Language：English   Publishing type：Research paper (scientific journal)   Publisher：{SAGE} Publications  

<jats:p> The composition of root chemicals was studied for 7 samples of Ligularia tongolensis collected in Yunnan and Sichuan Provinces of China. The structures of 2 new 3β-angeloyloxy-6β-acyloxyfuranoeremophilan-15-oic acids were determined. It was found that the plant harbors chemical diversity in the acyloxy groups in 3,6-bis(acyloxy)eremophilan-15-oic acids. The presence of a 3-methylpentanoate moiety at C-3 appears geographically differentiated to a degree. Consistent with this low diversity, results of DNA analysis indicated little genetic differentiation, although introgression was inferred for one of the samples. </jats:p>

DOI： 10.1177/1934578x19878937

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：{SAGE} Publications  

<jats:p> The chemical composition and the DNA sequence of an evolutionarily neutral region were studied for three samples of L. tsangchanensis, three samples of L. muliensis, and one sample of a hybrid between L. tsangchanensis and L. vellerea. L. tsangchanensis and L. muliensis were indistinguishable in the chemical composition and the ITS sequence, supporting a recent taxonomic study that merged the two species into L. tsangchanensis. Cacalol (1) was isolated as the most major component from both L. tsangchanensis and L. muliensis, while 6-acetoxy-9-oxofuranoeremophilane (10), a putative cacalol precursor, was isolated from the hybrid. </jats:p>

DOI： 10.1177/1934578x1801300402

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Pharmaceutical Society of Japan  

Chemical constituents of Ligularia hookeri (Asteraceae) collected in Yunnan and Sichuan Provinces in China were examined for the first time. Seven furanoeremophilanes, five of which were new, as well as known bisabolane- and eudesmane-type sesquiterpenoids, were isolated. Spectroscopic evidence indicates that the previously reported 3β-(2′-methylpropenoyloxy)furanoeremophilan-15,6β-olide should be revised to 3β-(2′-methylpropenoyloxy)furanoeremophilan-15,6α-olide.

DOI： 10.1248/cpb.c18-00191

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The diversity in root chemicals and evolutionally neutral DNA regions in the complex of Ligularia duciformis, L. kongkalingensis, and L. nelumbifolia (the d/k/n complex) was studied using eight samples collected in central and northern Sichuan Province of China. Cacalol (14) and epicacalone (15), rearranged eremophilanes, were isolated from the complex for the first time. Two new phenylpropanoids were also obtained. Seven of the eight samples produced phenylpropanoids and the other produced lupeol alone. Two of the seven samples also produced furanoeremophilanes or their derivatives and one produced oplopanes. The geographical distribution of the sesquiterpene-producing populations suggests that the production of sesquiterpenes evolved independently in separate regions. L. limprichtii collected in northern Sichuan was also analyzed and its chemical composition and the sequence of internal transcribed spacers (ITSs) in the ribosomal RNA gene cluster were found to be similar to that in the d/k/n complex and L. yunnanensis, which are morphologically similar.

DOI： 10.3390/molecules22122062

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Three natural hybrids and an introgressed individual of Ligularia were evaluated based on a combination of morphology, root chemicals, and nucleotide sequences of evolutionally neutral regions to understand the chemical outcomes of hybridization and introgression. Six previously undescribed eremophilane sesquiterpenes were isolated from hybrids between L cyathiceps and L. lamarum/L subspicata, and benzofurans were isolated from L subspicata for the first time. Their structures were elucidated based on spectroscopic analyses. Some compounds produced by hybrids have not been detected in either parental species, indicating that the metabolic profile was altered by hybridization and introgression. (C) 2017 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2017.04.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

The chemical composition of a sample of the title species collected in Pianma, Lushui County, Yunnan Province of China, was examined. Two 3,6,10 oxygenated and two 3,6-oxygenated furanoeremophilanes were isolated. Structures of two new compounds were elucidated. The 3,6-oxygenated furanoeremophilanes were not detected in a sample of the same species previously collected in Laojunshan, Jianchuan County, Yunnan, indicating chemical diversity in the species.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Chemical compositions and internal transcribed spacer (ITS) sequences of five samples of Ligularia lamarum collected in Sichuan Province, China, were analyzed. Fourteen compounds, including four new eremophilanes and one new seco-eremophilane, were isolated and their structures were elucidated by spectroscopic methods. Intra-specific diversity in the chemical composition was found to be higher than previously known. The result of DNA analysis suggested that one of the samples was introgressed, although its chemical composition was typical of L. lamarum.

DOI： 10.1016/j.phytol.2017.04.026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

The composition of root chemicals was examined for samples of L. cyathiceps x L. duciformis, L. duciformis x L. yunnanensis, and L. yunnanensis. Various furanoeremophilanes were isolated from a sample of L. cyathiceps x L. duciformis and found to be very similar to those isolated from L. cyathiceps. Lupeol and docosyl ferulate were isolated from L. yunnanensis, L. duciformis, and L. duciformis x L. yunnanensis.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

Three New Eremophilanes from a Ligularia Hybrid Collected in China

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

The chemical constituents of Ligularia wilsoniana were studied for the first time, using four samples collected in Chongqing City, China. Two new compounds, 6 beta-(2',3'-Lepoxy-2'-methylpropanoyloxy)-1 beta,10 beta-epoxyfuranoeremophilane and 11 alpha H-6 beta-(2'-hydroxymethylacryloyloxy)-1 beta,10 beta;7 beta,8 beta-diepoxyeremophilanolide, as well as eight known compounds, were isolated from one of the samples, while three to four known furanoeremophilanes were isolated from the other three samples. No compound was common to the two classes of the samples, demonstrating the presence of at least two chemotypes of this species.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

Root constituents of Ligularia longihastata were examined for the first time. Fourteen eremophilane sesquiterpenes, including two new eremophilanes identified as 3a-isobutyroyloxy-7 beta H-eremophila-9,11-dien-8-one and 3 beta-angeloyloxy-1 beta,10 beta-epoxyfuranoeremophilan-9-one, and three eudesmanes were isolated. From samples collected in Baiyu County, Sichuan Province, furanoeremophilane derivatives were isolated, while various eremophilan-8-ones were isolated from samples collected in Kangding County, Sichuan. The results suggest chemical diversity in the species.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

