bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vitamin B12 partners the CarH repressor to downregulate a photoinducible promoter in Myxococcus xanthus
Authors: Pérez‐Marín Mari Cruz , Padmanabhan S. , Polanco María Carmen , Murillo Francisco José , Elías‐Arnanz Montserrat 
Published: August 2010

SummaryA light‐inducible promoter, PB, drives expression of the carB operon in Myxococcus xanthus. Repressed by CarA in the dark, PB is activated when CarS, produced in the light, sequesters CarA to prevent operator‐CarA binding. The MerR‐type, N‐terminal domain of CarA, which mediates interactions with both operator and CarS, is linked to a C‐terminal oligomerization module with a predicted cobalamin‐binding motif. Here, we show that although CarA does bind vitamin B12, mutating the motif involved has no effect on its ability to repress PB. Intriguingly, PB could be repressed in the dark even with no CarA, so long as B12 and an intact CarA operator were present. We have discovered that this effect of B12 depends on the gene immediately downstream of carA. Its product, CarH, also consists of a MerR‐type, N‐terminal domain that specifically recognizes the CarA operator and CarS, linked to a predicted B12‐binding C‐terminal oligomerization module. The B12‐mediated repression of PB in the dark is relieved by deleting carH, by mutating the DNA‐ or B12‐binding residues of CarH, or by illumination. Our findings unveil parallel regulatory circuits that control a light‐inducible promoter using a transcriptional factor repertoire that includes a paralogous gene pair and vitamin B12.
The Bacillus subtilis	σM regulon and its contribution to cell envelope stress responses
Authors: Eiamphungporn Warawan , Helmann John D. 
Published: August 2010

SummaryThe Bacillus subtilis extracytoplasmic function (ECF) σM factor is activated by cell envelope stress elicited by antibiotics, and by acid, heat, ethanol and superoxide stresses. Here, we have used several complementary approaches to identify genes controlled by σM. In many cases, expression is only partially dependent on σM because of both overlapping promoter recognition with other ECF σ factors and the presence of additional promoter elements. Genes regulated by σM have a characteristic pattern of induction in response to cell envelope‐acting antibiotics as evidenced by hierarchical clustering analysis. σM also contributes to the expression of the Spx transcription factor and thereby indirectly regulates genes of the Spx regulon. Cell envelope stress responses also include regulons controlled by σW, σB and several two‐component regulatory systems (e.g. LiaRS, YycFG, BceRS). Activation of the σM regulon increases expression of proteins functioning in transcriptional control, cell wall synthesis and shape determination, cell division, DNA damage monitoring, recombinational repair and detoxification.
Pneumocystis PCINT1, a molecule with integrin‐like features that mediates organism adhesion to fibronectin
Authors: Kottom Theodore J. , Kennedy Cassie C. , Limper Andrew H. 
Published: August 2010

Summary Pneumocystis species cause severe pneumonia during chronic immunosuppression, especially in patients with AIDS or malignancy. Adhesion of Pneumocystis to extracellular matrix proteins, particularly fibronectin, associated with alveolar epithelial cell surfaces, triggers organism proliferative pathways. Herein, we report the characterization of a novel Pneumocystis molecule with considerable structural features of an integrin‐like extracellular matrix adhesion receptor. A PCINT1115 bp probe was initially identified from partial sequence present within the Pneumocystis genome project database. A full‐length 3018 bp cDNA was subsequently obtained with extensive homology to the C‐terminal region of Candida albicans INT1 (31% blastx), a gene originally described as encoding an integrin‐like molecule implicated in adhesion, growth, and virulence. Sequence analysis of PCINT1 indicated that the Pneumocystis molecule contained both a putative internal RGD motif and four Metal Ion‐Dependent Attachment Sites (MIDAS) motifs required for coordination of divalent cations, as well as a specific tyrosine residue found in the cytoplasmic tails of some integrin receptors and C. albicans INT1. Northern, Western and immunofluorescence studies demonstrated that the trophic forms of Pneumocystis, known to be the life cycle forms that tightly adhere to lung epithelium, expressed the molecule to a substantially greater degree than cystic forms. Heterologous expression of PCINT1 in yeast followed by application to human fibronectin‐coated surfaces demonstrated these yeast display PCINT1 on their surfaces and subsequently gain the ability to bind fibronectin in a cation dependent fashion. Taken together, these results indicate that Pneumocystis expresses a novel integrin‐like PCINT1 molecule sufficient to mediate interactions with extracellular matrix fibronectin, an integral component of host–cell organism interactions during this infection.
GTP‐dependent polymerization of the tubulin‐like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis
Authors: Anand Syam P. , Akhtar Parvez , Tinsley Eowyn , Watkins Simon C. , Khan Saleem A. 
Published: August 2010

