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BackgroundElucidating the Transcription Factors (TFs) that drive the gene expression changes in a given experiment is a common question asked by researchers. The existing methods rely on the predicted Transcription Factor Binding Site (TFBS) to model the changes in the motif activity. Such methods only work for TFs that have a motif and assume the TF binding profile is the same in all cell types.ResultsGiven the wealth of the ChIP-seq data available for a wide range of the TFs in various cell types, we propose that gene expression modeling can be done using ChIP-seq “signatures” directly, effectively skipping the motif finding and TFBS prediction steps. We presentxcore, an R package that allows TF activity modeling based on ChIP-seq signatures and the user's gene expression data. We also providexcoredataa companion data package that provides a collection of preprocessed ChIP-seq signatures. We demonstrate thatxcoreleads to biologically relevant predictions using transforming growth factor beta induced epithelial-mesenchymal transition time-courses, rinderpest infection time-courses, and embryonic stem cells differentiated to cardiomyocytes time-course profiled with Cap Analysis Gene Expression.Conclusionsxcoreprovides a simple analytical framework for gene expression modeling using linear models that can be easily incorporated into differential expression analysis pipelines. Taking advantage of public ChIP-seq databases,xcorecan identify meaningful molecular signatures and relevant ChIP-seq experiments.

Elucidating the Transcription Factors (TFs) that drive the gene expression changes in a given experiment is one of the most common questions asked by researchers. The existing methods rely on the predicted Transcription Factor Binding Site (TFBS) to model the changes in the motif activity. Such methods only work for TFs that have a motif and assume the TF binding profile is the same in all cell types. Given the wealth of the ChIP-seq data available for a wide range of the TFs in various cell types, we propose that the modeling can be done using ChiP-seq “signatures” directly, effectively skipping the motif finding and TFBS prediction steps. We present xcore, an R package that allows Transcription Factor activity modeling based on their ChiP-seq signatures and user’s gene expression data. We also provide xcoredata a companion data package that provides a collection of preprocessed ChiP-seq signatures. We demonstrate that xcore leads to biologically relevant predictions using TGF-beta induced epithelial-mesenchymal transition and rinderpest infection time-course CAGE data as examples.Availability and Implementationxcore and xcoredata R packages are freely available at www.github.com/bkaczkowski/xcore and www.github.com/mcjmigdal/xcoredata.xcore user guide is available www.bkaczkowski.github.io/xcore/articles/xcore_vignette.html.Contactb.kaczkowski@gmail.com

Street trees are one of the important components of green infrastructure in urban area. This study surveyed the relationship between introduction trends of street trees in Japan and factors such as latitude and climatic conditions. The results showed that Japanese street trees tended to be mainly broad-leaved rather than coniferous, and many of these were native species. In addition, we found that street trees not only fulfill the roles of disaster prevention and disaster reduction, but also contribute to citizen welfare and recreation because a relatively large number were flowering trees (generally planted to enjoy flowers). Also, it was suggested that Cerasus sp. are the street trees that represent Japan, because could be confirmed in all 47 prefectures. Meanwhile, Japan is categorized into four groups with characteristic street trees introduced under the influence of the latitude and temperature in each group. Particularly, while the introduction of street trees is conducted by the individual municipalities (prefectures, cities, towns, villages, and areas), similarities in the response in terms of latitude and temperature regarding the street trees in Japan are being observed beyond the administrative borders. Incidentally, weeds which invade at the base of street trees were managed by periodic weeding, instead of any special treatment, is the most common weed management strategy in Japan. In addition, installation of shrubs, artificial inorganic cover such as concrete, and equipment, etc. has also been observed and may lead to a reduction in the weeding previously mentioned.

Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.

Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in theTP53gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Furtherin vitroandin vivoassays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.

RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.

Owing to safety concerns or insufficient efficacy, few drug candidates are approved for marketing. Drugs already on the market may be withdrawn due to adverse effects (AEs) discovered after market introduction. Comprehensively investigating the on-/off-target effects of drugs can help expose AEs during the drug development process. We have developed an integrative framework for systematic identification of on-/off-target pathways and elucidation of the underlying regulatory mechanisms, by combining promoter expression profiling after drug treatment with gene perturbation of the primary drug target. Expression profiles from statin-treated cells and HMG-CoA reductase knockdowns were analyzed using the framework, allowing for identification of not only reported adverse effects but also novel candidates of off-target effects from statin treatment, including key regulatory elements of on- and off-targets. Our findings may provide new insights for finding new usages or potential side effects of drug treatment.

Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp. no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest. However, it is still unclear whether pre-mRNAs are uniformly exported from the nucleus following SSA treatment. We performed RNA-seq analysis on nuclear and cytoplasmic fractions of SSA-treated cells. Our statistical analyses showed that intron retention is the major consequence of SSA treatment, and a small number of intron-containing pre-mRNAs leak into the cytoplasm. Using a series of reporter plasmids to investigate the roles of intronic sequences in the pre-mRNA leakage, we showed that the strength of the 5′ splice site affects pre-mRNA leakage. Additionally, we found that the level of pre-mRNA leakage is related to transcript length. These results suggest that the strength of the 5′ splice site and the length of the transcripts are determinants of the pre-mRNA leakage induced by SF3b inhibitors.

To present the clinical and genetic findings in two siblings with autosomal recessive retinitis pigmentosa (RP) and their non-symptomatic parents.We studied two siblings, a 48-year-old woman and her 44-year-old brother, and their parents. They had general ophthalmic examinations including ophthalmoscopy, perimetry, and electroretinography (ERG). Their whole exomes were analyzed by the next-generation sequence technique.The two siblings had night blindness for a long time, and clinical examinations revealed diffuse retinal degeneration with bone spicule pigmentation, constriction of the visual field, and non-recordable ERGs. Their parents were non-symptomatic and had normal fundi; however, their rod ERGs were reduced. Genetic examination revealed compound heterozygous mutations of I535N and H557Y in the PDE6B gene in the siblings, and the parents were heterozygous carriers of the mutations.Heterozygous mutation in the PDE6B gene can cause a reduction in the rod function to different degrees. The retinal function of non-symptomatic carriers of autosomal recessive RP should be evaluated with care.

To report novel mutations in the CRB1 gene in two patients with early-onset retinal dystrophy (EORD) and the longitudinal clinical course of EORD. The patients were two unrelated Japanese children. Standard ophthalmic examinations including perimetry, electroretinography, and optical coherence tomography were performed on both patients. Whole exomes of the patients and their nonsymptomatic parents were analyzed using a next-generation sequence (NGS) technique. Patient 1 was noted to have esotropia and hyperopia at age 3. His decimal best-corrected visual acuity (BCVA) was 0.6 OD and 0.3 OS at age 6 with de-pigmentation of the retinal pigment epithelium (RPE). At age 19, his central vision was still preserved; however, numerous pigment granules were present in the retina. NGS analysis revealed a p.R632X nonsense and c.652 + 1_652 + 4delGTAA splice site mutations in the CRB1 gene. Patient 2 was noted to have hyperopia at age 3. His decimal BCVA at age 6 was 0.3 OD and 0.4 OS with de-pigmented RPE. The degree of retinal pigmentation was increased but his BCVA was good until the age of 14 years. NGS analysis revealed c.652 + 1_652 + 4delGTAA and c.652 + 1_652 + 2insT splice site mutations in the CRB1 gene. The phenotypes of these novel mutations for EORD are typical of CRB1-associated EORD (LCA8). They were slowly progressive until the second decade of life.

To report the longitudinal clinical course of three Japanese patients from two families with Leber congenital amaurosis/early-onset retinal dystrophy (LCA/EORD), and the results of next-generation DNA sequences on them. The patients were three Japanese children: a 4-year-old girl, a 6-year-old boy, and a 3-year-old girl. Patients 1 and 2 were siblings, and patient 3 was from an unrelated family. Standard ophthalmic examinations including perimetry, electroretinography, optical coherence tomography, and ultrasonography were performed on each patient. The patients were observed for 28, 16, and 10 years. Whole exomes of the patients and their non-symptomatic parents were analyzed using a next-generation sequence technique. The decimal visual acuity varied between 0.07 and 0.6 at the initial visit and decreased to counting finger to hand motion in their teens. Funduscopy showed diffuse retinal and macular degeneration. During the follow-up period, a posterior staphyloma developed and the macular area became atrophic. Patient 1 developed cataracts in her early twenties. Genetic analysis revealed a homozygous A126V substitution in the RDH12 gene in all patients. The three patients with LCA/EORD had a progressive decrease of their vision with the formation of a posterior staphyloma. This is the first report of Japanese patients with LCA/EORD with a RDH12 mutation.