Six Ligularia fischeri samples, two from Sichuan (samples 1 and 2) and four from Chongqing (samples 3-6), were examined for root chemicals and the DNA sequence of the internal transcribed spacers of the ribosomal RNA gene. Samples 2 and 3 contained benzofurans. The isolation of benzofurans shows that the chemical diversity in L. fischeri is higher than previously reported. Samples 1, 4, 5, and 6 contained eremophilanes. However, the compounds were different between sample 1 and samples 4-6, indicating variation within eremophilane producers. DNA data indicated that introgression could be a mechanism of benzofuran production in sample 2 and that sample 1 and samples 4-6 were genetically separate.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

Ribosome dimers are a translationally inactive form of ribosomes found in Escherichia coli and many other bacterial cells. In this study, we found that the 70S ribosomes of Bacillus subtilis dimerized during the early stationary phase and these dimers remained in the cytoplasm until regrowth was initiated. Ribosome dimerization during the stationary phase required the hpf gene, which encodes a homologue of the E. coli hibernation-promoting factor (Hpf). The expression of hpf was induced at an early stationary phase and its expression was observed throughout the rest of the experimental period, including the entire 6 h of the stationary phase. Ribosome dimerization followed the induction of hpf in WT cells, but the dimerization was impaired in cells harbouring a deletion in the hpf gene. Although the absence of ribosome dimerization in these Hpf-deficient cells did not affect their viability in the stationary phase, their ability to regrow from the stationary phase decreased. Thus, following the transfer of stationary-phase cells to fresh LB medium, Δhpf mutant cells grew slower than WT cells. This observed lag in growth of Δhpf cells was probably due to a delay in restoring their translational activity. During regrowth, the abundance of ribosome dimers in WT cells decreased with a concomitant increase in the abundance of 70S ribosomes and growth rate. These results suggest that the ribosome dimers, by providing 70S ribosomes to the cells, play an important role in facilitating rapid and efficient regrowth of cells under nutrient-rich conditions.

DOI： 10.1099/mic.0.000234

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

Ligularia hodgsonii was found to be diverse in China. Furanoeremophilanes were isolated from samples collected in Yunnan Province, while such compounds were absent from samples from Sichuan, Gansu, and Chongqing. DNA sequencing showed that the Yunnan samples were also genetically distinct. gamma-Humulene and a new bisabolane sesquiterpene were isolated.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Active equi-paritioning of the F plasmid is achieved by its sopABC gene. SopA binds to the sopAB promoter region and SopB binds to sopC. SopA also polymerizes in the presence of ATP and Mg(II), which is stimulated by SopB. Non-specific DNA is known to inhibit SopA polymerization and disassemble SopA polymer. This study followed kinetics of polymerization and de-polymerization of SopA by turbidity measurement and found new effects by DNA and SopB. Plasmid DNA, at low concentrations, shortened the lag (nucleation) phase of SopA polymerization and also caused an initial 'burst' of turbidity. Results with two non-specific 20-bp DNAs indicated sequence/length dependence of these effects. sopAB operator DNA only showed inhibition of SopA polymerization. Results of turbidity decrease of pre-formed SopA polymer in the presence of ethylenediaminetetraacetic acid showed that SopB also accelerates disassembly of the SopA polymer. The steady-state level of turbidity in the presence of SopB and plasmid DNA indicated synergy between SopB and DNA in the disassembly. SopB protein showed no effect on SopA polymerization, when SopB was specifically bound to DNA. This result and others with truncation mutants of SopB suggested that a proper configuration of the domains of SopB is important for SopA-SopB interactions.

DOI： 10.1093/jb/mvv003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

Ligztlaria vellerea and L. melanothyrsa have been found to share chemical characteristics based on geography. In one chemotype (Shangrila type), the major components were either 15-oxygenated furanoeremophilanes or eremophilanolides; and in another (Luguhu type), either 1-angeloyloxyfuranoeremophilanes or corresponding lactones. The two L. vellerea samples were found to have experienced hybridization with either L. cyathiceps or L. kanaitzensis; however, compounds typical of L. vellerea alone were isolated from them.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

To elucidate the biological functions of the ribosomal protein L34, which is encoded by the rpmH gene, the rpmH deletion mutant of Bacillus subtilis and two suppressor mutants were characterized. Although the Delta rpmH mutant exhibited a severe slow-growth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the Delta rpmH strain. Either the disruption of yhdP, which is thought to be involved in the efflux of Mg2+, or overexpression of mgtE, which plays a major role in the import of Mg2+, could suppress defects in both the formation of the 70S ribosome and growth caused by the absence of L34. Interestingly, the Mg2+ content was lower in the Delta rpmH cells than in the wild type, and the Mg2+ content in the Delta rpmH cells was restored by either the disruption of yhdP or overexpression of mgtE. In vitro experiments on subunit association demonstrated that 50S subunits that lacked L34 could form 70S ribosomes only at a high concentration of Mg2+. These results showed that L34 is required for efficient 70S ribosome formation and that L34 function can be restored partially by Mg2+. In addition, the Mg2+ content was consistently lower in mutants that contained significantly reduced amounts of the 70S ribosome, such as the Delta rplA (L1) and Delta rplW (L23) strains and mutant strains with a reduced number of copies of the rrn operon. Thus, the results indicated that the cellular Mg2+ content is influenced by the amount of 70S ribosomes.

