Comparative Studies of Genome-Wide Maps of Nucleosomes between Deletion Mutants of elp3 and hos2 Genes of Saccharomyces cerevisiae

In order to elucidate the influence of histone acetylation upon nucleosomal DNA length and nucleosome position, we compared nucleosome maps of the following three yeast strains; strain BY4741 (control), the elp3 (one of histone acetyltransferase genes) deletion mutant, and the hos2 (one of histone deactylase genes) deletion mutant of Saccharomyces cerevisiae. We sequenced mononucleosomal DNA fragments after treatment with micrococcal nuclease. After mapping the DNA fragments to the genome, we identified the nucleosome positions. We showed that the distributions of the nucleosomal DNA lengths of the control and the hos2 disruptant were similar. On the other hand, the distribution of the nucleosomal DNA lengths of the elp3 disruptant shifted toward shorter than that of the control. It strongly suggests that inhibition of Elp3-induced histone acetylation causes the nucleosomal DNA length reduction. Next, we compared the profiles of nucleosome mapping numbers in gene promoter regions between the control and the disruptant. We detected 24 genes with low conservation level of nucleosome positions in promoters between the control and the elp3 disruptant as well as between the control and the hos2 disruptant. It indicates that both Elp3-induced acetylation and Hos2-induced deacetylation influence the nucleosome positions in the promoters of those 24 genes. Interestingly, in 19 of the 24 genes, the profiles of nucleosome mapping numbers were similar between the two disruptants

Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase δ

A novel mutagenesis technique using error-prone DNA polymerase δ (polδ), the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the polδMKII mutator, which expresses the proofreading-deficient and low-fidelity polδ, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS). This mutator strain possesses exogenous mutant polδ supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the polδMKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis

ビセイブツ ゲノム ノ カイドク ト キノウカイセキ

ワトソン・クリックのDNA二重らせんモデル以来，遺伝子の本体であるDNAの塩基配列は分子生物学の発展と共に生物学者の大きな関心の的となった。1980年，サンガーとギルバートが塩基配列決定法でノーベル賞を受賞するが，筆者は1978年にいち早くサンガー法，マクサム・ギルバート法を試みた。その後，キット化によりサンガー法が世の主流になり，蛍光試薬の利用による非RI化，自動化，さらにキャピラリー化による大量解読の時代に入った。21世紀に入り，桁違いの解読量を誇ることから次世代型と呼ばれる革新的シーケンサーの登場で生物学の様相は一変する。モデル生物に関してはゲノム情報を共有した上でその機能解析を行うことが基盤となり，ゲノム生物学という新分野も誕生した。このような時代の流れの中，筆者は形質転換能の高い枯草菌とシアノバクテリアを研究対象としてきたが，要所要所で塩基配列を決定し，その情報から得られる恩恵に預かってきた。その背景には，常に1塩基の持つ意味を追求してきた姿勢がある。本稿では，始めにその端緒となった成果について紹介し，その後のゲノム解読関連の成果について述べる。次いで，次世代シーケンサーを用いた微生物の新規研究法に関する取り組みを紹介する。最後に，このような背景の下に得られた研究成果から，シアノバクテリアに関して光依存，DnaA非依存のDNA複製系の発見について，そして枯草菌に関して脂質合成系必須遺伝子plsXの相互作用解析から見えてきたいくつかの細胞機能ネットワークについて解説する。Since a double helix model of nucleic acid structure appeared, nucleotide sequences have been the focus of biologists’ attention as molecular biology has developed. After the Nobel prize for sequencing technique by Sanger and Gilbert, nucleotide sequencing has been gradually improving and finally burst into an era of massive decoding. Present-day, for most biology targets, it has become essential to have their own sequence information to analyze and breed each biological material. Particularly in microbiology fields, genetic strategy was fundamentally reformed and a new field, “genome microbiology”, has been born. The significance of one-nucleotide difference is more and more meaningful, and many past ambiguous results have been clearly elucidated by disclosing whole genome sequences. Here I present some unique techniques using next generation sequencer (NGS) and apply them to determining the bacterial origin of replication. For the analysis of the cyanobacterial study in which basic biological principle, i.e. DNA replication or transcription, has not been elucidated, NBS has been powerful tool and I introduce light-dependent DNA replication as well as DnaA dependency among various cyanobacteria. Alternatively, several newly identified networks involved in essential lipid synthetic enzyme, PlsX, in Bacillus subtilis are described. Based on our post-genome project, we identified several interactions between PlsX and cell division machinery. Besides, from the temperature sensitive mutant of plsX, we obtained many suppressor mutants and therefore analyzed functional interactions among these genes. These analyses revealed the global cellular function which, especially during transient growth phase, senses nutritional availability and regulates cell growth and divisions. It is important to describe cellular rational and intelligent functions at the level of molecular mechanisms