Gomafu (also referred to as RNCR2/MIAT) was originally identified as a noncoding RNA expressed in a particular set of neurons. Unlike protein-coding mRNAs, the Gomafu RNA escapes nuclear export and stably accumulates in the nucleus, making a unique nuclear compartment. Although recent studies have revealed the functional relevance of Gomafu in a series of physiological processes, the underlying molecular mechanism remains largely uncharacterized. In this report, we identified a chicken homologue of Gomafu using a comparative genomic approach to search for functionally important and conserved sequence motifs among evolutionarily distant species. Unexpectedly, we found that all Gomafu RNA examined shared a distinctive feature: tandem repeats of UACUAAC, a sequence that has been identified as a conserved intron branch point in the yeast Saccharomyces cerevisiae. The tandem UACUAAC Gomafu RNA repeats bind to the SF1 splicing factor with a higher affinity than the divergent branch point sequence in mammals, which affects the kinetics of the splicing reaction in vitro. We propose that the Gomafu RNA regulates splicing efficiency by changing the local concentration of splicing factors within the nucleus.

Summary The generation of new blood vessels via angiogenesis is critical for meeting tissue oxygen demands. A role for adult stem cells in this process remains unclear. Here, we identified CD157 (bst1, bone marrow stromal antigen 1) as a marker of tissue-resident vascular endothelial stem cells (VESCs) in large arteries and veins of numerous mouse organs. Single CD157 + VESCs form colonies in vitro and generate donor-derived portal vein, sinusoids, and central vein endothelial cells upon transplantation in the liver. In response to injury, VESCs expand and regenerate entire vasculature structures, supporting the existence of an endothelial hierarchy within blood vessels. Genetic lineage tracing revealed that VESCs maintain large vessels and sinusoids in the normal liver for more than a year, and transplantation of VESCs rescued bleeding phenotypes in a mouse model of hemophilia. Our findings show that tissue-resident VESCs display self-renewal capacity and that vascular regeneration potential exists in peripheral blood vessels.

Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process.

We have previously reported clinical features of two siblings, a sister with complete achromatopsia (ACHM) and a brother with incomplete ACHM, in a consanguineous Japanese family. With the current study, we intended to identify a disease-causing mutation in the siblings and to investigate why the phenotypes of the siblings differed.We performed a comprehensive ophthalmic examination for each sibling and parent. Whole-exome and Sanger sequencing were performed on genomic DNA. Molecular modeling was analyzed in an in silico study.The ophthalmic examination revealed severe macular atrophy in the older female sibling at 30 years of age and mild macular atrophy in the brother at 26 years of age. The genetic analysis identified a novel homozygous PDE6C mutation (p.E591K) as the disease-causing allele in the siblings. Each parent was heterozygous for the mutation. Molecular modeling showed that the mutation could cause a conformational change in the PDE6C protein and result in reduced phosphodiesterase activity. We also identified an OPN1SW mutation (p.G79R), which is associated with congenital tritan deficiencies, in the sister and the father but not in the brother.A novel homozygous PDE6C mutation was identified as the cause of ACHM. In addition, we identified an OPN1SW mutation in the sibling with complete ACHM, which might explain the difference in phenotype (complete versus incomplete ACHM) between the siblings.

The objective of this research is to evaluate the importance of vegetation of retaining walls made of natural stones (i.e., masonry revetment) in plant biodiversity. In this paper, plant compositions and the characteristics of masonry revetments were surveyed in terraced fields in Toho Village, southern Japan. In total, 43 families and 88 species were recorded in the spaces of the masonry revetments. Of these 88 species, 68 (77.3%) were herbaceous, excluding 13 (14.8%) ferns, and 7 (8.0%) species were woody plants. Native species accounted for 69 (78.4%) of the 88 species. Furthermore, numerous species not found in the horizontal environments around the terraced fields were also seen in the spaces of the masonry revetments. From these results, the authors consider that masonry revetments provide a habitat for plants and therefore contribute toward the conservation of plant biodiversity on a local scale.

