topic, we mentioned that gene duplication and alternative splicing (AS) were the two major evolutionary mechanisms that could bring the functional variation through the diversification of genes and gene products. The purpose of this research is to understand the evolutionary relationship between the two different mechanisms, utilizing the available data resources. The results of this study showed that the proportion of the AS loci in the singleton gene group was less than that in the duplicated gene group. Moreover, we found that the duplicated genes tended to have more AS isoforms than singleton genes. These results suggest that gene duplication would induce more alternative splicing events on duplicated copies than on singletons possibly by reducing the functional constraints on the duplicates. For the second topic, we pointed out that the RNA-mediated gene silencing pathways were evolutionarily conserved processes. It highlights a fundamental role of short RNAs in eukatyotic gene regulation and antiviral defense. Recently, three distinct small RNA-directed silencing pathways were observed, such as the destruction of mRNA via siRNA, inhibition of mRNA translation via miRNA, and epigenetic gene silencing via siRNA. It was also found that in these pathways, the members of ribonclease III family played important roles in diverse RNA maturation and decay. Here we investigated the evolution of Rnase III nucleases, Dicer as representative, to further figure out the evolutionary relationship among the three gene silencing pathways. With the advantage of using genomic sequences as the subjects of homology search in the annotated and un-annotated genomic regions, we were able to detect possible candidates for a gene of dicer and its two functional domains. Moreover, we found that representative prokaryotes including eubacteria and archbacteria lacked completely the PAZ domain of Dicer. These results clearly show the taxonomy-dependent evolution of the RNA-mediated gene silencing pathways. The results obtained in this study provide the information for the understanding of the evolutionary origin and relationships of the three pathways. The information may also be useful for the conducting of the relevant experiments. As a summary, these results suggested that changes in the genome and regulatory network were closely related evolutionary events, implying that gene multiplication and functional constraints were the two important sources for the change in gene function in the networks of transcription and gene regulation in evolution.

(12) The bioinformatics challenges in comparative analysis of cereal genomes-an overview

Matthew Bellgard, Jia Ye, Takashi Gojobori and Rudi Appels

--Comparative genomic analysis is the cornerstone of in silico-based approaches to understanding biological systems and processes across cereal species, such as rice, wheat and barley, in order to identify genes of agronomic interest. The size of the genomic repositories is nearly doubling every year, and this has significant implications on the way bioinformatics analyses are carried out. The concepts and technology underpinning bioinformatics as applied to comparative genomic analysis are discusse as the overview.

(13) Various adaptations for the perpetual darkness in the diversification process from an eyed surface-dwelling form to an eyeless cave-dwelling form of Mexican tetra, Astyanax mexicanus, by cDNA microarrays

Nobuhiko Tanaka, Shozo Yokoyama, Kazuho Ikeo and Takashi Gojobori

--Mexican tetra (Astyanax mexicanus), a single species with eyed surface and eyeless cave dwelling forms, gives a unique opportunity of the evolutionary process of gene diversification during a short period of time. In this study, we tried to find genes related to the evolution of eyeless cave form from the eyed surface form by cDNA microarrays, each of which has 3,070 non-redundant clones from the entire body of an eyed adult fish. Target genes for the microarrays were made from the entire body of each an eyed fish and an eyeless fish. On the basis of the results of comparative microarray analysis for 716 functionally known genes, relatively highly expressed genes in the surface fish and in the cavefish were listed up, respectively. In the surface fish, we for at that there were relatively highly expression of eye formation-related genes, digestive system-related genes, energy metabolism-related genes, lipocalin-type prostaglandin D synthase gene, elastase genes. The eye formation-related genes may be highly expressed because surface fish have well-developed eyes whereas cavefish have degenerated eyes. The digestive system-related and energy metabolism-related genes may be also highly expressed because the surface fish are more active than the cavefish. The lipocalin-type prostaglandin D synthase protein is related to the regulation of circadian sleep-wake cycles. The highly expression of the protein gene corresponds to the regressive circadian activity in the cavefish. The elastase digests elastin fibers in the skin, when the ultraviolet (UV) light reaches the dermis in the skin. It is suggested that the elastase genes is highly expressed because the surface fish has been exposed to sufficiently sunny conditions compared with the cavefish. On the other hand, in the cavefish, relatively highly expression of transferrin genes, lipid metabolism-related genes, sex differentiation-related genes were observed. Transferrin proteins are related to the oxygen transportation in the blood. To effectively transport oxygen in the blood of the cavefish living in the water with the lower concentration of oxygen at the dark cave environments, transferrin genes may be required to have relatively highly expression in the cavefish. The highly expression of lipid metabolism-related genes would be due to efficient acquirement of the energy under circumstances of at least temporary shortness in food. The highly expressions of the sex differentiation-related genes (diazepam binding inhibitor gene and granulin 1 gene) in the cavefish would be related to negative sex differentiation because of the lack of any kind of display/courtship in the cavefish. In conclusion, various adaptations for the perpetual darkness were evolutionarily raised in the diversification process from the surface fish to the cavefish. This is the first report that differences of gene expression between the surface fish and the cavefish are extensively examined.

