DNA Research - Advance Access

Characteristics and Prediction of RNA Editing Sites in Transcripts of the Moss Takakia lepidozioides Chloroplast

Yura, K., Miyata, Y., Arikawa, T., Higuchi, M., Sugita, M. — 2008-07-23

RNA editing in land plant organelles is a process primarily involving the conversion of cytidine to uridine in pre-mRNAs. The process is required for gene expression in plant organelles, because this conversion alters the encoded amino acid residues and improves the sequence identity to homologous proteins. A recent study uncovered that proteins encoded in the nuclear genome are essential for editing site recognition in chloroplasts; the mechanisms by which this recognition occurs remain unclear. To understand these mechanisms, we determined the genomic and cDNA sequences of moss Takakia lepidozioides chloroplast genes, then computationally analyzed the sequences within –30 to +10 nucleotides of RNA editing sites (neighbor sequences) likely to be recognized by trans-factors. As the T. lepidozioides chloroplast has many RNA editing sites, the analysis of these sequences provides a unique opportunity to perform statistical analyses of chloroplast RNA editing sites. We divided the 302 obtained neighbor sequences into eight groups based on sequence similarity to identify group-specific patterns. The patterns were then applied to predict novel RNA editing sites in T. lepidozioides transcripts; ~60% of these predicted sites are true editing sites. The success of this prediction algorithm suggests that the obtained patterns are indicative of key sites recognized by trans-factors around editing sites of T. lepidozioides chloroplast genes.

Soybean Seed Extracts Preferentially Express Genomic Loci of Bradyrhizobium japonicum in the Initial Interaction with Soybean, Glycine max (L.) Merr

2008-07-17

Initial interaction between rhizobia and legumes actually starts via encounters of both partners in the rhizosphere. In this study, the global expression profiles of Bradyrhizobium japonicum USDA 110 in response to soybean (Glycine max) seed extracts (SSE) and genistein, a major soybean-released isoflavone for nod genes induction of B. japonicum, were compared. SSE induced many genomic loci as compared with genistein (5.0 µM), nevertheless SSE-supplemented medium contained 4.7 µM genistein. SSE markedly induced four predominant genomic regions within a large symbiosis island (681 kb), which include tts genes (type III secretion system) and various nod genes. In addition, SSE-treated cells expressed many genomic loci containing genes for polygalacturonase (cell-wall degradation), exopolysaccharide synthesis, 1-aminocyclopropane-1-carboxylate deaminase, ribosome proteins family and energy metabolism even outside symbiosis island. On the other hand, genistein-treated cells exclusively showed one expression cluster including common nod gene operon within symbiosis island and six expression loci including multidrug resistance, which were shared with SSE-treated cells. Twelve putatively regulated genes were indeed validated by quantitative RT-PCR. Several SSE-induced genomic loci likely participate in the initial interaction with legumes. Thus, these results can provide a basic knowledge for screening novel genes relevant to the B. japonicum- soybean symbiosis.

Novel DNA Microarray System for Analysis of Nascent mRNAs

2008-07-16

Transcriptional activation and repression are a key step in the regulation of all cellular activities. The development of comprehensive analysis methods such as DNA microarray has advanced our understanding of the correlation between the regulation of transcription and that of cellular mechanisms. However, DNA microarray analysis based on steady-state mRNA (total mRNA) does not always correspond to transcriptional activation or repression. To comprehend these transcriptional regulations, the detection of nascent RNAs is more informative. Although the nuclear run-on assay can detect nascent RNAs, it has not been fully applied to DNA microarray analysis. In this study, we have developed a highly efficient method for isolating bromouridine-labeled nascent RNAs that can be successfully applied to DNA microarray analysis. This method can linearly amplify small amounts of mRNAs with little bias. Furthermore, we have applied this method to DNA microarray analysis from mouse G₂-arrested cells and have identified several genes that exhibit novel expression profiles. This method will provide important information in the field of transcriptome analysis of various cellular processes.