DOI： 10.1128/JB.01896-14

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Two morphologically ambiguous Ligularia samples (samples A and B), and samples with morphology of Ligularia subspicata (sample C), Ligularia lamarum (sample D), or Ligularia cyathiceps (sample E), were collected at Tianchi Pond, Shangrila County, Yunnan Province, China. Analysis of the nucleotide sequence of the internal transcribed spacers (ITSs) in the nuclear ribosomal RNA gene cluster indicated that not only sample B but also sample D was a hybrid of L. cyathiceps and L. lamarum/ L. subspicata. Although the morphology of sample A suggested that it was also a hybrid, the ITS sequence of sample Awas that of L. cyathiceps. Twenty compounds were isolated from the five samples, and the structures of two new compounds 7 and 14 were determined. Furanoeremophilanes typical of L. lamarum/ L. subspicata were detected not only in samples C and D, but also in samples A and B. These results indicate that the ability to produce root chemicals can spread through introgression.

DOI： 10.1002/cbdv.201400133

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Language：Japanese   Publisher：SOC SYNTHETIC ORGANIC CHEM JPN  

A series of our studies on the chemical and genetic diversity of Ligularia species in the Hengduan Mountains area of China is reviewed. Diversities were detected in various species in terpenoid compositions and DNA sequences of the ITS region. Furanoeremophilanes were isolated from many species together with eremophilan-8-ones. Samples of some species such as L. virgaurea were grouped into two or more chemotypes, while L. cymbulifera and L. cyathiceps were uniform. Complex diversity was found in L. kanaitzensis. A hypothesis that the production of furanoeremophilanes is ecologically advantageous was proposed. Production of terpenoids in L. duciformis and related species were suggested to be acquired by hybridization which may be one of the major pathways of the evolution of Ligularia. Some taxonomically close species, such as L. lamarum and L. subspicata, were indistinguishable in both the root chemicals and the ITS sequences.

DOI： 10.5059/yukigoseikyokaishi.72.717

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The sesquiterpenoid composition in the root and the DNA sequences of evolutionarily neutral regions were studied in Ligularia anoleuca and Ligularia fischeri (Asteraceae) collected in the Sichuan Province of China. LC-MS analysis showed that L anoleuca populations from different localities had different chemical compositions. However, the isolated compounds were similar to each other, indicating that the differences in chemical composition were not large. The DNA analysis suggested that the two species were indistinguishable. Seventeen furanoeremophilanes and an eremophilane acetal were isolated. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2014.03.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

Ligularol (1), a furanoeremophilane, was isolated together with eremophil-1(10)-en-11-ol (2) from one of eight collected samples of Ligularia nelumbifolia and L. kongkalingensis, a closely related species. It is proposed that the ability to produce the furanoeremophilane has been acquired through hybridization.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Chemical constituents and evolutionally neutral DNA sequences of six samples of Cremanthodium lineare Maxim., collected in the Sichuan Province of China, were studied. Three samples produced furanoeremophilanes and the other three, eremophilan-8-ones. The chemotypes were found to be correlated with DNA sequence types, suggesting that the chemical diversity observed has a genetic origin. Production of furanoeremophilanes by a Cremanthodium species suggests an evolutionary relationship between Cremanthodium and Ligularia species, and possibly to related genera. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2013.08.009

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Language：English   Publishing type：Research paper (scientific journal)  

Formation of RNA-DNA hybrid, or R-loop, was studied in vitro by transcribing an AGGAG repeat with T7 RNA polymerase. When ribonuclease T1 was present, R-loop formation in cis was diminished, indicating that the transcript was separated from the template and reassociated with it. The transcript was found to form an R-loop in trans with DNA comprising the AGGAG repeat, when the DNA was supercoiled. Results of chemical modification indicated that the duplex opened at the AGGAG repeat under negative supercoiling. © 2013 Kazuya Toriumi et al.

DOI： 10.1155/2013/629218

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Root chemicals and DNA sequences were analyzed for Ligularia virgaurea collected in the northern Sichuan Province and adjacent areas of China. Thirty-eight samples were found to contain five chemotypes, two known and three new ones. From samples of the new chemotypes, 13 new compounds were isolated, including 3 sesquiterpene dimers and some rearranged eremophilanes. The DNA sequences suggested that the chemotype differentiation had a genetic origin. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2012.09.042

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Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Chemical constituents of roots and evolutionarily neutral DNA base sequences of L. virgaurea collected in Hengduan Mountains and neighboring areas of China were studied. Fifty-one samples were classified into five chemotypes (types 1-5) on the basis of TLC analyses and isolated compounds. The major component(s) in each chemotype were virgaurenones A (1) and B (2) (type 1), ligularol (7) (type 2), cacalol (18) (type 3), 6-hydroxyeuryopsin (36) (type 4), and neoadenostylone (48) and its 1,10-epoxy derivative (50) (type 5), respectively. Fifty-nine terpenoids were identified, thirteen of which including seco-eremophilane derivatives (44 and 59) and sesquiterpene dimer (42) were new. The presence of genetic diversity was also suggested from the base sequences of evolutionarily neutral region in DNA. Two-types of base sequences in atpB-rbeL intergenic region were found, while three-types in ITSs region. These genetic types showed good correspondence to the chemotypes, although the latter one is a little more complex than the other. The collection sites of five chemotypes of L. virgaurea were overlapping, indicating that the environmental effects are not nearly concerned with the intra-specific diversity.

DOI： 10.24496/tennenyuki.54.0_471

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the Delta relA Delta yjbM Delta ywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG-induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.

DOI： 10.1002/mbo3.16

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the Delta relA Delta yjbM Delta ywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG-induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.

DOI： 10.1002/mbo3.16

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The chemical constituents of the root extracts and the evolutionarily neutral DNA base sequences were studied for 28 samples of Ligularia duciformis, L. kongkalingensis, and L. nelumbifolia collected in Sichuan and Yunnan Provinces of China. The samples could be classified into four chemotypes (14). Sesquiterpenoids having eremophilane and oplopane skeletons were isolated from two (Chemotype 1) and three (Chemotype 2) samples, respectively. Two new oplopane derivatives were isolated and their structures were determined. In 18 samples, phenylpropenoids were the major components (Chemotype 3). In five samples, neither phenylpropenoids nor sesquiterpenoids were found (Chemotype 4). Despite this large chemical variety, no correlation was found between the chemotype and the morphological criteria of species identification. The analysis of the evolutionarily neutral DNA regions also indicated that the samples were not separated into distinct clades and that introgression was extensive.