(14) Evolutionary analysis of transcriptional coactivator MBF1

Qing-Xin Liu, Kazuho Ikeo, Susumu Hirose and Takashi Gojobori

--Transcriptional coactivators play a crucial role in gene expression by communicating between regulatory factors and the basal transcription machinery. How a coactivator evolves was poorly understood. We have taken a phylogenetic approach to analyze an evolutionarily history of coactivator MBF1 and TATA element-binding protein TBP. We found the following points. (1) MBF1 and TBP are evolutionarily conserved from Archaea to human to achieve transcription initiation. (2) Archaeal MBF1 carries a DNA-binding domain consisting of a Zn-ribbon which is absent from its eukaryotic counterparts. Thus, Eukaryotic MBF1 can interact with various regulatory factors while archaeal MBF1 has a single partner. (3) The interaction between MBF1 and TBP is also conserved from Archaea to human. In Archaea, MBF1 binds to TBP through Lysine or Asparagine to Glutamic acid interaction. In eukaryotes, MBF1 binds to TBP through Asparatic acid or Glutamic acid to Glutamine interaction. (4) The phylogenetic tree of MBF1 is very similar to that of TBP, indicating that MBF1 coevolved with TBP.

(15) Pigment cell lineage-specific expression activity of the ascidian tyrosinase-related gene

Reiko Toyoda, Akiko Kasai, Shigeru Sato, Shuichi Wada, Hidetoshi Saiga, Kazuho Ikeo, Takashi Gojobori, Takaharu Numakunai and Hiroaki Yamamoto

--Solitary ascidian tadpole larvae develop two types of black pigment cells in the major sensory organs of the brain. Such pigment cells have been demonstrated to express the melanogenic genes, tyrosinase and Tyrp/TRP (tyrosinase-related protein). To understand the genetic and developmental mechanisms underlying the differentiation of chordate pigment cells, we examined the function of the promoter region of Tyrp/TRP gene, an ascidian (Halocynthia roretzi) tyrosinase family gene. The expression of the gene in pigment cell lineage starts at the early-mid gastrula stages. To identify the transcriptional regulatory region of the gene allowing cell-type-specific expression, a deletion series of the HrTyrp 5' flanking region fused to a lacZ reporter gene was constructed and microinjected into ascidian fertilized eggs. The region of 73 bp in HrTyrp was identified as sufficient for expression in pigment cell-precursors of tailbud stage embryos. It is noteworthy that there is no M-box element highly conserved in the promoters for vertebrate tyrosinase family genes such as tyrosinase, Tyrp1/TRP-1 and Tyrp2/TRP-2 (Dct). Although the regulatory system of ascidian pigment-cell development is likely to contain most factors critical to vertebrate pigment-cell development, there might be critical differences in the mode of regulation, such as the developmental timing of interactions of factors, proteins and genes, involved in pigment cell differentiation and pigmentation.

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