Several recent studies indicate that mammals and other organisms produce large numbers of RNA transcripts that do not correspond to known genes. It has been suggested that these transcripts do not encode proteins, but may instead function as RNAs. However, discrimination of coding and non-coding transcripts is not straightforward, and different laboratories have used different methods, whose ability to perform this discrimination is unclear. In this study, we examine ten bioinformatic methods that assess protein-coding potential and compare their ability and congruency in the discrimination of non-coding from coding sequences, based on four underlying principles: open reading frame size, sequence similarity to known proteins or protein domains, statistical models of protein-coding sequence, and synonymous versus non-synonymous substitution rates. Despite these different approaches, the methods show broad concordance, suggesting that coding and non-coding transcripts can, in general, be reliably discriminated, and that many of the recently discovered extra-genic transcripts are indeed non-coding. Comparison of the methods indicates reasons for unreliable predictions, and approaches to increase confidence further. Conversely and surprisingly, our analyses also provide evidence that as much as approximately 10% of entries in the manually curated protein database Swiss-Prot are erroneous translations of actually non-coding transcripts.

Manual curation has long been held to be the “gold standard” for functional annotation of DNA sequence. Our experience with the annotation of more than 20,000 full-length cDNA sequences revealed problems with this approach, including inaccurate and inconsistent assignment of gene names, as well as many good assignments that were difficult to reproduce using only computational methods. For the FANTOM2 annotation of more than 60,000 cDNA clones, we developed a number of methods and tools to circumvent some of these problems, including an automated annotation pipeline that provides high-quality preliminary annotation for each sequence by introducing an “uninformative filter” that eliminates uninformative annotations, controlled vocabularies to accurately reflect both the functional assignments and the evidence supporting them, and a highly refined, Web-based manual annotation tool that allows users to view a wide array of sequence analyses and to assign gene names and putative functions using a consistent nomenclature. The ultimate utility of our approach is reflected in the low rate of reassignment of automated assignments by manual curation. Based on these results, we propose a new standard for large-scale annotation, in which the initial automated annotations are manually investigated and then computational methods are iteratively modified and improved based on the results of manual curation.

The Mouse Genome Sequencing Consortium and the RIKEN Genome Exploration Research grouphave generated large sets of sequence data representing the mouse genome and transcriptome, respectively. These data provide a valuable foundation for genomic research. The challenges for the informatics community are how to integrate these data with the ever-expanding knowledge about the roles of genes and gene products in biological processes, and how to provide useful views to the scientific community. Public resources, such as the National Center for Biotechnology Information (NCBI; http://www.ncbi.nih.gov), and model organism databases, such as the Mouse Genome Informatics database (MGI; http://www.informatics.jax.org), maintain the primary data and provide connections between sequence and biology. In this paper, we describe how the partnership of MGI and NCBI LocusLink contributes to the integration of sequence and biology, especially in the context of the large-scale genome and transcriptome data now available for the laboratory mouse. In particular, we describe the methods and results of integration of 60,770 FANTOM2 mouse cDNAs with gene records in the databases of MGI and LocusLink.

The identification of coding sequences (CDS) is an important step in the functional annotation of genes. CDS prediction for mammalian genes from genomic sequence is complicated by the vast abundance of intergenic sequence in the genome, and provides little information about how different parts of potential CDS regions are expressed. In contrast, mammalian gene CDS prediction from cDNA sequence offers obvious advantages, yet encounters a different set of complexities when performed on high-throughput cDNA (HTC) sequences, such as the set of 60,770 cDNAs isolated from full-length enriched libraries of the FANTOM2 project. We developed a CDS annotation strategy that uses a variety of different CDS prediction programs to annotate the CDS regions of FANTOM2 cDNAs. These include rsCDS, which uses sequence similarity to known proteins; ProCrest; Longest-ORF and Truncated-ORF, which are ab initio based predictors; and finally, DECODER and NCBI CDS predictor, which use a combination of both principles. Aided by graphical displays of these CDS prediction results in the context of other sequence similarity results for each cDNA, FANTOM2 CDS inspection by curators and follow-up quality control procedures resulted in high quality CDS predictions for a total of 14,345 FANTOM2 clones.