SummaryRepX protein encoded by the pXO1 plasmid of Bacillus anthracis is required for plasmid replication. RepX harbours the tubulin signature motif and contains limited amino acid sequence homology to the bacterial cell division protein FtsZ. Although replication proteins are not known to polymerize, here we show by electron microscopy that RepX undergoes GTP‐dependent polymerization into long filaments. RepX filaments assembled in the presence of GTPγS were more stable than those assembled in the presence of GTP, suggesting a role for GTP hydrolysis in the depolymerization of the filaments. Light scattering studies showed that RepX underwent rapid polymerization, and substitution of GTP with GTPγS stabilized the filaments. RepX exhibited GTPase activity and a mutation in the tubulin signature motif severely impaired its GTPase activity and its polymerization in vitro. Unlike FtsZ homologues, RepX harbours a highly basic carboxyl‐terminal region and exhibits GTP‐dependent, non‐specific DNA binding activity. We speculate that RepX may be involved in both the replication and segregation of the pXO1 plasmid.
An aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos
Authors: Bougourd Sue , Marrison Jo , Haseloff Jim 
Published: August 2010

SummaryA new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high‐resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild‐type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three‐dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short‐root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.
Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators
Authors: Sakai Hiroe , Aoyama Takashi , Oka Atsuhiro 
Published: August 2010

SummaryThe genes coding for the response regulators ARR1 and ARR2 have previously been identified by in silico screening of an expression sequence tag database and subsequent cloning from both Arabidopsis cDNA and genomic libraries. Their structures, in which the N‐terminal signal receiver domain is followed by the output domain, are characteristic of typical bacterial response regulators of the two‐component regulatory systems that control responses to a variety of environmental stimuli. Here we present evidence that these response regulators actually work as transcription factors. ARR1 and ARR2 were localized in the nuclei of plant cells regardless of the presence or absence of their signal receiver domain. Their middle segments, which faintly resemble the mammalian oncogene product Myb, were capable of binding double‐stranded DNA in a sequence‐specific manner in vitro. Their C‐terminal halves functioned as transactivation domains in plant cells when combined with the DNA‐binding domain of yeast GAL4. They thus possess all the essential components of a transcriptional activator. Both ARR1 and ARR2 promoted expression of a reporter gene in plant cells through their own target sequence. Truncation of their N‐terminal signal receiver domain led to an increase in transactivation. An as yet unidentified phospho‐relay signal may modulate the capability for transactivation and/or DNA binding through the signal receiver domain.
Temperature‐dependent translational regulation of the ER ω‐3 fatty acid desaturase gene in wheat root tips
Authors: Horiguchi Gorou , Fuse Takuichi , Kawakami Naoto , Kodama Hiroaki , Iba Koh 
Published: August 2010

SummaryThe proportion of α‐linolenic acid (18:3) among the total fatty acids in root tissue increases as the growth temperature decreases. Endoplasmic reticulum ω‐3 fatty acid desaturase is responsible for the production of most of the 18:3 in root tissue. In this study, the effect of temperature on expression of the TaFAD3 gene, which encodes endoplasmic reticulum ω‐3 desaturase in wheat (Triticum aestivum L. cv. Chihoku), was analysed at the mRNA and protein levels. In wheat root tips grown at 30 and 10°C, the proportions of 18:3 among total fatty acids were 22% and 55%, respectively. The level of TaFAD3 protein in microsomal preparations of the root tips grown at 10°C was approximately 7.5 times higher than that of the root tips grown at 30°C. The increased level of TaFAD3 protein at the lower temperature was accompanied by enhanced association of TaFAD3 mRNA with polyribosomes. In contrast, the level of TaFAD3 mRNA in root tips grown at 10°C was slightly higher than in those grown at 30°C. These results suggest that, in root tips, the increase in the 18:3 level at low temperature is achieved directly by an increase in the amount of TaFAD3 protein, and that temperature‐dependent translational regulation of the TaFAD3 gene, rather than its transcriptional regulation, contributes to modulation of the TaFAD3 protein accumulation.
The MuDR transposon terminal inverted repeat contains a complex plant promoter directing distinct somatic and germinal programs
Authors: Raizada Manish N. , Benito Maria‐Ines , Walbot Virginia 
Published: August 2010