Algal protein kinase, Triacylglycerol Accumulation Regulator 1, modulates cell viability and gametogenesis in carbon/nitrogen imbalanced conditions

Nutrient-deprived microalgae accumulate triacylglycerol (TAG) in lipid droplets. A dual-specificity tyrosine phosphorylation-regulated kinase, TAG accumulation regulator 1 (TAR1) has been shown to be required for acetate-dependent TAG accumulation and the degradation of chlorophyll and photosynthesis-related proteins in photomixotrophic nitrogen (N)-deficient conditions (Kajikawa et al. 2015). However, this previous report only examined particular condition. Here, we report that in photoautotrophic N-deficient conditions, tar1-1 cells, with a mutation in the TAR1 gene, maintained higher levels of cell viability and lower levels of hydrogen peroxide generation and accumulated higher levels of TAG and starch compared with those of wild-type (WT) cells with bubbling of air containing 5% carbon dioxide. Transcriptomic analyses suggested that genes involved in the scavenging of reactive oxygen species are not repressed in tar1-1 cells. In contrast, the mating efficiency and mRNA levels of key regulatory genes for gametogenesis, MID, MTD, and FUS, were suppressed in tar1-1 cells. Among the TAR1-dependent phosphopeptides deduced by phosphoproteomic analysis, protein kinases and enzymes related to N assimilation and carbon (C) metabolism are of particular interest. Characterization of these putative downstream factors may elucidate the molecular pathway whereby TAR1 mediates cellular propagation and C and N metabolism in C/N-imbalanced stress conditions

Discovery of Genome-Wide DNA Polymorphisms in a Landrace Cultivar of Japonica Rice by Whole-Genome Sequencing

Molecular breeding approaches are of growing importance to crop improvement. However, closely related cultivars generally used for crossing material lack sufficient known DNA polymorphisms due to their genetic relatedness. Next-generation sequencing allows the identification of a massive number of DNA polymorphisms such as single nucleotide polymorphisms (SNPs) and insertions–deletions (InDels) between highly homologous genomes. Using this technology, we performed whole-genome sequencing of a landrace of japonica rice, Omachi, which is used for sake brewing and is an important source for modern cultivars. A total of 229 million reads, each comprising 75 nucleotides of the Omachi genome, was generated with 45-fold coverage and uniquely mapped to 89.7% of the Nipponbare genome, a closely related cultivar. We identified 132,462 SNPs, 16,448 insertions and 19,318 deletions between the Omachi and Nipponbare genomes. An SNP array was designed to validate 731 selected SNPs, resulting in validation rates of 95 and 88% for the Omachi and Nipponbare genomes, respectively. Among the 577 SNPs validated in both genomes, 532 are entirely new SNP markers not previously reported between related rice cultivars. We also validated InDels on a part of chromosome 2 as DNA markers and successfully genotyped five japonica rice cultivars. Our results present the methodology and extensive data on SNPs and InDels available for whole-genome genotyping and marker-assisted breeding. The polymorphism information between Omachi and Nipponbare is available at NGRC_Rice_Omachi (http://www.nodai-genome.org/oryza_sativa_en.html)

Characteristics of nucleosomes and linker DNA regions on the genome of the basidiomycete Mixia osmundae revealed by mono- and dinucleosome mapping

We present findings on the nucleosomal arrangement in the genome of the basidiomycete Mixia osmundae, focusing on nucleosomal linker DNA regions. We have assembled the genomic sequences of M. osmundae, annotated genes and transcription start sites (TSSs) on the genome, and created a detailed nucleosome map based on sequencing mono- and dinucleosomal DNA fragments. The nucleosomal DNA length distribution of M. osmundae is similar to that of the filamentous ascomycete Aspergillus fumigatus, but differs from that of ascomycetous yeasts, strongly suggesting that nucleosome positioning has evolved primarily through neutral drift in fungal species. We found clear association between dinucleotide frequencies and linker DNA regions mapped as the midpoints of dinucleosomes. We also describe a unique pattern found in the nucleosome-depleted region upstream of the TSS observed in the dinucleosome map and the precursor status of dinucleosomes prior to the digestion into mononucleosomes by comparing the mono- and dinucleosome maps. We demonstrate that observation of dinucleosomes as well as of mononucleosomes is valuable in investigating nucleosomal organization of the genome