bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rpc25, a conserved RNA polymerase III subunit, is critical for transcription initiation
Authors: Zaros Cécile , Thuriaux Pierre 
Published: December 2004

SummaryRpc25 is a strongly conserved subunit of RNA polymerase III with homology to Rpa43 in RNA polymerase I, Rpb7 in RNA polymerase II and the archaeal RpoE subunit. A central domain of Rpc25 can replaced the corresponding region of Rpb7 with little or no growth defect, underscoring the functional relatedness of these proteins. Rpc25 forms a heterodimer with Rpc17, another conserved component of RNA polymerase III. A conditional mutant (rpc25‐S100P) impairs this interaction. rpc25‐S100P and another conditional mutant obtained by complementation with the Schizosaccharomyces pombe subunit (rpc25‐Sp) were investigated for the properties of their purified RNA polymerase III. The mutant enzymes were defective in the specific synthesis of pre‐tRNA transcripts but acted at a wild‐type level on poly[d(A‐T)] templates. They were also indistinguishable from wild type in transcript elongation, cleavage and termination. These data indicate that Rpc25 is needed for transcription initiation but is not critical for the elongating properties of RNA polymerase III.
Distinct segregation dynamics of the two Vibrio cholerae chromosomes
Authors: Fogel Michael A. , Waldor Matthew K. 
Published: December 2004

SummaryThe study of prokaryotic chromosome segregation has focused primarily on bacteria with single circular chromosomes. Little is known about segregation in bacteria with multipartite genomes. The human diarrhoeal pathogen Vibrio cholerae has two circular chromosomes of unequal sizes. Using static and time‐lapse fluorescence microscopy, we visualized the localization and segregation of the origins of replication of the V. cholerae chromosomes. In all stages of the cell cycle, the two origins localized to distinct subcellular locations. In newborn cells, the origin of chromosome I (oriCIvc) was located near the cell pole while the origin of chromosome II (oriCIIvc) was at the cell centre. Segregation of oriCIvc occurred asymmetrically from a polar position, with one duplicated origin traversing the length of the cell towards the opposite pole and the other remaining relatively fixed. In contrast, oriCIIvc segregated later in the cell cycle than oriCIvc and the two duplicated oriCIIvc regions repositioned to the new cell centres. DAPI staining of the nucleoid demonstrated that both origin regions were localized to the edge of the visible nucleoid and that oriCIvc foci were often associated with specific nucleoid substructures. The differences in localization and timing of segregation of oriCIvc and oriCIIvc suggest that distinct mechanisms govern the segregation of the two V. cholerae chromosomes.
Exploring prokaryotic diversity: there are other molecular worlds
Authors: Fernández Luis Angel 
Published: December 2004

SummaryProkaryotes are the major source of biological diversity on earth. This is not simply because of the large number of species present, or because of their diverse growth conditions and environmental niches populated by them, but because of the wealth of genes, metabolic pathways and molecular processes that are only found in prokaryotic cells. Therefore, Bacteria and Archaea (and their phages) cannot be considered any longer as miniaturized models of Eukaryotes, but as a genuine source of unique biological processes that are mediated by unique sets of genes and molecular devices. A true understanding of complex biological phenomena will require a deeper knowledge of this vast prokaryotic world. The second European Molecular Biology Organization (EMBO) conference on Molecular Microbiology entitled ‘Exploring Prokaryotic Diversity’ explored many aspects of this newly emerging interest in the prokaryotic world.
Differential ability of σs and σ70 of Escherichia coli to utilize promoters containing half or full UP‐element sites
Authors: Typas Athanasios , Hengge Regine 
Published: December 2004

