生化学反応による計算能力の研究

早大学位記番号:新6514早稲田大

T-DNA integration patterns in transgenic maize lines mediated by Agrobacterium tumefaciens

To explore transfer deoxyribonucleic acid (T-DNA) integration patterns in the maize genome, we improved the protocol of thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), and amplified the flanking sequences around T-DNA integration sites from 70 independent transgenic maize lines mediated by Agrobacterium tumefaciens. Out of 64 specific amplified fragments, 32 and 9 are homologous to the sequences of the maize genome and the expression plasmid, respectively. For 26 of them, a filler sequence was found flanking the cleavage sites. These results demonstrate that cleavage occurs not only during the T-DNA borders but also inside or outside the borders. The border sequences and some inside sequences can be deleted, and filler sequences can be inserted. Illegitimate recombination is a major pattern of T-DNA integration, while some hot spots and preference are present on maize chromosomes.Key words: Agrobacterium tumefaciens, maize, thermal asymmetric interlaced PCR, transfer DNA,transgenics

Difference in real-time magnetic image analysis of colonic looping patterns between males and females undergoing diagnostic colonoscopy.

Abstract Background and study aim Magnetic imaging technology is of proven benefit to trainees in colonoscopy, but few studies have examined its benefits in experienced hands. There is evidence that colonoscopy is more difficult in women. We set out to investigate (i) associations between the looping configurations in the proximal and distal colon and (ii) differences in the looping prevalence between the sexes. We have examined their significance in terms of segmental intubation times and position changes required for the completion of colonoscopy. Patients and methods We analyzed 103 consecutive synchronized luminal and magnetic image videos of diagnostic colonoscopies with normal anatomy undertaken by a single experienced operator. Results Deep transverse loops and sigmoid N-loops were more common in females. A deep transverse loop was more likely to be present if a sigmoid alpha-loop or N-loop had formed previously. Patients with sigmoid N-loops were turned more frequently from left lateral to supine before the sigmoid-descending junction was reached, but there was no statistical correlation between completion time and looping pattern. Conclusions This study has reexamined the prevalence of the common looping patterns encountered during colonoscopy and has identified differences between the sexes. This finding may offer an explanation as to why colonoscopy has been shown to be more difficult in females. Although a deep transverse loop following a resolved sigmoid alpha-loop was the most commonly encountered pattern, no statistical correlation between completion time and looping pattern could be shown. It is the first study to examine segmental completion times using a magnetic imager in expert hands.</jats:p

Endoscopy International Open

Background and study aim Magnetic imaging technology is of proven benefit to trainees in colonoscopy, but few studies have examined its benefits in experienced hands. There is evidence that colonoscopy is more difficult in women. We set out to investigate (i) associations between the looping configurations in the proximal and distal colon and (ii) differences in the looping prevalence between the sexes. We have examined their significance in terms of segmental intubation times and position changes required for the completion of colonoscopy. Patients and methods We analyzed 103 consecutive synchronized luminal and magnetic image videos of diagnostic colonoscopies with normal anatomy undertaken by a single experienced operator. Results Deep transverse loops and sigmoid N-loops were more common in females. A deep transverse loop was more likely to be present if a sigmoid alpha-loop or N-loop had formed previously. Patients with sigmoid N-loops were turned more frequently from left lateral to supine before the sigmoid-descending junction was reached, but there was no statistical correlation between completion time and looping pattern. Conclusions This study has reexamined the prevalence of the common looping patterns encountered during colonoscopy and has identified differences between the sexes. This finding may offer an explanation as to why colonoscopy has been shown to be more difficult in females. Although a deep transverse loop following a resolved sigmoid alpha loop was the most commonly encountered pattern, no statistical correlation between completion time and looping pattern could be shown. It is the first study to examine segmental completion times using a magnetic imager in expert hands

Temporal regulation of the Mus81-Mms4 endonuclease ensures cell survival under conditions of DNA damage

The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damagedSpanish Ministry of Economy and Competitiveness [BFU2010-16989 and Consolider Ingenio CSD2007-00015 to J.A.T.]; Fundación Ramón Areces (Institutional Grant to the Centro de Biologıa Molecular Severo Ochoa); Spanish Ministry of Economy and Competitiveness (predoctoral fellowships to M.V.V and M.A.O-B.); Universidad Autónoma de Madrid (predoctoral fellowship to M.G-F.); Consejo Superior de Investigaciones Cientıficas (JAE-Doc contract to M.S.). Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2010-16989 and Consolider Ingenio CSD2007-00015]Peer Reviewe

Design, Synthesis, And Conformational Studies Of Peptides Containing α,α-Disubstituted amino acids