Purpose. To investigate genetic and clinical features of patients with rhodopsin (RHO) mutations in two Japanese families with autosomal dominant retinitis pigmentosa (adRP).Methods. Whole-exome sequence analysis was performed in ten adRP families. IdentifiedRHOmutations for the cosegregation analysis were confirmed by Sanger sequencing. Ophthalmic examinations were performed to evaluate the RP phenotypes. The impact of theRHOmutation on the rhodopsin conformation was examined by molecular modeling analysis.Results. In two adRP families, we identified twoRHOmutations (c.377G>T (p.W126L) and c.1036G>C (p.A346P)), one of which was novel. Complete cosegregation was confirmed for each mutation exhibiting the RP phenotype in both families. Molecular modeling predicted that the novel mutation (p.W126L) might impair rhodopsin function by affecting its conformational transition in the light-adapted form. Clinical phenotypes showed that patients with p.W126L exhibited sector RP, whereas patients with p.A346P exhibited classic RP.Conclusions. Our findings demonstrated that the novel mutation (p.W126L) may be associated with the phenotype of sector RP. Identification ofRHOmutations is a very useful tool for predicting disease severity and providing precise genetic counseling.

Objective The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population. Methods In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed. Results Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation. Conclusions This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin.

Polar flagellum-defective mutants (Pof- Laf-) have been isolated from a lateral flagella-defective mutant (Pof+ Laf-). Among these Pof- Laf- mutants, polar-filamentless mutants, which have the hook structure but not the filament, were identified by electron microscopy. Their hooks were covered with a sheath structure which is contiguous to the outer membrane. The filament proteins, flagellins, were shed into the culture medium of these mutants. These flagellins could be sedimented by high-speed centrifugation even after heat or low pH treatment whereas the depolymerized flagellin of the Pof+ strain was degraded by these treatments. After Triton X-100 treatment, most flagellin of the filamentless mutants could no longer be sedimented, and was degraded. We observed vesicle-like structures on the tips of the hooks and in the flagellin fraction sedimented by high speed centrifugation. These results suggest that flagellin of the filamentless mutants is not assembled into the tip of the hook, but is excreted together with a membrane structure which is probably the sheath of polar flagella.

Vibrio alginolyticus has two types of flagella, polar (Pof) and lateral (Laf). From a Laf-defective mutant (Pof+Laf-), polar-flagellar-length mutants which have short Pof and long Pof were isolated. The mean lengths of the helical axis in wild-type, short and long Pof were 5.5.0.9 μm, 2.5.0.6 μm and 11.2.3.6 μm, respectively. The swimming speeds of the short- and long-Pof mutants were slower than that of the wild-type strain. The relationship between swimming speed and flagellar length in a population of mutant cells was examined. In the short-Pof mutant, the decrease of swimming speed seemed to be derived from the decrease in flagellar length. In the long-Pof mutant, there was almost no correlation between swimming speed and flagellar length, and the slow swimming was explained by the helical shape of the flagella, whose pitch and radius were 1.4 μm and 0.062 μm, respectively, whereas those of the wild-type flagella were 1.5 μm and 0.16 μm. The relative amounts of the various molecular components of the long Pof were different from those of the wild-type or the short Pof. This seems to be the reason for the difference in flagellar shape and length, though the mutation may be pleiotropic and affect flagellar function or regulation.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

