bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of interferon α and cell cycle progression on translation mediated by the hepatitis C virus 5′ untranslated region: a study using a transgenic mouse model
Authors: Takeda Y. , Okoshi S. , Suzuki K. , Yano M. , Gangemi J. D. , Jay G. , Asakura H. , Aoyagi Y.
Published: January 2004

Summary.  The effect of interferon α (IFNα) and the progression of the cell cycle on translation mediated by the 5′ untranslated region (5′UTR) of hepatitis C virus (HCV) was evaluated in a transgenic mouse model containing the β‐galactosidase (β‐gal) gene under the control of the mouse albumin promoter and HCV 5′UTR. The transgene was exclusively expressed in the liver and specifically in hepatocytes around the periportal area. IFNα significantly suppressed the expression of both the β‐gal gene product and its enzymatic activity at 6 h after the treatment of the mice. The mRNA level of the transgene and endogenous albumin gene expression were not affected, so this suppression was considered to be specific to 5′UTR‐directed translation. Phosphorylation of the Stat1 protein was observed in the liver extract 20 min after the treatment, thus confirming a specific known effect of IFNαin vivo. We suggest that suppression of 5′UTR‐directed translation may be one of the mechanisms whereby IFNα exerts its anti‐viral activity. We further investigated whether the restriction of 5′UTR‐directed translation in periportal hepatocytes may be explained by the proliferative state of the cell. Transgene expression was slightly enhanced in the liver 48 h after partial hepatectomy when a substantial number of hepatocytes entered cell cycle progression. However, 5′UTR‐directed translation could not be detected in hepatocellular carcinoma lesions in transgenic mice that were induced to develop such tumours. We suggest that the state of differentiation of the cell, and not its proliferative capacity, is important for supporting HCV expression. This animal model may be a useful tool to dissect the control of HCV expression and to search for ways to block viral replication.
Detection and quantitation of HCV core protein in single hepatocytes by means of laser capture microdissection and enzyme‐linked immunosorbent assay
Authors: Sansonno D. , Lauletta G. , Dammacco F.
Published: January 2004

Summary.  Immunohistochemistry provides valuable information concerning the localization and distribution of hepatitis C virus (HCV)‐related proteins in histological sections of liver tissue, but does not readily permit their quantitation in individual cells and the staining intensity of cell immunodeposits cannot be calibrated with the current number of antigen molecules. We specifically detected and quantitated HCV core protein in single hepatocytes by coupling laser capture microdissection (LCM) with a sensitive enzyme‐linked immunosorbent assay (ELISA). Quantitation of HCV core protein per cell was carried out on liver tissue cells obtained by LCM from fixed and stained frozen sections of 10 HCV‐positive patients with chronic active hepatitis (CAH). Macromolecules from captured cells were solubilized in an extraction buffer and directly assayed for core protein using a sandwich ELISA. Calibration was achieved by developing a standard curve based on known concentrations of HCV core protein. Precision, linearity and sensitivity were verified for known numbers of microdissected tissue cells. In this study, the concentration of HCV core protein in single hepatocytes ranged from 7 to 56 pg/cell. Specificity was verified on 10 replicates of 10 HCV‐negative liver tissues. Immunohistochemical staining of HCV core protein was compared with the results of the soluble immunoassay for the adjacent liver tissue sections. Independent scoring of HCV immunostaining failed to parallel the LCM quantitative immunoassay. LCM‐based immunoassay significantly expands our ability to investigate function‐related antigens in apparently pure cell populations in HCV infection.
Evidence that plasma concentration rather than dose per kilogram body weight predicts ribavirin‐induced anaemia
Authors: Lindahl K. , Schvarcz R. , Bruchfeld A. , Ståhle L.
Published: January 2004