DOI： 10.1002/cbdv.201100240

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

The chemical constituents of the roots of two Ligularia lankongensis samples collected in Yunnan and Sichuan Provinces, China, were investigated, together with the DNA sequence of the atpB-rbcL and ITS regions. Four new highly oxygenated bisabolane-type sesquiterpenes (1 - 4) were obtained. Intraspecific diversity in the DNA sequence was found to be limited.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

Two samples of Ligularia kanaitzensis collected in the Shizhongshan Mountain region of Yunnan Province, as well as one sample collected in Sichuan Province, were analyzed with regard to root constituents and neutral DNA sequences. The two Shizhongshan samples were found to be different from each other and from other samples analyzed previously, indicating that the intra-specific diversity in the species was highly complex.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

The chemical constituents of the roots of two Ligularia lankongensis samples collected in Yunnan and Sichuan Provinces, China, were investigated, together with the DNA sequence of the atpB-rbcL and ITS regions. Four new highly oxygenated bisabolane-type sesquiterpenes (1 - 4) were obtained. Intraspecific diversity in the DNA sequence was found to be limited.

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Language：English   Publishing type：Research paper (scientific journal)  

Four Ligularia individuals possessing morphological characters of both L. nelumbifolia and L. subspicata were discovered in Shangrila County, Yunnan Province, China. DNA sequencing showed that the specimens were hybrids of the two species and their maternal parent was L. subspicata. The chemical composition of the root extract was examined for three of them as well as L. nelumbifolia and L. subspicata collected sympatrically. Nelumol was isolated from L. nelumbifolia, and furanoeremophilanes from L. subspicata. While nelumol and furanoeremophilanes were detected in two of the hybrid samples, only furanoeremophilanes were detected in the others.

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Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Chemical constituents in root and evolutionarily neutral DNA base sequences were studied for L. duciformis, L. kongkaligensis, and L. nelumbifolia collected in Sichuan and Yunnan Provinces of China. The three species were indistinguishable genetically and morphologically. Twenty-eight samples were classified into four chemo-types (types 1-4). Sesquiterpenoids having eremophilane 1-3 and oplopane skeletons 4-7 were isolated from two (type 1) and three (type 2) samples, respectively. The structure of two new oplopane compounds 6 and 7 were determined spectroscopically. In 18 samples, phenylpropenoids 8-11 were the major component (type 3). In five samples, neither phenylpropenoids nor sesquiterpenoids were found (type 4). DNA analysis suggested that hybridization and introgression are extensive. All the present samples were not separated from one another or from L. franchetiana with the exception of sample 5. Since the sesquiterpenoid-producing samples (types 1 and 2) consist of minor group, the genetic analysis suggests that the ability of the production of eremophilane and oplopane sesquiterpenes was obtained by introgression. These samples were collected in the east or south edge of the distribution area. Thus the present results gave supports on the hypothesis that introgression occurs more frequently in the outer regions.

DOI： 10.24496/tennenyuki.53.0_271

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The binding of SopA to the promoter region of its own gene, in which four copies of SopA's recognition sequence, 5'-CTTTGC-3', are arrayed asymmetrically, was examined in vitro. Titration using electrophoretic mobility shift assay showed that the stoichiometry of SopA protomers to the promoter-region DNA is 4 and that the binding is highly co-operative. The co-operativity was corroborated by EMSA and DNase I footprinting for a number of mutant DNA fragments in which 5'-CTTTGC-3' was changed to 5'-CTTACG-3'. EMSA in the style of circular permutation showed that SopA bends DNA. Mutation at either outermost binding site had a different effect on DNA bending by SopA, reflecting the asymmetry in the arrangement of the binding sites, for which the results of DNase I footprinting were in agreement. Gel filtration chromatography and analytical ultracentrifugation of free SopA showed that the protein can exist as a monomer and oligomers in the absence of ATP. Hence, the results indicate that the co-operativity in SopA's DNA binding is based on its intrinsic protein-protein interaction modified by DNA interaction.

DOI： 10.1093/jb/mvq151

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LTD  

Introduction - There are numerous Ligularia species in the eastern Qinghai-Tibet Plateau and adjacent areas. L. virgaurea has been used as a traditional folk medicine for the treatment of stomachache and nausea.
Objective - To analyse chemical constituents of L. virgaurea, grown in Yunnan and Sichuan provinces of China.
Methodology - The direct MS measurement of the crude extract of plant samples was used for grouping of this species. As the main compounds were available in pure form, the main peaks were analysed by LCMS.
Results - An easy and speedy method of analysis using MS spectra was developed. On the basis of the findings, L. virgaurea could be divided into two groups. The genetic studies also supported this grouping. Type 1 mainly includes virgaurenones and virgaurenolides. The MS of type 2 is quite different because it includes mainly ligularol and its congeners. Both MS were easily distinguished.
Conclusion - The crude extracts of 11 L. virgaurea samples already collected in recent years were analysed and it was possible to identify them as type 1 or 2. This method was applied to three samples collected in 2009 to successfully classify them as either type 1 or 2. Copyright (C) 2010 John Wiley & Sons, Ltd.

DOI： 10.1002/pca.1219

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Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Chemical composition and neutral DNA sequences of three pairs of morphologically close Ligularia species, L. subspicata/L. lamarum, L. anoleuca/L. veitchiana, and L. franchetiana/L. oligonema, were studied. Samples L. subspicata and L. lamarum were collected at 21 locations (Table 1 and Fig. 1). Both L. subspicata and L. lamarum samples could be grouped into two based on the presence or absence of furanoeremophilanes, but the two species were not distinguished in their chemical components. The major components of the Ehrlich-negative type were eremophilane-8-one derivative(s), while ligularol, subspicatins, and their derivatives were isolated from Ehrlich-positive type. The two species were not separate in the base sequences of ITS region as well. Subspicatins(5-7) were isolated as the characteristic new compounds of the species, and their structure was determined by spectroscopic methods. Chemical and genetic study of L. anoleuca and L. veitchiana revealed that the two species are not close to each other despite morphological similarities. From L. veitchiana, euparin and its derivatives were isolated, while furanoeremophilanes were isolated from L. anoleua. Furanoeremophilanes were isolated from both L. franchetiana and L. oligonema, belonging to the section Corymbosae series Retusae. Howeverk alignment of the ITS sequences of the sepcies showed that L. oligonema was separated from the other species belonging to the same section/series. These results suggest that chemical evolution in this series is complex.

