Comparative genomics study reveals Red Sea Bacillus with characteristics associated with potential microbial cell factories (MCFs).

Recent advancements in the use of microbial cells for scalable production of industrial enzymes encourage exploring new environments for efficient microbial cell factories (MCFs). Here, through a comparison study, ten newly sequenced Bacillus species, isolated from the Rabigh Harbor Lagoon on the Red Sea shoreline, were evaluated for their potential use as MCFs. Phylogenetic analysis of 40 representative genomes with phylogenetic relevance, including the ten Red Sea species, showed that the Red Sea species come from several colonization events and are not the result of a single colonization followed by speciation. Moreover, clustering reactions in reconstruct metabolic networks of these Bacillus species revealed that three metabolic clades do not fit the phylogenetic tree, a sign of convergent evolution of the metabolism of these species in response to special environmental adaptation. We further showed Red Sea strains Bacillus paralicheniformis (Bac48) and B. halosaccharovorans (Bac94) had twice as much secreted proteins than the model strain B. subtilis 168. Also, Bac94 was enriched with genes associated with the Tat and Sec protein secretion system and Bac48 has a hybrid PKS/NRPS cluster that is part of a horizontally transferred genomic region. These properties collectively hint towards the potential use of Red Sea Bacillus as efficient protein secreting microbial hosts, and that this characteristic of these strains may be a consequence of the unique ecological features of the isolation environment.

DDBJ with new system and face.

DDBJ (http://www.ddbj.nig.ac.jp) collected and released 1 880 115 entries or 1 134 086 245 bases in the period from July 2006 to June 2007. The released data contains the high-throughput cDNAs of cricket and high-quality draft genome of medaka among others. Our computer system has been upgraded since March 2007. Another new aspect is an efficient data retrieval tool that has recently been equipped and served at DDBJ. It is called All-round Retrieval for Sequence and Annotation, which enables the user to search for keywords also in the Feature/Qualifier of the International Nucleotide Sequence Database Collaboration (http://www.insdc.org/). We will also replace our home page with a more efficient one by the end of 2007.

Evolution of protein-protein interaction network.

Protein-protein interactions (PPIs) are one of the most important components of biological networks. It is important to understand the evolutionary process of PPIs in order to elucidate how the evolution of biological networks has contributed to diversification of the existent organisms. We focused on the evolutionary rates of proteins involved with PPIs, because it had been shown that for a given protein-coding gene the number of its PPIs in a biological network was one of the important factors in determining the evolutionary rate of the gene. We studied the evolutionary rates of duplicated gene products that were involved with PPIs, reviewing the current situation of this subject. In addition, we focused on how the evolutionary rates of proteins were influenced by the characteristic features of PPIs. We, then, concluded that the evolutionary rates of the proteins in the PPI networks were strongly influenced by their PPI partners. Finally, we emphasized that evolutionary considerations of the PPI proteins were very important for understanding the building up of the current PPI networks.

Molecular characterization of the developmental gene in eyes: through data-mining on integrated transcriptome databases.

OBJECTIVES: Our aim was to utilize publicly available and proprietary sources to discover candidate genes important for ocular development. DESIGN AND METHODS: The collated information on our 5092 non-redundant clusters was grouped and functional annotation was conducted using gene ontology (FatiGO) for categorizing them with respect to molecular function. The web-based viewer technological platform (H-InvDB) was employed for transcription analyses of in-house high quality fetal eye Expressed Sequence Tags (ESTs). Eye-specific ESTs were also analyzed across species by using EMBEST. RESULTS: According to adult eye cDNA libraries, nucleic acid binding and cell structure/cytoskeletal protein genes were the most abundant among the ESTs of fetal eyes. Using cDNA assembly in H-InvDB, 20 (80%) of the 25 most commonly expressed genes in the human eye are also expressed in extraocular tissues. The crystalline gamma S gene is highly expressed in the eye, but not in other tissues. We used EMBEST to compare human fetal eye and octopus eye ESTs and the expression similarity was low (1.6%). This indicated that our fetal eye library contains genes necessary for the developmental process and biological function of the eye, which may not be expressed in the fully developed octopus eyes. The human fetal eye cDNA library also contained highly abundant eye tissue genes, including alphaA-crystallin, eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), bestrophin (VMD2), cystatin C, and transforming growth factor, beta-induced (BIGH3). CONCLUSIONS: Our annotated EST set provides a valuable resource for gene discovery and functional genomic analysis. This display will help to appreciate the strengths and weaknesses of the different technological platforms, so that in future studies the maximum amount of beneficial information can be derived from the appropriate use of each method.

