Genome-wide Analysis of Chlamydophila pneumoniae Gene Expression at the Late Stage of Infection

Chlamydophila pneumoniae, an obligate intracellular eubacterium, changes its form from a vegetative reticulate body into an infectious elementary body during the late stage of its infection cycle. Comprehension of the molecular events in the morphological change is important to understand the switching mechanism between acute and chronic infection, which is deemed to relate to the pathogenesis of atherosclerosis. Herein, we have attempted to screen genes expressed in the late stage with a genome-wide DNA microarray, resulting in nomination of 17 genes as the late-stage genes. Fourteen of the 17 genes and six other genes predicted as late-stage genes were confirmed to be up-regulated in the late stage with a quantitative reverse transcriptase–polymerase chain reaction. These 20 late-stage genes were classified into two groups by clustering analysis: ‘drastically induced’ and ‘moderately induced’ genes. Out of eight drastically induced genes, four contain σ28 promoter-like sequences and the other four contain an upstream common sequence. It suggests that besides σ28, there are certain up-regulatory mechanisms at the late stage, which may be involved in the chlamydial morphological change and thus pathogenesis

Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus

Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multi-phenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and five genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9-Mb chromosome and six plasmids. There were three single nucleotide mutations and five transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature-resistant strain grown at an unviable high-temperature (42°C). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92-kb deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

ゲノム情報解析に基づく酢酸菌の遺伝的不安定性要因の解析

[Abstract] Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multi-phenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and 5 genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9 Mb chromosome and 6 plasmids. There were 3 single nucleotide mutations and 5 transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature resistant strain grown at an unviable high-temperature (42℃). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92 kb deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria. [要旨] 酢酸菌はα-proteobacteriaに属する細菌群で、食酢醸造槽から主要発酵菌として分離される。自然界では糖が蓄積するニッチ(花や果実など)に酵母などと共棲し広く分布する。一般にアルコールや酢酸に対する高い耐性、多様な有用二次代謝産物の生産、バイオポリマー生成など非常に多彩な特徴を持つ。一方で、酢酸菌の形質は継代培養によって変化しやすく、発酵産業や研究施設においては有用形質を維持するために経験的な対策が取られてきた。我々は酢酸菌が有する多様な有用代謝経路を明らかにするとともに、その多様性を生み出す遺伝的背景を明らかにすることを目的として、本邦伝統的酢酸発酵菌Acetobacter pasteurianusの全ゲノムDNA配列を詳細に解読した。ゲノム解析の試料には20年にも及ぶ継代培養により複数の形質を示す変異体複合菌系を用いた。そのゲノム解析からその複合菌系に共通する特徴として、2.9Mbpの環状ゲノムDNAと6つの環状プラスミドを有し、合計3107遺伝子をコードすることが明らかとなった。形質の変化しやすさ、つまりゲノムの易変異性はそのプラスミドの多さに加え、トランスポゾンが全遺伝子の9％にも及ぶ280存在することが原因すると考えられる。さらに、細菌ゲノムとしては稀な遺伝子内部に存在するマイクロサテライとが5ヶ所発見された。一方、複合菌系に存在する差異を明らかにするために複合菌系から32株を分離し詳細に解析したところ、少なくとも3ヶ所の一塩基多型とトランスポゾンの転移による遺伝子の破壊が5ヶ所観察された。これは20年間の継代培養から考慮するとき、酢酸菌での変異発生率は大腸菌で報告されている変異発生率よりも有意に高いことが示された。酢酸菌のマイクロサテライの伸長・短縮は20世代くらいで1反復配列程度を示した。酢酸菌が有する易変異性を利用し通常では生育不可能な高温(42℃)で増殖が可能となる菌株の育種を試みた。40℃ での長期の培養の末、42℃ で増殖する株の獲得に成功した。その育種株の高温耐性形質が遺伝することを確認の後、全ゲノムDNA配列を次世代シークエンサーによって決定した。3カ所の一塩基置換と複製の集結店付近における92kbpにおよぶゲノムの欠落が観察された。育種途中の株の解析から高温耐性にはその92kbpの欠落のみが関与することが明らかとなった。その欠落領域には72遺伝子が存在し、そのいずれかの遺伝子の欠落によって生育限界を超えた高い温度で増殖が可能となったと考えられる。一方、欠落自体によりゲノムDNA複製のストレスが軽減され高い温度でも増殖が可能となった可能性もある。今後の解析を待つ必要がある。酢酸菌が属するα-proteobacteriaにはリケッチア菌群や根粒細菌群が含まれる。これらリケッチア菌群や根粒細菌群は動物や植物の細胞中をその増殖のニッチとして選択した菌群であり、顕著なゲノム縮重を進化の特徴としている。酢酸菌は環境中で自由に増殖が可能な菌ではあるが、リケッチア菌群や根粒細菌群と同様に高い易変異性や大胆なゲノムDNAの欠落を許容しえる機構を有するのかもしれない