SummaryThe σs subunit of RNA polymerase (RNAP) is the master regulator of the general stress response in Escherichia coli. Nevertheless, the selectivity of promoter recognition by the housekeeping σ70‐containing and σ5‐containing RNAP holoenzymes (Eσ70 and Eσs respectively) is not yet fully clarified, as they both recognize nearly identical −35 and −10 promoter consensus sequences. In this study, we show that in a subset of promoters, Eσs favours the presence of a distal UP‐element half‐site, and at the same time is unable to take advantage of a proximal half‐site or a full UP‐element. This is reflected by the frequent occurrence of distal UP‐element half‐sites in natural σs‐dependent promoters and the absence of proximal half‐sites. Eσ70, however, exhibits the opposite preference. The presence of the −35 element is a prerequisite for this differential behaviour. In the absence of the −35 element, half or full UP‐element sites play no role in sigma selectivity, but the distal subsite leads to an equivalent, if not greater, transcriptional stimulation than the proximal one for both sigma factors. Finally, experiments using single amino acid substitutions of σs indicate that the foundation for this preference lies in an inability of σs to interact with the a subunit C‐terminal domain.
Disulphide trapping of an in vivo energy‐dependent conformation of Escherichia coli TonB protein
Authors: Ghosh Joydeep , Postle Kathleen 
Published: December 2004

SummaryIn Escherichia coli, the TonB system transduces the protonmotive force (pmf) of the cytoplasmic membrane to support a variety of transport events across the outer membrane. Cytoplasmic membrane proteins ExbB and ExbD appear to harvest pmf and transduce it to TonB. Experimental evidence suggests that TonB shuttles to the outer membrane, apparently to deliver conformationally stored potential energy to outer membrane transporters. In the most recent model, discharged TonB is then recycled to the cytoplasmic membrane to be re‐energized by the energy coupling proteins, ExbB/D. It has been suggested that the carboxy‐terminal 75 amino acids of active TonB could be represented by the rigid, strand‐exchanged, dimeric crystal structure of the corresponding fragment. In contrast, recent genetic studies of alanine substitutions have suggested instead that in vivo the carboxy‐terminus of intact TonB is dynamic and flexible. The biochemical studies presented here confirm and extend those results by demonstrating that individual cys substitution at aromatic residues in one monomeric subunit can form spontaneous dimers in vivo with the identical residue in the other monomeric subunit. Two energized TonBs appear to form a single cluster of 8–10 aromatic amino acids, including those found at opposite ends of the crystal structure. The aromatic cluster requires both the amino‐terminal energy coupling domain of TonB, and ExbB/D (and cross‐talk analogues TolQ/R) for in vivo formation. The large aromatic cluster is detected in cytoplasmic membrane‐, but not outer membrane‐associated TonB. Consistent with those observations, the aromatic cluster can form in the first half of the energy transduction cycle, before release of conformationally stored potential energy to ligand‐loaded outer membrane transporters. The model that emerges is one in which, after input of pmf mediated through ExbB/D and the TonB transmembrane domain, the TonB carboxy‐terminus can form a meta‐stable high‐energy conformation that is not represented by the crystal structure of the carboxy‐terminus.
The TyrR regulon
Authors: Pittard James , Camakaris Helen , Yang Ji 
Published: December 2004

SummaryThe TyrR protein of Escherichia coli can act both as a repressor and as an activator of transcription. It can interact with each of the three aromatic amino acids, with ATP and, under certain circumstances, with the C‐terminal region of the α‐subunit of RNA polymerase. TyrR protein is a dimer in solution but in the presence of tyrosine and ATP it self‐associates to form a hexamer. Whereas TyrR dimers can, in the absence of any aromatic amino acids, bind to certain recognition sequences referred to as ‘strong TyrR boxes’, hexamers can bind to extended sequences including lower‐affinity sites called ‘weak TyrR boxes’, some of which overlap the promoter. There is no single mechanism for repression, which in some cases involves exclusion of RNA polymerase from the promoter and in others, interference with the ability of bound RNA polymerase to form open complexes or to exit the promoter. When bound to a site upstream of certain promoters, TyrR protein in the presence of phenylalanine, tyrosine or tryptophan can interact with the α‐subunit of RNA polymerase to activate transcription. In one unusual case, activation of a non‐productive promoter is used to repress transcription from a promoter on the opposite strand. Regulation of individual transcription units within the regulon reflects their physiological function and is determined by the position and nature of the recognition sites (TyrR boxes) associated with each of the promoters. The intracellular levels of the various forms of the TyrR protein are also postulated to be of critical importance in determining regulatory outcomes. TyrR protein remains a paradigm for a regulator that is able to interact with multiple cofactors and exert a range of regulatory effects by forming different oligomers on DNA and making contact with other proteins. A recent analysis identifying putative TyrR boxes in the E. coli genome raises the possibility that the TyrR regulon may extend beyond the well‐characterized transcription units described in this review.
Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome
Authors: Hashimoto Masayuki , Ichimura Toshiharu , Mizoguchi Hiroshi , Tanaka Kimie , Fujimitsu Kazuyuki , Keyamura Kenji , Ote Tomotake , Yamakawa Takehiro , Yamazaki Yukiko , Mori Hideo , Katayama Tsutomu , Kato Jun‐ichi 
Published: December 2004