Genomic annotation of 15,809 ESTs identified from pooled early gestation human eyes.

To complement cDNA libraries from the human eye at early gestation and to discover candidate genes associated with early ocular development, we used freshly dissected human eyeballs from week 9-14 of gestation to construct the early human fetal eye cDNA library. A total of 15,809 clones were isolated and sequenced from the unamplified and unnormalized library. We screened 11,246 good-quality ESTs, leading to the identification of 5,534 nonredundant clusters. Among them, 4,010 (72%) genes matched in the human protein database (Ensembl). The remaining 28% (1,524) corresponded to potentially novel or previously unidentified ESTs. We used BLASTX to compare our EST data with eight organisms and found common expression of a high portion of genes: Caenorhabditis briggsae (26%), Caenorhabditis elegans (27%), Anopheles gambiae (37%), Drosophila melanogaster (32%), Danio rerio (42%), Fugu rubripes (49%), Rattus norvegicusvalitus (52%), and Mus musculus (59%). Nevertheless, 48% (2,680 of 5,534) of the genes expressed in the early developing eye were not shared with current NEIBank human eye cDNA data. In addition, eight known retinal disease genes existed in our ESTs. Among them, six (COL11A1, BBS5, PDE6B, OAT, VMD2, and PGK1) were conserved among the genomes of other organisms, indicating that our annotated EST set provides not only a valuable resource for gene discovery and functional genomic analysis but also for phylogenetic analysis. Our foremost early gestation human eye cDNA library could provide detailed comparisons across species to identify physiological functions of genes and to elucidate evolutionary mechanisms.

DDBJ in collaboration with mass-sequencing teams on annotation.

In the past year, we at DDBJ (DNA Data Bank of Japan; http://www.ddbj.nig.ac.jp) collected and released 1,066,084 entries or 718,072,425 bases including the whole chromosome 22 of chimpanzee, the whole-genome shotgun sequences of silkworm and various others. On the other hand, we hosted workshops for human full-length cDNA annotation and participated in jamborees of mouse full-length cDNA annotation. The annotated data are made public at DDBJ. We are also in collaboration with a RIKEN team to accept and release the CAGE (Cap Analysis Gene Expression) data under a new category, MGA (Mass Sequences for Genome Annotation). The data will be useful for studying gene expression control in many aspects.

Proteomic signatures and aberrations of mouse embryonic stem cells containing a single human chromosome 21 in neuronal differentiation: an in vitro model of Down syndrome.

Neurodegeneration in fetal development of Down syndrome (DS) patients is proposed to result in apparent neuropathological abnormalities and to contribute to the phenotypic characteristics of mental retardation and premature development of Alzheimer disease. In order to identify the aberrant and specific genes involved in the early differentiation of DS neurons, we have utilized an in vitro neuronal differentiation system of mouse ES cells containing a single human chromosome 21 (TT2F/hChr21) with TT2F parental ES cells as a control. The paired protein extracts from TT2F and TT2F/hChr21 cells at several stages of neuronal differentiation were subjected to two-dimensional polyacrylamide gel electrophoresis protein separation followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to identify the proteins differentially expressed between TT2F and TT2F/hChr21 cells. We provide here a novel set of specific gene products altered in early differentiating DS neuronal cells, which differs from that identified in adult or fetal brain with DS. The aberrant protein expression in early differentiating neurons, due to the hChr21 gene dosage effects or chromosomal imbalance, may affect neuronal outgrowth, proliferation and differentiation, producing developmental abnormalities in neural patterning, which eventually leads to formation of a suboptimal functioning neuronal network in DS.