SummaryThe Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near‐identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20‐fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell‐cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.
Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action
Authors: Yoshioka Keiko , Nakashita Hideo , Klessig Daniel F. , Yamaguchi Isamu 
Published: August 2010

SummaryProbenazole (PBZ; 3‐allyloxy‐1,2‐benzisothiazole‐1,1‐dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2‐benzisothiazol‐3 (2H)‐one 1,1‐dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild‐type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis‐related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1‐1 and coi1‐1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR‐1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1‐1 and coi 1‐1 mutant plants. Thus SA and NPR1 appear to be required for PBZ‐ and BIT‐mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1‐mediated defense signaling pathway upstream of SA.
Substrate preferences of O‐methyltransferases in alfalfa suggest new pathways for 3‐O‐methylation of monolignols
Authors: Parvathi Kota , Chen Fang , Guo Dianjing , Blount Jack W. , Dixon Richard A. 
Published: August 2010

SummaryMeasurement of relative O‐methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5‐hydroxyferulic acid > caffeoyl aldehyde > 5‐hydroxyconiferyl alcohol > 5‐hydroxyferuloyl CoA > 5‐hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O‐methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O‐methyltransferase (COMT) reduced activities against the non‐esterifed substrates in the order: 5‐hydroxyconiferyl alcohol > 5‐hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5‐hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest Vmax/Km values with 5‐hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.
REVOLUTA regulates meristem initiation at lateral positions
Authors: Otsuga Denichiro , DeGuzman Bernadette , Prigge Michael J. , Drews Gary N. , Clark Steven E. 
Published: August 2010

SummaryWhile the shoot apical meristem (SAM) is indirectly responsible for the initiation of all above‐ground postembryonic organs, in most plants the vast majority of these organs are directly initiated by lateral meristems. In Arabidopsis thaliana, the lateral meristems include flower meristems (FMs), which form on the flanks of the SAM, and lateral shoot meristems (LSMs), which develop in leaf axils. While significant progress has been made on the molecular genetic basis of SAM initiation during embryo development, relatively little is known about the initiation of meristems at lateral positions. Here we have characterized the phenotypic consequences and genetic interactions of mutations in the REVOLUTA (REV) gene, with an emphasis on the role of REV in lateral meristem initiation. Our observations indicate that REV is required for initiation of both LSMs and FMs, and likely acts in the same pathway as, and upstream of, known meristem regulators. We identified the REV gene and found it encodes a predicted homeodomain/leucine zipper transcription factor that also contains a START sterol‐lipid binding domain. REV is the same as the IFL gene. REV was expressed at the earliest stages of LSM and FM formation. Within the inflorescence shoot meristem, REV expression appeared to predict 3–5 incipient flower primordia on the flanks of the SAM, and REV expression at stage 1 and stage 2 matched that of WUS and STM, respectively. We propose that REV acts at lateral positions to activate the expression of known meristem regulators.
A FORTRAN IV E AMINO ACID ANALYZER COMPUTER PROGRAM
Authors: Gerding J. J. T. 
Published: August 2010

A computer program has been written in basic Fortran IV (E level subset) which can be used on many computers and which enables calculation of amino acid analyser data in a short time. Special care has been taken to simplify the data entry. For every analysis (long or short column) only two data cards are required. Surveyable data entry forms, including typing instructions for the key punch operator, have been printed. These data entry forms can also be used for the standard calibrations runs. The program is specially suited for laboratories where many routine amino acid analyses have to be performed on a two‐column system and where the apparatus is not yet fully automated. The data input can be given in integrator units or in H X W units. The output is rendered as surveyable as possible in order to avoid human errors in the steps that are not yet automated. The data input is reprinted together with the calculated results on the data output sheet. The correctness of the results can then easily be checked.
THE OXIDATION WITH N‐BROMOSUCCINIMIDE OF THE SINGLE TRYPTOPHAN RESIDUE IN RIBONUCLEASE T1
Authors: Kawashima Seiichi , Ando Toshio 
Published: August 2010