SummaryThe minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large‐scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.
cAMP blocks MAPK activation and sclerotial development via Rap‐1 in a PKA‐independent manner in Sclerotinia sclerotiorum
Authors: Chen Changbin , Dickman Martin B. 
Published: December 2004

Summary Sclerotinia sclerotiorum is a filamentous ascomycete phytopathogen able to infect an extremely wide range of cultivated plants. Our previous studies have shown that increases in cAMP levels result in the impairment of the development of the sclerotium, a highly differentiated structure important in the disease cycle of this fungus. cAMP also inhibits the activation of a S. sclerotiorum mitogen‐activated protein kinase (MAPK), which we have previously shown to be required for sclerotial maturation; thus cAMP‐mediated sclerotial inhibition is modulated through MAPK. However, the mechanism(s) by which cAMP inhibits MAPK remains unclear. Here we demonstrate that a protein kinase A (PKA)‐independent signalling pathway probably mediates MAPK inhibition by cAMP. Expression of a dominant negative form of Ras, an upstream activator of the MAPK pathway, also inhibited sclerotial development and MAPK activation, suggesting that a conserved Ras/MAPK pathway is required for sclerotial development. Evidence from bacterial toxins that specifically inhibit the activity of small GTPases, suggested that Rap‐1 or Ras is involved in cAMP action. The Rap‐1 inhibitor, GGTI‐298, restored MAPK activation in the presence of cAMP, further suggesting that Rap‐1 is responsible for cAMP‐dependent MAPK inhibition. Importantly, inhibition of Rap‐1 is able to restore sclerotial development blocked by cAMP. Our results suggest a novel mechanism involving the requirement of Ras/MAPK pathway for sclerotial development that is negatively regulated by a PKA‐independent cAMP signalling pathway. Cross‐talk between these two pathways is mediated by Rap‐1.
A cis‐acting sequence involved in chromosome segregation in Escherichia coli
Authors: Fekete Richard A. , Chattoraj Dhruba K. 
Published: December 2004

SummaryEukaryotic chromosomes contain a locus, the centromere, at which force is applied to separate replicated chromosomes. A centromere analogue is also found in some bacterial plasmids and chromosomes, although not yet identified in the well‐studied Escherichia coli chromosome. We aimed to identify centromere‐like sequences in E. coli with the premise that such sequences would be the first to migrate towards the cell poles, away from the cell centre where DNA replication is believed to occur. We have labelled different loci on the chromosome by integrating arrays of binding sites for LacI–EYFP and phage λcI–ECFP and supplying these fusion proteins in trans. Comparison of such pairs of loci suggests the presence of a centromere‐like site close to the origin of replication. Polar migration of the site was dependent on migS, a locus recently implicated in chromosome migration, thus providing strong support for migS being the E. coli centromere.
The human malaria parasite Plasmodium falciparum possesses two distinct dihydrolipoamide dehydrogenases
Authors: McMillan Paul J. , Stimmler Luciana M. , Foth Bernardo J. , McFadden Geoffrey I. , Müller Sylke 
Published: December 2004