Structural Studies of Schizosaccharomyces pombe ＲＮＡ polymerase II

　　The molecular composition of DNA‐dependent RNA polymerase II from Schizosaccharomyces pombe was studied.First,RNA polymerase II was purified from the wild type S.pombe, in five steps:precipitation with Polymin P;elution from the Polymin P precipitates; separation of RNA polymerase II from RNA polymerases I and III by DEAE-Sephadex column chromatography; purification by Q Sepharose FF; and by Superose 6 gel filtration column chromatogfaphy.The highly purified S.pombe RNA polymerase II contained more than eleven polypeptides as analyzed by SDS-po1yacrylamide gel electrophoresis.The molar ratio of the three large subunit,RPB1,2 and 3,was 1:1:2,which is identical with that of the E.coli and S.cerevisiae enzymes.　　In order to establish in vitro reconstitution system of RNA polymerase II and to decide the subunit composition of RNA polymerase II,preliminary attempts were made to find conditions for reversible denaturation of RNA polymerase II by treatment with urea.RNA polymerase II was not inactivated up to 0.5M urea.RNA polymerase II 1ost activity from 1.0M to 2.5M urea,but the activity was recovered perfectly simply by dilution of urea. Above 3.0M urea,howevef,RNA polymerase II activity was irreversibly inactivated.From the result of gel filtration,multi‐subunit complexes of RNA polymerase II were observed but several small components were missing from the complexes after denaturation in 4M urea.　　The S.pombe gene coding for the largest subunit(subunit 1) of RNA polymerase II was cloned using the S.cerevisiae corresponding gene,RPB1,as a probe in cross‐hybridization.The sequence determination of both the entire genomic DNA and parts of cDNA indicated that this rpb1 gene has six introns in the N-terminal region and encodes the subunit 1 of 1,752 amino acid residues with the molecular mass of 194kDa.From Southern analysis and gene disruption experiments,it was found that this rpb1 gene exists as a single copy in the S.pombe genome and is essential for cell viability. Northern analysis and sequence determination of 3´-and 5´-terminal regions of rpb1 transcript indicated that the size of the rpb1 transcript is about 5.6kb in length.　　Among the subunit 1 of S,pombe RNA polymerase II and other b´homo1ogues,nine structurally conserved domains(domain A to H and CTD)were identified:domain A,aputative zinc‐binding site with the consensus sequence of CX2CX9HX2H;domains C and D,the conserved sequences within E.coli DNA polymerase I and T7 DNA polymerase;Domain C,a single two‐helix motif for putative DNA binding:domain F,the putative a‐amanitin binding site:CTD,highly conserved unique repetition with the unit sequence of YSPTSPS among the largest subunits of RNApolymerase II.ln the subunit 1 of S.pombe RNA polymerase II,29 repeats exist in CTD.　　The cDNA fragment coding for the third largest subunit(subunit 3) of S.pombe RNA polymerase II was cloned by RT‐PCR using primers designed from the amino acid sequences of V8 fragments of subunit 3.A genomic DNA fragment carrying the entire subunit 3 gene(rpb3) was isolated by hybridization using this cDNA fragment as a probe.The sequence determination indicated that the coding frame of rpb3 is interrupted by two introns and this gene encodes subunit 3 of 297 amino acids in length.Southern and Nothern analyses indicated that the rpb3 gene is present as a single copy in haploid S.pombe cells and the size of rpb3 transcript is about 1.2kb in length.　　Among the RNApolymerase subunit 3 from various organisms,four structural conserved domains(domains A to D) were found:domains A and D exist even in the a subunit of E.coli RNA polymerase; and domains B and C are conserved only in eukaryotic RNA polymerases. Domain A may play a role in subunit‐subunit contact of RNA polymerase.Domain B with a putative meta1‐binding sequence,CXCX3CX2C,exists only in RNA polymerase II,but not in RNA polymerase I nor III.Domain D with a leucine zipper-like motif may be required for the formation and/or stability of RNA polymerase.Subunit 3 of eukaryotic RNA polymerases lacks the sequence corresponding to the C‐terminal region of E.coli RNA polymerase a subunit carrying the contact site I for some transcription activators.　　To identify the function(s) of subunit 3,the rpb3 gene was mutagenized by lower fidelity PCR and transferred into S.pombe using either spheroplast method or electroporation method.　　Total 178 temperature‐sensitive ts mutants were isolated from about nine thousand transformants. PCR analysis and Southern analysis were carried out for 68 stable ts mutants.Most of the mutants tested carried a single copy of full length DNA fragment integrated in the genomic DNA,With nine mutants,the mutation sites were determined after cloning and sequencing.All the mutant rpb3 genes carried multiple mutations,but many mutations were clusteredin the N-terminal region of RPB3 polypeptides.Upon temperaturc shift from permissive temperaturc,25-C to non‐permissive temperature,37-C,some of the mutants stop growing immediately,while other mutants stop growing s1owly,implying that the assembly of RNA polymerase II is defective in these mutants.Some mutants show abnormal cell shapes(fof example,7 times 1onger in cell size) at the non‐permissive temperature.None of the sixteen mutants survived after one day incubation at non‐permissive temperature.Even in these non‐viable cells,the nuclei still appeared intact