DOI： 10.24496/tennenyuki.52.0_529

CiNii Article

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

From the roots of Ligularia oligonema and an unidentified sample of Ligularia collected in Yunnan Province of China, two new sesquiterpenoids, 3 beta-acetoxy-6 beta-(2-methylbutyryloxy)furanoereinophilan-10 beta-ol and 3 beta-acetoxy-6 beta-isobutyryloxyfuranoeremophilan-10 beta-ol, were isolated. DNA sequencing showed that the unidentified sample was likely to be L. oligonema. In addition, the DNA data suggested that the chemical evolution among L. oligonema and related Ligularia species is rather complex.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Chemical and genetic study of Ligularia anoleuca and L. veitchiana, which belong to section Ligularia, series Speciosae, was carried out. From L. anoleuca samples, collected in Yunnan and Sichuan Provinces of China, a new compound, furanoeremophil-1(10)-en-6 alpha-ol, was isolated together with known 6 beta-{[2-(hydroxymethyl)prop-2-enoyl]oxy}furanoeremophil-1(10)-ene and 1 beta,10 beta-epoxy-6 beta-{[2-(hydroxymethyl)prop-2-enoyl]oxy}furanoeremophilane. From L. veitchiana samples, collected in Yunnan Province, euparin, 2-isopropenyl-5,6-dimethoxybenzofuran, and 6-hydroxy-3 beta-methoxytrementone were isolated. DNA Sequencing of the internal transcribed spacers of the ribosomal RNA gene showed that the two species are not particularly close despite morphological similarities, in agreement with the chemical results.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Root chemicals and evolutionarily neutral DNA regions in L. cyathiceps samples collected in the Zhongdian (Shangrila) County of Yunnan, P. R. China, were examined. Twenty compounds were isolated, including three new ones, 1 beta,10 beta-epoxy-6 beta-(propionyloxy)furanoeremophilan-9-one (6), 1 beta,10 beta-epoxy-8 alpha-ethoxyeremophila-6,11-diene (14), and 11 alpha H-6 beta-isobutyryloxy-1 beta,10 beta,7 beta,8 beta-diepoxyeremophilan-12,8 alpha-olide (15). The chemical diversity was found to be limited, with cacalol (1) and 6(acyloxy)furanoeremophilan-9-ones (4 and/or5) being major components in all the samples. The nuclear ribosomal RNA gene was also found to harbor little variation, although two distinct sequence types were found for the plastid atp B-rbcL intergenic region.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The chemical composition of the root extract and neutral base sequences of L. hodgsonii samples collected in Japan and China were examined. From the Japanese and the Chinese samples, 6 beta-(angeloyloxy)furanoeremophilan-15-oic acid (2) and 3 beta-acetoxy-6 beta-[(2-methyl butanoyl)oxy]furaneremophilan-10 beta-ol (3) were isolated as the major component, respectively. The sequence of the internal transcribed spacers (ITSs) of the ribosomal RNA gene was different in the Japanese and the Chinese samples. In contrast to the significant genetic difference, chemical diversity was limited to the positions of O-functionalities on the furanoeremophilane skeleton, supporting our hypothesis that the production of furanocremophilanes is ecologically advantageous,

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Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Cremanthodium plants grow in the high mountain area of northwest and southwest region of China. They are used as Tibetan traditional herbs. Due to the difficulty of the plant collection only a few reports have been published so far. Chemical constituents, as well as their base sequences in atpB-rbcL intergenic region, of Cremanthodium helianthus, C. angustifolium, C. lineare var. lineare, and C. campanulatum, are investigated. Thirteen secondary metabolites were isolated and their structures were established by spectroscopic methods, especially 2D NMR spectra. All of seven compounds (1-7) isolated from C. helianthus, C. angustifolium, and C. lineare var. lineare were known eremophilane-type sesquiterpenoids, which were quite often discovered in Ligularia plant, closely related to genus Cremanthodium and widely distributed around the Hengduan Mountains. On the other hand, C. campanulatum contained four bisabolane-type sesquiterpenoids (8-11), one oplopane derivative (12), and one lignan compound (13), four of which were new. C. campanulatum seems to be related to L. lankongensis because it also produces the bisabolane-type compounds but not eremophilanes. In the base sequence analysis of five Cremanthodium samples, different genetic types were observed even in two C. angustifolium samples. Although the examples are few, intra-specific diversity, along with inter-specific diversity may be present in Cremanthodium species.

DOI： 10.24496/tennenyuki.50.0_327

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Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Intra-specific diversity in L. tsangchanensis, L. latihastata, L. lankongensis, L. duciformis, and their related species growing in Yunnan and Sichuan provinces of China was studied by chemical and genetic approaches. L. tsangchanensis samples collected in Yunnan and in Sichuan could be distinguished based on the root constituents and the base sequences of the internal transcribed spacers (ITSs) of the ribosomal RNA gene. The Yunnan samples were found to contain cacalol (1) as the major component, while samples from Sichuan contained eremophila-9, 11-dien-8-ones (5,6) and their derivative as the major components. Intra-specific diversity in L. latihastata was found to be small in chemical composition and large in the atpB-rbcL sequence. In all collected L. latihastata and L. villosa samples, two major components, 2-isopropenyl-5, 6-dimethoxybenzofuran (11) and 2-isopropenyl-5-acetyl-6-methoxybenzofuran (euparin; 12) were detected, indicating that the two species are chemically close to each other. Five highly oxygenated bisabolane-type sesquiterpenes were isolated from L. lankongensis, including four new compounds 15, 17-19. A small intra-specific diversity was observed in the composition of the root chemicals but not in the DNA sequence. L. duciformis collected in Sichuan was found to contain sinapyl or coniferyl alcohols (20-23), while the species in Yunnan contained oplopanes (24,25) or eremophilane sesquiterpenes (8,9). Diversity was observed in the atpB-rbcL sequence as well.