SummaryThe Plasmodium falciparum genome contains genes encoding three α‐ketoacid dehydrogenase multienzyme complexes (KADHs) that have central metabolic functions. The parasites possess two distinct genes encoding dihydrolipoamide dehydrogenases (LipDH), which are indispensable subunits of KADHs. This situation is reminiscent of that in plants, where two distinct LipDHs are found in mitochondria and chloroplasts, respectively, that are part of the organelle‐specific KADHs. In this study, we show by reverse transcription polymerase chain reaction (RT‐PCR) that the genes encoding subunits of all three KADHs, including both LipDHs, are transcribed during the erythrocytic development of P. falciparum. Protein expression of mitochondrial LipDH and mitochondrial branched chain α‐ketoacid dihydrolipoamide transacylase in these parasite stages was confirmed by Western blotting. The localization of the two LipDHs to the parasite's apicoplast and mitochondrion, respectively, was shown by expressing the LipDH N‐terminal presequences fused to green fluorescent protein in erythrocytic stages of P. falciparum and by immunofluorescent colocalization with organelle‐specific markers. Biochemical characterization of recombinantly expressed mitochondrial LipDH revealed that the protein has kinetic and physicochemical characteristics typical of these flavo disulphide oxidoreductases. We propose that the mitochondrial LipDH is part of the mitochondrial α‐ketoglutarate dehydrogenase and branched chain α‐ketoacid dehydrogenase complexes and that the apicoplast LipDH is an integral part of the pyruvate dehydrogenase complex which occurs only in the apicoplast in P. falciparum.
The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast
Authors: Foth Bernardo J. , Stimmler Luciana M. , Handman Emanuela , Crabb Brendan S. , Hodder Anthony N. , McFadden Geoffrey I. 
Published: December 2004

SummaryThe relict plastid (apicoplast) of apicomplexan parasites synthesizes fatty acids and is a promising drug target. In plant plastids, a pyruvate dehydrogenase complex (PDH) converts pyruvate into acetyl‐CoA, the major fatty acid precursor, whereas a second, distinct PDH fuels the tricarboxylic acid cycle in the mitochondria. In contrast, the presence of genes encoding PDH and related enzyme complexes in the genomes of five Plasmodium species and of Toxoplasma gondii indicate that these parasites contain only one single PDH. PDH complexes are comprised of four subunits (E1α, E1β, E2, E3), and we confirmed four genes encoding a complete PDH in Plasmodium falciparum through sequencing of cDNA clones. In apicomplexan parasites, many nuclear‐encoded proteins are targeted to the apicoplast courtesy of two‐part N‐terminal leader sequences, and the presence of such N‐terminal sequences on all four PDH subunits as well as phylogenetic analyses strongly suggest that the P. falciparum PDH is located in the apicoplast. Fusion of the two‐part leader sequences from the E1α and E2 genes to green fluorescent protein experimentally confirmed apicoplast targeting. Western blot analysis provided evidence for the expression of the E1α and E1β PDH subunits in blood‐stage malaria parasites. The recombinantly expressed catalytic domain of the PDH subunit E2 showed high enzymatic activity in vitro indicating that pyruvate is converted to acetyl‐CoA in the apicoplast, possibly for use in fatty acid biosynthesis.
Crustal structure of the southern Dabie ultrahigh‐pressure orogen and Yangtze foreland from deep seismic reflection profiling
Authors: Dong Shuwen , Gao Rui , Cong Bolin , Zhao Zhongyan , Liu Xiaochun , Li Sanzhong , Li Qiusheng , Huang Dongding 
Published: December 2004

AbstractA new 140‐km‐long seismic reflection profile provides a high‐resolution crustal‐scale image of the southern Dabieshan high‐pressure (HP) metamorphic belt and the Yangtze foreland fold‐and‐thrust belt. The seismic image of the stacked section shows that the southern Dabieshan metamorphic terrane and Yangtze foreland belt are separated by a large north‐dipping fault. In the foreland the upper crust is dominated by a series of folds and thrusts formed during the collisional stage in the mid‐Triassic; it was reworked by crustal extension resulting in the formation of a late Jurassic and Cretaceous red‐bed basin. The southern Dabieshan profile shows stacked crustal slabs developed along the margin of the collisional orogenic belt. The Moho reflectors at 10–11 s (∼30–33 km) are seismically prominent and segmented by a number of south‐verging thrusts that were probably developed by foreland‐directed thrusting of the deeply subducted continental crust during exhumation. The seismic reflection profile suggests that structures related to the Triassic–Jurassic subduction and exhumation of the Yangtze plate are preserved despite the severe crustal extension superimposed during the late Mesozoic and Cenozoic.
Complete Tertiary exhumation history of the Menderes massif, western Turkey: an alternative working hypothesis
Authors: Seyitoǧlu Gürol , Işık Veysel , Çemen İbrahim 
Published: December 2004