Cα,α-disubstituted amino acids (ααAAs) are widely utilized to conformationally constrain peptides. Several pentapeptides containing dipropylglycine (Dpg) at alternating positions and their α-amino acid counterpart L-norvaline (Nva) analogues were synthesized to fully investigate the impact of Dpg on peptide backbone structure in aqueous solution. CD, VCD and NMR spectral analysis suggest that Dpg containing peptides adopt more ordered structures relative to their Nva containing analogues. The central residues (Ala, Thr, Tyr, Val) and the charged side-chains of Glu and Lys play important roles in the degree of peptide folding. Hydrophobic and branched residues (Val, Tyr) at the central position of the peptide produce greater folding as judged by CD and NMR. Temperature-dependent NMR analysis (Δδ/ ΔΤ ΝΗ) of Ac-Glu-Dpg-Tyr-Dpg-Lys-NH2 suggests a series of i→i+3 hydrogen bonds between the N-terminal acetyl carbonyl and the Tyr3NH, and the Glu1 carbonyl and the Dpg4NH. The solution conformation of Ac-Glu-Dpg-Tyr-Dpg-Lys-NH2 calculated from NMR-derived constraints shows a 310-helical structure (two repetitive type-III β-turns) at residues 1-4, which is supported by 2D NMR, CD and VCD spectra. Analysis of NMR-derived models of these peptides suggest that there is a strong hydrophobic interaction of the pro-S propyl side chain of Dpg2 and the Tyr3 side-chain that may be a strong stabilizing force of the peptide folding in water. Cα,α-diisobutylglycine(Dibg) was synthesized via palladium catalyzed allylation reaction with an excellent overall yield and incorporated into various positions of a model β-hairpin peptide GHP in order to determine the effectiveness of the ααAAs as design elements in both the strand and turn portions of β-hairpins. CD and NMR data of Dibg containing peptides showed Dibg residue can contribute to the stability of the strand portion of a β-hairpin peptide and destabilize the β-turn in the GHP. The sheet stabilizing effect of Dibg may be due to the strong propensity of Dibg to have a fully extended conformation(φ/ψ = 180°). Several chiral amino esters were prepared with high enantioselectivity by alkylation of the corresponding Schiff bases under chiral phase-transfer condition. The enantiomeric excess of these chiral amino esters was efficiently determined by 19F-NMR analysis of the corresponding diastereomeric Mosher amides

Functionally distinct Botrytis cinerea Argonaute proteins in plant-microbe interaction