Non-protein-coding RNAs (ncRNAs) are increasingly being recognized as having important regulatory roles. Although much recent attention has focused on tiny 22- to 25-nucleotide microRNAs, several functional ncRNAs are orders of magnitude larger in size. Examples of such macro ncRNAs include Xist and Air, which in mouse are 18 and 108 kilobases (Kb), respectively. We surveyed the 102,801 FANTOM3 mouse cDNA clones and found that Air and Xist were present not as single, full-length transcripts but as a cluster of multiple, shorter cDNAs, which were unspliced, had little coding potential, and were most likely primed from internal adenine-rich regions within longer parental transcripts. We therefore conducted a genome-wide search for regional clusters of such cDNAs to find novel macro ncRNA candidates. Sixty-six regions were identified, each of which mapped outside known protein-coding loci and which had a mean length of 92 Kb. We detected several known long ncRNAs within these regions, supporting the basic rationale of our approach. In silico analysis showed that many regions had evidence of imprinting and/or antisense transcription. These regions were significantly associated with microRNAs and transcripts from the central nervous system. We selected eight novel regions for experimental validation by northern blot and RT-PCR and found that the majority represent previously unrecognized noncoding transcripts that are at least 10 Kb in size and predominantly localized in the nucleus. Taken together, the data not only identify multiple new ncRNAs but also suggest the existence of many more macro ncRNAs like Xist and Air., Synopsis The human genome has been sequenced, and, intriguingly, less than 2% specifies the information for the basic protein building blocks of our bodies. So, what does the other 98% do? It now appears that the mammalian genome also specifies the instructions for many previously undiscovered “non protein-coding RNA” (ncRNA) genes. However, what these ncRNAs do is largely unknown. In recent years, strategies have been designed that have successfully identified hundreds of short ncRNAs—termed microRNAs—many of which have since been shown to act as genetic regulators. Also known to be functionally important are a handful of ncRNAs orders of magnitude larger in size than microRNAs. The availability of complete genome and comprehensive transcript sequences allows for the systematic discovery of more large ncRNAs. The authors developed a computational strategy to screen the mouse genome and identify large ncRNAs. They detected existing large ncRNAs, thus validating their approach, but, more importantly, discovered more than 60 other candidates, some of which were subsequently confirmed experimentally. This work opens the door to a virtually unexplored world of large ncRNAs and beckons future experimental work to define the cellular functions of these molecules.

FANTOM DB, the database of Functional Annotation of RIKEN Mouse cDNA Clones, is designed to store sequence information of RIKEN full-length enriched mouse cDNA clones, graphical views of sequence analysis results, curated functional annotation information and additional descriptions, including Gene Ontology terms. RIKEN’s Mouse Gene Encyclopedia Project aims to collect full-length enriched cDNA clones from various mouse tissues, determine the full-length nucleotide sequences, infer their chromosomal locations by computer and characterize gene expression patterns. FANTOM DB has been developed to facilitate this work and to facilitate functional genomic studies such as positional candidate cloning, cDNA microarrays and protein interaction analyses. FANTOM DB contains 21 076 full-length cDNA sequences with rich functional annotations and is publicly available. FANTOM DB thus provides curated functional annotation to RIKEN full-length enriched mouse clones, and has links to other public resources. FANTOM DB can be accessed at http://fantom.gsc.riken.go.jp/db/.

The axial length of the polar flagellum (Pof) of Vibrio alginolyticus is about 5 microm. We previously isolated mutants that make abnormally long flagella. The swarm sizes of these mutants in a soft agar plate are smaller than that of a wild-type strain. We cloned a DNA fragment into the pMF209 plasmid that restored the swarming ability of the long-Pof strain V10578. The swimming speed and flagellar length of these transformants were almost equal to the wild-type values. The amounts of PF47 flagellin and PF60 sheath-associated protein, which increased in the long-Pof mutants, were retrieved to almost the wild-type level in the transformants. The plasmid pMF209 contained only a 143 bp chromosomal fragment whose sequence is about 80% similar to that of the motX promoter region of V parahaemolyticus. We speculate that this sequence interacts with a regulatory protein that controls Pof expression. The mutation causing the long-Pof phenotype may be in the gene encoding this protein or in the control region of a structural gene that is regulated by this protein.

A bombyxin gene encoding precursor molecule for bombyxin-IV, one of the insulin-related neurosecretory peptide of the silkmoth Bombyx mori, has been cloned and characterized. The nucleotide sequence of this gene and its deduced amino acid sequence deviate moderately from those characterized previously for the family A, B, C and D bombyxin genes. The gene encoding the bombyxin-IV precursor was therefore defined into a novel family E and designated as gene E1. The bombyxin E1 transcript in Bombyx brain was shown to locate in four pairs of medial neurosecretory cells, which also produce other bombyxin family mRNAs, and the amount of the E1 transcript did not change markedly during the fifth larval instar. Genomic Southern hybridization indicated that the Bombyx haploid genome contained a single copy of the bombyxin family E gene.