Notes on the maximum likelihood estimation of haplotype frequencies.

The maximum likelihood estimation (MLE) is one of the most popular ways to estimate haplotype frequencies of a population with genotype data whose linkage phases are unknown. The MLE is commonly implemented in the use of the Expectation-Maximization (EM) algorithm. It is known that the EM algorithm carries the risk that an estimator may converge erroneously to one of the local maxima or saddle points of the likelihood surface, resulting in serious errors in the MLE of haplotype frequencies. In this note, by theoretical treatments we present the necessary and sufficient conditions that the local maxima or saddle points on the likelihood surface appear. As a rule of thumb, that the difference between the coupling and repulsive haplotype frequencies in phase known individuals is 3/2 times larger than the frequency of phase ambiguous individuals is the sufficient condition that the likelihood surface is unimodal. Moreover, we present the analytic solution to the biallelic two-locus problem, and construct a general algorithm to obtain the global maximum.

The bioinformatics challenges in comparative analysis of cereal genomes-an overview.

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. In this overview the concepts and technology underpinning bioinformatics as applied to comparative genomic analysis are considered in the context of other manuscripts appearing in this issue of Functional and Integrative Genomics.

DDBJ in the stream of various biological data.

In the past year we at DDBJ (http://www.ddbj.nig. ac.jp) have made a steady increase in the number of data submissions with a 50.6% increment in the number of bases or 46.5% increment in the number of entries. Among them the genome data of man, ascidian and rice hold the top three. Our activity has extended to providing a tool that enables sequence retrieval using regular expressions, and to launching our SOAP server and web services to facilitate the acquisition of proper data and tools from a huge number of biological data resources on websites worldwide. We have also opened our public gene expression database, CIBEX.

Phylogenetic analysis of Vietnamese isolates of feline immunodeficiency virus: genetic diversity of subtype C.

Phylogenetic relationships of novel Vietnamese strains of feline immunodeficiency virus (FIV) were analysed. One Vietnamese strain was found to cluster with subtype D, which was previously known only in Japan, while the other seven strains were placed with members of subtype C. Calculation of the relative numbers of mutations resulting in amino acid and silent changes in FIV env subtypes suggested that subtype C isolates may be less structurally constrained (potentially more pathogenic) than subtype B.

Human cytomegalovirus glycoprotein N (gpUL73-gN) genomic variants: identification of a novel subgroup, geographical distribution and evidence of positive selective pressure.

Human cytomegalvirus (HCMV) ORF UL73 is a polymorphic locus, encoding the viral glycoprotein gpUL73-gN, a component of the gC-II envelope complex. The previously identified gN genomic variants, denoted gN-1, gN-2, gN-3 and gN-4, were further investigated in this work by analysing a large panel of HCMV clinical isolates collected from all over the world (223 samples). Sequencing and phylogenetic analysis confirmed the existence of the four gN genotypes, but also allowed the identification of a novel subgroup belonging to the gN-3 genotype, which was designated gN-3b. The number of non-synonymous (d(N)) and synonymous (d(S)) nucleotide substitutions and their ratio (d(N)/d(S)) were estimated among the gN genotypes to evaluate the possibility of positive selection. Results showed that the four variants evolved by neutral (random) selection, but that the gN-3 and gN-4 genotypes are maintained by positive selective pressure. The 223 HCMV clinical isolates were subdivided according to their geographical origin, and four main regions of gN prevalence were identified: Europe, China, Australia and Northern America. The gN variants were found to be widespread and represented within the regions analysed without any significant difference, and no new genotype was detected. Finally, for clinical and epidemiological purposes, a rapid and low-cost method for genetic grouping of the HCMV clinical isolates was developed based on the RFLP revealed by SacI, ScaI and SalI digestion of the PCR-amplified UL73 sequence. This technique enabled us to distinguish all four gN genomic variants and also their subtypes.