DOI： 10.24496/tennenyuki.50.0_285

CiNii Article

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

New C-1 oxidized eremophilane-type sesquiterpenoids, subspicatins A, B, C, and D, and subspicatolide, were isolated from the root of Ligularia subspicata and their structures were established by spectroscopic and X-ray analyses. Subspicatins A and B were the major components. The species were found to be diverse both in the composition of the root chemicals and in the nucleotide sequences of the internal transcribed spacers (ITS) of the ribosomal RNA gene and the atpB-rbcL intergenic region, but the difference among the samples was not distinct but continuous. (C) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2008.06.077

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Twenty-six eremophilane-type sesquiterpenoids, including six new compounds, were isolated from the title species. One of the new compounds, kanaitzensol, suggested the presence of an enzyme for the conversion of eremophil-7(11)-en-8-one derivative to furanoeremophilanes. The plant was collected at 15 locations in Yunnan Province of China and found to be diverse with respect to its sesquiterpenoid composition. DNA sequencing also revealed a large intraspecific diversity of the plant. (c) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2008.02.036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The chemical constituents of the roots of Ligularia lankongensis collected at seven different places in Yunnan Province, China, were investigated together with the DNA sequence of the atpB-rbcL intergenic region. All the samples contained a new, highly oxygenated bisabolane-type sesquiterpene (1). Four other oxygenated bisabolanes (2 and the new 3, 4, and 5) were also obtained. Intraspecific diversity was observed in the composition of the compounds, but not in the DNA sequence.

DOI： 10.1021/np070195p

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Four Ligularia species, L. kanaitzensis, L. vellerea, L. subspicata, and L. lamarum collected in Yunnan province of China were examined and 64 compounds were isolated. Compounds 1-15 were new. The samples of L. kanaitzensis could be divided into two groups, based on chemical composition of the root extract. Furanoeremophilane-6β,10β-diol and its derivatives were isolated from one group, while eremophilan-8-one derivatives were isolated from the other group. In contrast, intra-specific diversity was found to be small in the atpB-rbcL intergenic sequence. The samples of L. vellerea could be divided into three groups. The major components isolated from types A, B, and C were 6,15-dioxygenated furanoeremophilanes, 1,6-dioxygenated furanoeremophilanes, and 6,15-dioxygenated eremophilanolides. The difference in chemical composition is consistent with geographic distribution. Intra-specific diversity in the atpB-rbcL sequence was small. The samples of L. subspicata were grouped into two, that is, a group A producing 1-angeloyloxy derivatives and a group B producing ligularol (35) and its derivatives. Diversity was found to be present in the nucleotide sequences as well. 1,10-Dioxygenated derivatives from L. lamarum were isolated. The chemical composition was diverse. However, the DNA sequence of atpB-rbcL is currently under investigation.

DOI： 10.24496/tennenyuki.49.0_247

CiNii Article

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURAL PRODUCTS INC  

The chemical constituents of the root of Ligularia duciformis, collected in Yunnan Province of China, was studied and two new oplopane-type sesquiterpenoids were isolated, whose structures were determined on the basis of spectroscopic evidence. Quite recently, oplopane-type compounds were found in Ligularia narynensis, and this is the second example to isolate oplopane-type sesquiterpenoids from Ligularia plants.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The intraspecific diversity in L. tsangchanensis collected in the Chinese Provinces Yunnan and southwestern Sichuan was studied by chemical and genetic approaches. ne samples collected in Yunnan were found to contain cacalol (1) as the sole major component, while samples from Sichuan contained 7 alpha- and 7 beta-eremophila-9,11-dien-8-one (5 and 6) as well as the 3 alpha-angeloyloxy derivative 7 as major components. In addition, the sequences of the internal transcribed spacers (ITSs) of the ribosomal RNA gene indicated that the Yunnan and the Sichuan samples constitute separate clades. These results demonstrate that L. tsangchanensis in Yunnan and Sichuan are distinct.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

ne chemical constituents of the root extracts and the nucleotide sequences of the atpB-rbcL intergenic region of Ligularia latihastata and L. villosa, collected in northwestern Yunnan Province, were studied. In the twelve collected samples of L. latihastata, two major benzofurans. 5,6-dimethoxy-2-(1-methylethenyl)-1-benzofuran (1) and euparin (2) were detected as major components. The minor compound (2R*,3S*)-5-acetyl-2,3-dihydro-6-hydroxy-2-(1-methylethenyl)-1-benzofuran-3-yl (2Z)-2[(acetoxy)methyl]but-2-enoate (4) was found to be susceptible to artifact formation upon extraction with EtOH. The intra-specific diversity in chemical composition of the samples was small, but the diversity in the atpB-rbcL sequence was fairly large. Compounds 1 and 2 were also found in the three collected samples of L. villosa, indicating that the two species are chemically close to each other, in agreement with morphological taxonomy

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Chemical constituents of root extract and the nucleotide sequences of the atpB-rbcL intergenic region and the internal transcribed spacers (ITSs) of the ribosomal RNA gene were studied for Ligularia virgaurea var. virgaurea collected in southwestern Sichuan Province of China. Eleven samples were collected. Four of them were found to contain four new furanoeremophilanes, virgaurenones A-D, as well as a new cremophilanolide, virgaurenolide A. The other samples contained different furanoeremophilanes and their derivatives including nor-type of compounds, two of which were new. Diversity was found to be present in the nucleotide sequences as well. The chemical composition was found to be correlated with the ITS variation but not with the geographic distribution of the samples. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2006.02.078