Expression Profiling of PBMC-based Diagnostic Gene Markers Isolated from Vasculitis Patients

2008-06-17

Vasculitis (angiitis) is a systemic autoimmune disease that often causes fatal symptoms. We aimed to isolate cDNA markers that would be useful for diagnosing not only vasculitis but also other autoimmune diseases. For this purpose, we used stepwise subtractive hybridization and cDNA microarray analyses to comprehensively isolate the genes whose expressions are augmented in peripheral blood mononuclear cells (PBMCs) pooled from vasculitis patients. Subsequently, we used quantitative real-time polymerase chain reaction (qRT–PCR) to examine the mRNA levels of each candidate gene in individual patients. These analyses indicated that seven genes exhibit remarkably augmented expression in many vasculitis patients. Of these genes, we analyzed G0/G1 switch gene 2 (G0S2) further because G0S2 expression is also enhanced in the PBMCs of patients with systemic lupus erythematodes (SLE). We generated G0S2 transgenic mice that ubiquitously overexpress human G0S2. Although we did not observe any obvious vasculitis-related histopathologic findings in these mice, these mice are unhealthy as they produce only few offspring and showed elevated serum levels of two autoimmunity-related antibodies, anti-nuclear antibody, and anti-double strand DNA antibody. Thus, our large-scale gene profiling study may help finding sensitive and specific DNA markers for diagnosing autoimmune diseases including vasculitis and SLE.

Determination of the Genome Sequence of Porphyromonas gingivalis Strain ATCC 33277 and Genomic Comparison with Strain W83 Revealed Extensive Genome Rearrangements in P. gingivalis

2008-06-03

The gram-negative anaerobic bacterium Porphyromonas gingivalis is a major causative agent of chronic periodontitis. Porphyromonas gingivalis strains have been classified into virulent and less-virulent strains by mouse subcutaneous soft tissue abscess model analysis. Here, we present the whole genome sequence of P. gingivalis ATCC 33277, which is classified as a less-virulent strain. We identified 2090 protein-coding sequences (CDSs), 4 RNA operons, and 53 tRNA genes in the ATCC 33277 genome. By genomic comparison with the virulent strain W83, we identified 461 ATCC 33277-specific and 415 W83-specific CDSs. Extensive genomic rearrangements were observed between the two strains: 175 regions in which genomic rearrangements have occurred were identified. Thirty-five of those genomic rearrangements were inversion or translocation and 140 were simple insertion, deletion, or replacement. Both strains contained large numbers of mobile elements, such as insertion sequences, miniature inverted-repeat transposable elements (MITEs), and conjugative transposons, which are frequently associated with genomic rearrangements. These findings indicate that the mobile genetic elements have been deeply involved in the extensive genome rearrangement of P. gingivalis and the occurrence of many of the strain-specific CDSs. We also describe here a very unique feature of MITE400, which we renamed MITEPgRS (MITE of P. gingivalis with Repeating Sequences).

The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution

2008-05-28

Scrub typhus (‘Tsutsugamushi’ disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Rickettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain Ikeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of ‘Tsutsugamushi’ disease.

Genome Structure of the Legume, Lotus japonicus

2008-05-28

The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L. japonicus genome. The 315.1-Mb sequences determined in this and previous studies correspond to 67% of the genome (472 Mb), and are likely to cover 91.3% of the gene space. Linkage mapping anchored 130-Mb sequences onto the six linkage groups. A total of 10 951 complete and 19 848 partial structures of protein-encoding genes were assigned to the genome. Comparative analysis of these genes revealed the expansion of several functional domains and gene families that are characteristic of L. japonicus. Synteny analysis detected traces of whole-genome duplication and the presence of synteny blocks with other plant genomes to various degrees. This study provides the first opportunity to look into the complex and unique genetic system of legumes.