Der nekrotrophe, filamentöse und phytopathogene Pilz Botrytis cinerea (B. cinerea) verursacht weltweit an mindestens 200 Eudikotyledonen und Monokotyledonen eine schwere Grauschimmelkrankheit. Neben verschiedenen Enzymen sezerniert Botrytis auch sRNAs als virulente Faktoren in Wirte, welche die Pflanzenphysiologie und Pflanzenimmunitätt über Cross-Kingdom-RNA-Interferenz (ckRNAi) manipulieren,. Dabei erfolgt die Translokation der sRNA-Effektoren bidirektional zwischen Pathogen und Wirt. Die Rolle der Ago-vermittelten Regulation auf die Pathogenität durch RNA-Interferenz (RNAi) in B. cinerea ist jedoch noch unklar. In dieser Doktorarbeit wurden vier Gene, Ago1, Ago2, Ago3 und Ago4 in B. cinerea identifiziert, die für Ago-Proteine kodieren. Die phylogenetische Analyse zeigte, dass Ago1, Ago3 und Ago4 zur Klade der Quelling gehören und Ago2 in die Gruppe der MSUD in Botrytis cinerea fällt. Das Expressionsmuster zeigte, dass Botrytis die endogene Ago2-Expression als Reaktion auf eine Infektion von Tomaten im Früh- und Spätstadium unterdrückt, was auf eine mögliche Funktion von Ago2 im Cross-Talk der Pilz-Wirt-Interaktion hinweist. Bei der Phänotypisierung homokaryotischer Mangel-Mutanten von Botrytis, denen Ago1, Ago2, Ago3 bzw. Ago4 fehlten, konnte ich beobachten, dass der Verlust der Ago-Gene keinen Einfluss auf die Myzelmorphologie, Konidiengröße und Konidienbildung hatte, was darauf hindeutet, dass Ago-Gene für die Entwicklung und das Wachstum von Botrytis in der vegetativen Phase nicht erforderlich sind. Darüber hinaus zeigten Ago2-Loss-of-Function-Mutanten eine verminderte Virulenz im Vergleich zu WT und die Komplementierung von Ago2 stellte die verminderte Pathogenität wieder her, was zeigt, dass Ago2 zur Pathogenität von Botrytis während der Infektion auf Tomate beiträgt. Die Abundanz von sRNAs war in den Ago2- und Ago3-Mutationsstämmen erhöht, was die Rolle der mRNA-Degradation für Ago2 und Ago3 anstelle der Stabilisierung im post-transcriptional silencing (PTGS) impliziert. In der Zwischenzeit waren die Transkripte des Retroelements LTR2 in der Ago1- oder Ago2-Loss-of-Function-Mutante signifikant erhöht und LTR1 wurde in der Ago3-Loss-of-Function-Mutante herunterreguliert, was darauf hindeutet, dass Ago2 ein dominanter Slicer in B. cinerea sein könnte, während Ago3 wahrscheinlich ein untergeordneter Slicer ist, der mit Ago2 konkurriert, indem er sRNAs und interferierende RNAi bindet, und Ago4 möglicherweise nicht an PTGS beteiligt ist. Bemerkenswert ist, dass aus mRNA-seq von Ago2 Loss-of-Function-Mutanten acht differentiell exprimierte Gene (DEGs), die in zellulären Komponenten angereichert sind, und drei DEGs (BCIN_16g04050, die eine Glutamat-Dehydrogenase kodieren, BCIN_09g04410 kodiert eine Galaktose-Oxidase und BCIN_03g04090 kodiert eine Glycerin-Kinase), die im KEGG-Pfad angereichert sind, identifiziert wurden, die eine Rolle in der Arginin-Biosynthese, dem Galaktose-Stoffwechsel bzw. dem Lipid-Stoffwechsel spielen. Ein sRNA-Profiling aus diesen identifizierten Genen deckte auf, dass 24-28-nt sRNAs, wahrscheinlich microRNA-like RNAs, in Ago2-Mutationsstämmen dramatisch verloren gingen. Zusammengenommen reguliert Ago2 die Pathogenität positiv, möglicherweise über die Regulierung dieser Gene, die in GO- oder KEGG-Pfaden angereichert sind und die die Ziele der 24-28-nt milRNAs sein könnten, die eine positive Rückkopplungsschleife in B. cinerea bilden.A necrotrophic filamentous phytopathogenic fungus Botrytis cinerea causes severe grey mould disease on at least 200 eudicots and monocots worldwide. In addition to various enzymes, Botrytis also secretes sRNAs as virulent factors into hosts manipu-lating plant physiology and immunity via cross-kingdom RNA interference (ckRNAi) and the translocation of sRNA effectors is bidirectional between pathogen and hosts. However, the function of Ago-mediated regulation on pathogenicity by RNA interference (RNAi) in B. cinerea remains blank. In this doctoral project, four Botrytis genes encoding Ago proteins were identi-fied encompassing Ago1, Ago2, Ago3 and Ago4. Phylogenetic analysis showed that Ago1, Ago3 and Ago4 belong to the clade of quelling and Ago2 falls into the group of MSUD in Botrytis cinerea. The expression pattern indicated that Botrytis sup-pressed endogenous Ago2 expression in response to early- and late-stage infection on tomatoes, suggesting a potential function of Ago2 in the cross-talk of fungal-hosts interaction. Through phenotypical analyses with homokaryotic deficient mutants disrupting Botrytis Ago1, Ago2, Ago3 and Ago4, I observed that loss of Ago genes had no impact on mycelial morphology, conidial size and conidiation, implicating that Ago genes are not required for Botrytis development and growth in the vegeta-tive phase. Furthermore, Ago2 loss-of-function mutants displayed impaired virulence compared to WT and complementation of Ago2 restored reduced pathogenicity, demonstrating that Ago2 contributes to pathogenicity in Botrytis during infection on tomato. The abundance of sRNAs were elevated in Ago2 and Ago3 mutation strains, which suggests the role of mRNA degradation for Ago2 and Ago3 instead of stabili-zation in post-transcriptional gene silencing (PTGS). Meanwhile, transcripts of retro-element LTR2 was significantly increased in Ago1 or Ago2 loss-of-function mutant and LTR1 was down-regulated in Ago3 loss-of-function mutant, indicating that Ago2 might be a dominant slicer in B. cinerea, whereas Ago3 is likely a minor slicer com-peting with Ago2 binding with sRNAs and interfering RNAi, and Ago4 may not in-volve in PTGS. Notably, mRNA-seq from Ago2 loss-of-function mutants showed that eight differentially expressed genes (DEGs) enriched in cellular component and three DEGs (BCIN_16g04050 encoding a glutamate dehydrogenase, BCIN_09g04410 en-coding a galactose oxidase and BCIN_03g04090 encoding glycerol kinase) enriched in the KEGG pathway were identified, which played a role in arginine biosynthesis, galactose metabolism and lipid metabolism, respectively. sRNA profiling derived from these identified genes uncovered that 24-28-nt sRNAs, probably microRNA-like RNAs, dramatically lost in Ago2 mutation strains. Taken together, Ago2 posi-tively regulates pathogenicity, possibly via regulation on these genes enriched in GO or KEGG pathway that might be the targets of 24-28-nt milRNAs forming a positive feedback loop in B. cinerea

Around the Bend: The Curious Power of the Hills around Queenstown, Tasmania

This article traces a concern with excess and waste, landscape and identity in a town on the western periphery of Tasmania.&nbsp; Queenstown&rsquo;s bald hills have been a popular tourist drawcard since the turn of the century, sold in travel guides and brochures as a spectacular &lsquo;moonscape&rsquo;.&nbsp;A combination of a severe bushfire in 1896 and the emission of sulphurous gases from the pyritic smelting process at the Mt Lyell Copper Mine have resulted in Queenstown&rsquo;s weird denuded landscape.&nbsp;But&nbsp;the locals have grown attached to the hills and want to preserve their baldness.&nbsp; Here, the impulse to preserve a grotesque landscape registers a culture surviving on desecration to stave off its own disappearance