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

Intra-specific diversity of Ligularia pleurocaulis was examined from both chemical and genetic aspects, using furanoeremophilanes as the chemical index and the nucleotide sequences in the atpB-rbcL and the ITS1 regions as the genetic index. Four samples of L. pleurocaulis were collected in northwestern Yunnan, and six in southwestern Sichuan. From the ethanol extracts of the roots of all of the samples, four compounds, 6 beta-angeloyloxy- and 6 beta-tigloyloxy-furanoeremophil-1(10)-enes, and 6 beta-angeloyloxy- and 6 beta-tigloyloxy-furanoeremophil-1(10)-en-3 beta-ols were isolated. Further, 3 beta-angeloyloxyfuranoeremophil-1(10)-en-6 beta-ol and furanoligularenone were isolated from the Yunnan and the Sichuan samples, respectively. Two variants were found for the atpB-rbcL sequence. Base multiplicity in the ITSI region was observed at only a few positions in the Yunnan samples, whereas it was observed at about 10 positions for the Sichuan samples. These results show that L. pleurocaulis is diverse, both chemically and genetically, and that plants in northwestern Yunnan and those in southwestern Sichuan can be distinguished by their chemical and genetic indexes.

DOI： 10.1246/bcsj.79.300

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

Three Ligularia species (L. tongolensis, L. cymbulifera, and L. atroviolacea) were examined with respect to the chemical composition and nucleotide sequence. Furanoeremophilanes were found to be produced in the root of all samples of the three species collected in northwest Yunnan and southwest Sichuan. Eight furanoeremophilanes were identified, two of which were new. Most of the identified furanoeremophilanes were oxygenated at 3, 6, and 15-positions. The nucleotide sequence of the atpB-rbcL intergenic region was found to be essentially the same in the three species. These similarities imply that the three Ligularia species, all belonging to the section Corymbosae, are close to one another. The intra-specific diversity in the two widely distributed species in the Hengduan Mountains area, L. tongolensis and L. cymbulifera, were in contrast. Four out of 19 L. tongolensis samples contained a strongly Ehrlich-positive compound besides a number of positive compounds, and five variants of the atpB-rbcL sequence were found in these samples. In contrast, no variation was observed in 13 L. cymbulifera samples with respect to the furanoeremophilane composition or the atpB-rbcL sequence. The lack of diversity in L. cymbulifera probably resulted from the uniformity of its habitat.

DOI： 10.1246/bcsj.78.1302

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

A human gene previously identified as a partial cDNA homologous to the gene of RET. finger protein was characterized. Northern hybridization detected three messages of 3.3, 4.2, and 7.5 kb. The coding sequences of the more abundant of the three messages, the 4.2 and the 3.3 kb, were determined. The former encodes a 630 amino acid protein ( TRIM41&alpha;) and the latter a 518 amino acid protein ( TRIM41&beta;). Green. fluorescent protein ( GFP) fusions of full- length TRIM41&alpha; and TRIM41&beta; were both observed as speckles in the cytoplasm and the nucleus. The result was corroborated by Western analysis of cellular fractions. Results with GFP fusions of various segments of the TRIM41 proteins indicated that the nuclear transport of the proteins is mediated by an N- terminal segment common to both isoforms, but independent of a classical nuclear localization signal sequence.

DOI： 10.1007/s11033-004-6613-2

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Language：English   Publishing type：Research paper (international conference proceedings)  

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S138_1

CiNii Article

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Two cDNA forms were characterized for a human dnaJ homologue, HSJ2. Nucleotide sequencing showed that the gene product HSJ2 was longer than previously reported, extending its homology to other human DnaJ paralogues, and that the two cDNAs encoded two proteins as a result of alternative splicing. The products were 326 amino acids (designated as HSJ2a) and 241 amino acids (HSJ2b) in length, sharing the N-terminal 231 amino acids including the DnaJ homology region. When fused to green fluorescent protein and expressed in HeLa cells, HSJ2a was found to be localized to the nucleus, indicating that HSJ2a is a nuclear co-chaperone. HSJ2b, however, was observed throughout the cell, consistent with the elimination of a putative nuclear localization signal sequence as a result of the alternative splicing.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

The product of the sopB gene on the Escherichia coli F-plasmid has been shown to silence genes in the vicinity of its binding region, sopC, when overexpressed. We searched for mutants defective in SopB-dependent silencing by screening for a plasmid incompatibility phenotype, in order to examine the relationship between gene silencing and the intracellular localization of SopB, as revealed by a green fluorescent protein (GFP)-SopB fusion. Nine new mutants were isolated. One of them, in which leucine 92 is replaced by proline, was completely compatible with a sopC-carrying plasmid and was defective in other silencing activities. When expressed as a GFP fusion protein, the L92P mutant was found to be uniformly distributed in the cell. This implies a link between silencing and SopB localization, supporting the view that a high local concentration of SopB drives nonspecific DNA binding in segments of the plasmid adjacent to sopC. Despite the lack of apparent localization of GFP fluorescence, the mutant protein, like the wild-type SopB, was found mostly in the inner membrane fraction, indicating that the association with the inner membrane was retained.

DOI： 10.1007/s00438-001-0597-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

Two isoforms, 1 and 2, of human DNA topoisomerase III beta were expressed in HeLa cells as a fusion protein to the C-terminus of green fluorescent protein (GFP). The fusion protein of the isoform 1 was found to be localized to the nucleus, and to be associated with chromosomes during metaphase and anaphase. As yeast top3 mutants are known to exhibit phenotypes indicative of defective chromosome segregation, the result suggests that the isoform I of the human enzyme may also be involved in chromosome segregation. Two-hybrid screening for interaction partners of the isoform identified three candidate genes: CENP-F, a gene encoding a centromere protein and two genes of no known function, one of which was novel. The GFP fusion of the isoform 2 was found in the cytoplasm, indicating the nuclear localization signal sequence in the isoform I is in the C-terminal part that is different between the two isoforms. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2001.5580