AbstractThe main exhumation of the Menderes massif, western Turkey, occurred along an originally N‐dipping Datça–Kale main breakaway fault that controlled depositions in the Kale and the Gökova basins during the Oligocene – Early Miocene interval. The isostatically controlled upward bending of the main breakaway fault brings the lower plate rocks to the surface. In the Early Miocene, E–W‐trending N‐ and S‐dipping graben‐bounding faults fragmented the exhumed, dome‐shaped massif. The development of half grabens by rolling master fault hinges has allowed further exhumation of the central Menderes massif. After the Pliocene, high‐angle normal faults cut all of the previous structures. This model suggests that the Menderes massif is a single large metamorphic core complex that has experienced a two‐stage exhumation process.
Value of reflux diagnostic questionnaire in the diagnosis of gastroesophageal reflux disease
Authors:   CHINESE GERD STUDY GROUP
Published: December 2004

OBJECTIVE:  To evaluate the value of the reflux diagnostic questionnaire (RDQ) in the diagnosis of gastroesophageal reflux disease (GERD).METHODS:  One hundred and twenty‐eight patients with heartburn, substernal pain, acid reflux and regurgitation in 10 hospitals in China were assessed. Each symptom was scored according to severity and frequency (five scales). The highest score was 40. Upper gastrointestinal endoscopy and 24‐h esophageal pH monitoring were carried out in patients with symptom scores of more than six, and results were compared with the RDQ score to determine the critical value for diagnosis of GERD.RESULTS:  The RDQ score correlated positively with the severity of reflux esophagitis. Esophageal pH monitoring showed that patients with abnormal RDQ had higher DeMeester scores compared with those with normal RDQ scores (20.18 vs 16.84). Taking 12 as the critical RDQ score for GERD, achieved a maximal Youden index. The area under the receiver operating characteristic curve was 0.71, the true positive diagnostic rate was 88.07%, and the true negative diagnostic rate was 68.42% with a sensitivity of 94.12% and specificity of 50%.CONCLUSION:  The RDQ might be a useful screening test for GERD.
Risk factors for anastomotic leakage after anterior resection of the rectum
Authors: Matthiessen P. , Hallböök O. , Andersson M. , Rutegård J. , Sjödahl R. 
Published: December 2004

AbstractObjective  Surgical technique and peri‐operative management of rectal carcinoma have developed substantially in the last decades. Despite this, morbidity and mortality after anterior resection of the rectum are still important problems. The aim of this study was to identify risk factors for anastomotic leakage in anterior resection and to assess the role of a temporary stoma and the need for urgent re‐operations in relation to anastomotic leakage.Patients and methods  In a nine‐year period, from 1987 to 1995, a total of 6833 patients underwent elective anterior resection of the rectum in Sweden. A random sample of 432 of these patients was analysed (sample size 6.3%). The associations between death and 10 patient‐ and surgery‐related variables were studied by univariate and multivariate analysis. Data were obtained by review of the hospital files from all patients.Results  The incidence of symptomatic clinically evident anastomotic leakage was 12% (53/432). The 30‐day mortality was 2.1% (140/6833). The rate of mortality associated with leakage was 7.5%. A temporary stoma was initially fashioned in 17% (72/432) of the patients, and 15% (11/72) with a temporary stoma had a clinical leakage, compared with 12% (42/360) without a temporary stoma, not significant. Multivariate analysis showed that low anastomosis (≤ 6 cm), pre‐operative radiation, presence of intra‐operative adverse events and male gender were independent risk factors for leakage. The risk for permanent stoma after leakage was 25%. Females with stoma leaked in 3% compared to men with stoma who leaked in 29%. The median hospital stay for patients without leakage was 10 days (range 5–61 days) and for patients with leakage 22 days (3–110 days).Conclusion  In this population based study, 12% of the patients had symptomatic anastomotic leakage after anterior resection of the rectum. Postoperative 30‐day mortality was 2.1%. Low anastomosis, pre‐operative radiation, presence of intra‐operative adverse events and male gender were independent risk factors for symptomatic anastomotic leakage in the multivariate analysis. There was no difference in the use of temporary stoma in patients with or without anastomotic leakage.