DNA Data Bank of Japan (DDBJ) in XML.

The DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp) has collected and released more entries and bases than last year. This is mainly due to large-scale submissions from Japanese sequencing teams on mouse, rice, chimpanzee, nematoda and other organisms. The contributions of DDBJ over the past year are 17.3% (entries) and 10.3% (bases) of the combined outputs of the International Nucleotide Sequence Databases (INSD). Our complete genome sequence database, Genome Information Broker (GIB), has been improved by incorporating XML. It is now possible to perform a more sophisticated database search against the new GIB than the ordinary BLAST or FASTA search.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA.

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.

DNA Data Bank of Japan (DDBJ) for genome scale research in life science.

The DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp) has made an effort to collect as much data as possible mainly from Japanese researchers. The increase rates of the data we collected, annotated and released to the public in the past year are 43% for the number of entries and 52% for the number of bases. The increase rates are accelerated even after the human genome was sequenced, because sequencing technology has been remarkably advanced and simplified, and research in life science has been shifted from the gene scale to the genome scale. In addition, we have developed the Genome Information Broker (GIB, http://gib.genes.nig.ac.jp) that now includes more than 50 complete microbial genome and Arabidopsis genome data. We have also developed a database of the human genome, the Human Genomics Studio (HGS, http://studio.nig.ac.jp). HGS provides one with a set of sequences being as continuous as possible in any one of the 24 chromosomes. Both GIB and HGS have been updated incorporating newly available data and retrieval tools.

Early detection of G + C differences in bacterial species inferred from the comparative analysis of the two completely sequenced Helicobacter pylori strains.

Identifying the G + C difference between closely related bacterial species or between different strains of the same species is one of the first steps in understanding the evolutionary mechanisms accounting for the differences observed among bacterial species. The G + C content can be one of the most important factors in the evolution of genomic structures. In this paper, we describe a new method for detecting an initial stage of differentiation of the G + C content at the third codon base position between two strains of the same bacterial species. We apply this method to the two strains of Helicobacter pylori. A group of genes is detected with large variations of G + C in the third positions-apparently genes of early response to pressures of changing G + C. We discuss our findings from the viewpoint of genomic evolution.

Intragenic variation of synonymous substitution rates is caused by nonrandom mutations at methylated CpG.

It has been observed that synonymous substitution rates vary among genes in various organisms, although the cause of the variation is unresolved. At the intragenic level, however, the variation of synonymous substitutions is somewhat controversial. By developing a rigorous statistical test and applying the test to 418 homologous gene pairs between mouse and rat, we found that more than 90% of gene pairs showed a statistical significance in intragenic variation of synonymous substitution rates. Moreover, by examining all conceivable possibilities for the cause of the variation, we successfully found that intragenic variation of synonymous substitutions in mammalian genes is caused mainly by a nonrandom mutation due to the methylation of CpG dinucleotides rather than by functional constraints.

Positively selected amino acid sites in the entire coding region of hepatitis C virus subtype 1b.

To predict the amino acid sites important for the clearance of hepatitis C virus (HCV) subtype 1b in vivo, positively selected amino acid sites were detected by analyzing the sequence data collected from the international DNA databank. The rate of nonsynonymous substitutions per nonsynonymous site was compared with that of synonymous substitutions per synonymous site for each codon site in the entire coding region. As a result, 13 out of 3010 amino acid sites were found to be positively selected. Among the 13 positively selected amino acid sites, eight were located in the structural proteins and five were in the nonstructural proteins. Moreover, eight were located in B-cell epitopes and two were in T-cell epitopes. These observations suggest that both the antibody and the cytotoxic T lymphocyte are involved in the clearance of HCV subtype 1b in vivo. These positively selected amino acid sites represent candidate vaccination targets for HCV subtype 1b.