1. The single tryptophan residue‐59 present in RNase T1 was oxidized almost specifically by NBS in 8 M urea. The enzymatic activity of RNase T1 decreased with the increase in the amount of NBS used, and complete inactivation took place within 10 minutes when 2 equivalents of the reagent were used. 2. During the oxidation with NBS of RNase T1 no cleavage of peptide bonds was found and none of the amino acid residues other than tryptophan was affected except for oxidation of cystine to cysteic acid in a small amount (up to 20 %). The complete inactivation of the enzyme occurred when the tryptophan residue was completely oxidized. 3. The oxidized RNase T1 possessed a lower solubility than the native enzyme. Measurements of the optical rotatory dispersion and the circular dichroism indicated the occurrence, of some but not so great conformational changes in the enzyme by the oxidation. 4. These residts clearly demonstrated the importance of the single tryptophan residue in RNase T1 for the proper function of the enzyme.
THE CONFORMATION OF THE HIGH‐SULPHUR PROTEINS OF WOOL. I. THE PREPARATION AND PROPERTIES OF A WATER‐SOLUBLE METAKERATIN
Authors: Bhatnagar G. M. , Crewther W. G. 
Published: August 2010

Whereas the S‐cyanoethyl derivatives of the high‐sulphur kerateines extracted from Merino wool were virtually insoluble in aqueous buffer solutions, the disulphide form of a high‐sulphur fraction designated Metakeratin‐B(5,0‐8+4) (M‐B(5.0‐8+4)), obtained by dialysing the corresponding kerateine fraction in the presence of oxygen, was soluble in aqueous buffer solutions at pH 8.0. The material was highly aggregated in solution and could be partially disaggregated by 8 M urea. Gel filtration of the meta‐keratin fraction in 8 M urea on Sephadex G‐200 indicated that the molecular weights ranged from values exceeding 100,000 (excluded from column) to about 10,000. The latter value was confirmed by sedimentation measurements. When reduced and alkylated with iodoacetate the S‐carboxymethylkerateine fraction so formed (SCMK‐5(5.o 8.4)) contained chiefly components with molecular weights of about 10,000 as determined by gel filtration. M‐B(5,0‐8.4) showed a minimum solubility at about pH 4.4 in salt solutions, the pH of minimum solubility decreasing to about 3.0 with progressive S‐carboxymethylation of the protein. No evidence could be obtained for the presence in M‐B(5.0.8.4) of asymmetrical disulphide groups of the form CyS‐S‐CH2COOH. The incorporation of 8 M urea in solutions of the metakeratin at pH 8 gave rise to a positive difference spectrum, suggesting that few if any additional tyrosine residues become accessible in this solvent. Optical rotatory dispersion measurements gave a value of–50 for the parameter b0 in the Moffitt‐Yang equation. In the presence of 8 M urea the value of b0 became zero. M‐B(5,0‐8.4) was digested relatively slowly by Pronase P but the initial rate of digestion increased greatly as the disulphide bonds were converted progressively to S‐carboxy‐methyl groups, the greatest change in rate occurring over the range 10 to 20% conversion of the cystine residues to S‐carboxymethylcysteine residues. The wavelength of maximum fluorescence of M‐B(5.0‐8.4) was unaffected by the S‐carboxymethylation but the intensity of the fluorescence was greatly increased by this modification. The ultraviolet absorption curve for the metakeratin showed absorption maxima at about 276 and 330 nm. With progressive S‐carboxymethylation the ab‐sorbance over the whole range of wavelengths decreased, the absorbance peak at about 330 nm disappeared, and an additional peak at about 280 nm appeared. The results suggest that the reduced chains of the high‐sulphur protein fraction from wool, when allowed to oxidize to the disulphide form, tend to fold in a manner resembling native globular proteins. The unit structures so formed appear to be stabilized by intrachain disulphide bonds.