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Recombinant human DNA topoisomerase III alpha was expressed in mutant yeast cells devoid of both topoisomerases I and III, and the gene product was partially purified. The activity of the protein in supercoil removal was found to be limited and also biphasic: in the first phase it processively changed the linking-number of hypernegatively supercoiled DNA, but only to the superhelicity of a regular negative supercoil; in the second phase the enzyme relaxed the DNA further, but only slightly and slowly. The optimal solution conditions for the enzyme activity were found to be physiological, The assay results with a truncation mutant showed that the C-terminal 334 amino acids are unnecessary for the activity, suggesting that this region, and perhaps the entire protein, is involved in a function other than supercoil removal.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Purpose: To determine directly the minimal distance between two ssbs on complementary strands in circular DNA that are not observed as a dsb by electrophoresis.
Materials and methods: 3.2kbp DNAs with cohesive overhangs of various lengths were systematically generated by a newly devised method and electrophoresed in agarose slabs.
Results: At 4 degrees C, 3.2kbp DNA with cohesive overhangs larger than 6 bp migrated as circular DNA. The minimal overhang size for the DNA to migrate as circular DNA was larger at 25 degrees C. Whether the DNA migrated as a circular or a linear molecule depended also on the nucleotide sequence of its overhangs, most notably at the minimal size.
Conclusions: The minimal distance obtained in the present study agrees with the smaller values of previous indirect estimates. The dependence of the distance on experimental conditions suggests feasibility of obtaining the stagger-size distribution of radiation-induced dsbs.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing, Expressing the cloned human cDNA in yeast Delta top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA, On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12.

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Language：English   Publishing type：Research paper (scientific journal)  

Biochemical and genetic experiments were carried out to deduce the structural and functional domains of SopB protein involved in the equipartition of F plasmid. The protein is dimeric. Proteolytic and chemical footprinting studies support earlier genetic analyses that the binding of SopB to specific sites within the F plasmid sopC locus involves mainly the C- terminal region. In vivo, the expression of a high level of SopB protein is known to repress sopC-linked genes. This silencing activity is shown to be unaffected by the deletion of 35 N-terminal residues, but abolished when 71 or more were removed from the N terminus. An excess of SopB protein does not extend its in vitro binding outside sopC, implicating participation of a host factor(s) in SopB-mediated gene silencing. A data base search identified a number of SopB homologues, including both chromosomally encoded bacterial proteins and phage- and plasmid-encoded proteins known to be involved in partition. Sequence homology is limited to the N-terminal half, suggesting that the N-terminal regions of these proteins are conserved to interact with a conserved cellular structure(s), whereas the C-terminal regions have diverged to bind different nucleotide sequences.

DOI： 10.1074/jbc.271.29.17469

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

A two-step lysine-modification procedure has been devised to chemically footprint protein surfaces involved in macromolecular interactions. A protein tagged at one particular end, in the free state or in a complex, is first treated lightly with a reversible lysine-modifying reagent, The protein is then unfolded and treated extensively with an irreversible lysine reagent to block those lysines that did not react previously; next, the first lysine modification is reversed, and a lysine-specific endoproteinase is used to cleave the tagged polypeptide at the deblocked lysines. Separation of the proteolytic products by size and identification of the tagged fragments map the positions of these lysines. In this procedure, the reversible lysine reagent serves as the chemical footprinting agent, as cleavage of the polypeptide ensues only at the sites of reaction with this reagent. Lysines involved in macromolecular contacts are identified from differences in proteolytic patterns of the tagged protein when the first lysine modification is done with the protein in the free form and in a complex. Application of the method to vaccinia virus topoisomerase identifies a number of lysines that are involved in its binding to DNA.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CURRENT BIOLOGY LTD  

Background: Vaccinia virus, a cytoplasmically-replicating poxvirus, encodes a type I DNA topoisomerase that is biochemically similar to eukaryotic-like DNA topoisomerases I, and which has been widely studied as a model topoisomerase. It is the smallest topoisomerase known and is unusual in that it is resistant to the potent chemotherapeutic agent camptothecin.
Results: The crystal structure of a 9 kDa amino-terminal fragment of vaccinia virus DNA topoisomerase I has been determined at 1.6 Angstrom resolution. The fragment forms a five-stranded, antiparallel beta-sheet with two shore alpha-helices and connecting loops. Residues that are conserved between all eukaryotic-like type I topoisomerases are not clustered in particular regions of the structure.
Conclusions: This is the first atomic structure of any region of a eukaryotic-like DNA topoisomerase I, It has provided insights into the structural bases of the phenotypes of some single-site mutants of the intact topoisomerase. The structure has enabled us to study the interactions within a well-folded protein fragment and the camptothecin resistance of the viral topoisomerase.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have examined the biological role and catalytic function of two juxtaposed tyrosyl residues in the bacteriophage phiX174 gene A protein, Tyr-343 and Tyr-347, which have been implicated in the catalysis of sequence-specific DNA strand transfer. Site-directed mutagenesis changing either tyrosine to phenylalanine abolishes phage viability. The biochemical basis of this inviability was studied using purified A* protein containing the carboxyl-terminal 341 amino acids of the A protein, as well as purified A* protein with a Y343F or Y347F mutation. All three proteins can cleave the phiX174 replication origin and perform strand transfer between oligodeoxy-nucleotides bearing the recognition sequence of the A protein; however, both Tyr-343 and Tyr-347 appear to be required for coordinated DNA strand transfer by a single A* protein molecule. The chirality of a phosphorothioate group at the site of strand transfer in the DNA was found to be retained following the strand-transfer reaction, which argues against transfer of Tyr-343-linked DNA to Tyr-347 on the same protein molecule or vice versa. These results support the current model of gene A protein function in which the two tyrosines of a single protein molecule alternate in catalyzing DNA strand transfer at the viral replication origin.

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ADENINE PRESS INC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：The Chemical Society of Japan  

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：Humana Press  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：Shujunsha  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：Kyoritsu Press  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：The Biophysical Society of Japan  

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Language：Japanese   Publisher：一般社団法人日本物理学会  

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