Summary.  Ribavirin in combination with interferon alpha‐2 or pegylated interferon is the standard treatment for chronic hepatitis C. The current dosage recommendations for ribavirin are based on body weight (bw). Ribavirin is mainly eliminated by the kidneys and we have recently shown that ribavirin plasma concentrations are determined primarily by renal function. It is therefore reasonable to hypothesize that side‐effects of ribavirin, i.e. anaemia, should be more closely related to plasma concentrations of ribavirin than to the dose per kg bw. A total of 108 consecutive patients eligible for treatment of chronic hepatitis C were studied. Ribavirin concentrations in plasma were measured by high‐performance liquid chromatography (HPLC)‐UV after solid‐phase extraction in trough samples taken 4, 8 and 12 weeks after the treatment commenced. A total of 213 samples were obtained and the change in the haemoglobin level and the creatinine concentration was measured in addition to ribavirin. The dose of ribavirin per kg bw did not correlate with the drop in haemoglobin level induced by ribavirin. The concentration of ribavirin was non‐linearly related to the drop in the haemoglobin level as revealed by fitting a standard Hill equation type dose–response curve. The half maximal drop in haemoglobin was obtained at 4.4 μm. The results from this study suggest that the anaemia induced by ribavirin depends primarily on the concentration of ribavirin, and not on the dose per kg bw. This lends further support to the idea that ribavirin should be dosed according to renal function.
Consensus conference on chronic viral hepatitis and HIV infection: updated Spanish recommendations
Authors: Soriano V. , Miró J. M. , García‐Samaniego J. , Torre‐Cisneros J. , Núñez M. , Del Romero J. , Martín‐Carbonero L. , Castilla J. , Iribarren J. A. , Quereda C. , Santín M. , González J. , Arribas J. R. , Santos I. , Hernández‐Quero J. , Ortega E. , Asensi V. , Del Pozo M. A. , Berenguer J. , Tural C. , Clotet B. , Leal M. , Mallolas J. , Sánchez‐Tapias J. M. , Moreno S. , Gatell J. M. , Téllez M. J. , Rubio R. , Ledesma E. , Domingo P. , Barreiro P. , Pedreira J. , Romero M. , González‐Lahoz J. , Lissen E.
Published: January 2004

Summary. Chronic hepatitis B and C represent a leading cause of morbidity and mortality among human immunodeficiency virus (HIV)‐infected patients worldwide. New treatment options against both hepatitis B (HBV) and C (HCV) viruses have prompted us to update previous recommendations for the management of coinfected individuals. Fifteen topics (nine related to HCV, five to HBV and one to both viruses) were selected for this purpose. A panel of Spanish experts in the field was invited to review these areas and propose specific recommendations, which were scored according to the Infectious Disease Society of America (IDSA) grading system. These guidelines represent a comprehensive and updated overview on the management of hepatitis B and C in HIV‐infected patients.
MinD and role of the deviant Walker A motif, dimerization and membrane binding in oscillation
Authors: Lutkenhaus Joe , Sundaramoorthy M.
Published: January 2004

The ATPase activity of MinD is required for it to oscillate between the ends of the cell and spatially regulate cell division in Escherichia coli. It is a member of a functionally diverse subgroup of ATPases which are involved in activities ranging from nitrogen fixation (NifH) to plasmid segregation (ParA). All members of the subgroup have a deviant Walker A motif which contains a conserved ‘signature’ lysine that characterizes this subgroup. In the NifH homodimer the signature lysines make intermonomer contact with the bound nucleotides indicating a role in ATP hydrolysis. ATP binding to NifH leads to formation of an active dimer that associates with a partner that is also a dimer. Because ATP hydrolysis is coupled to formation of the complex, the complex is only transient. In the presence of ATP MinD binds MinC and goes to the membrane, however, the ATPase is not stimulated and the complex is stable. Subsequent interaction of this complex with MinE, however, leads to ATPase stimulation and release of the Min proteins from the membrane. The sequential interaction of MinD with these two proteins, which is dictated by the membrane, is critical to the oscillatory mechanism involved in spatial regulation of division.
A Streptomyces coelicolor functional orthologue of Escherichia coli RNase E shows shuffling of catalytic and PNPase‐binding domains
Authors: Lee Kangseok , Cohen Stanley N.
Published: January 2004

Previous work has detected an RNase E‐like endoribonucleolytic activity in cell extracts obtained from Streptomyces. Here, we identify a Streptomyces coelicolor gene, rns, encoding a 140 kDa protein (RNase ES) that shows endoribonucleolytic cleavage specificity characteristic of RNase E, confers viability on and allows propagation of Escherichia coli cells lacking RNase E and accomplishes RNase E‐like regulation of plasmid copy number in E. coli. However, notwithstanding its complementation of rne‐deleted E. coli, RNase ES did not accurately process 9S rRNA from E. coli. Additionally, whereas RNase E is normally required for E. coli survival, rns is not an essential gene in S. coelicolor. Deletion analysis mapped the   catalytic   domain   of   RNase   ES   near   its   centre and showed that regions located near the RNase ES termini interact with an S. coelicolor homologue of polynucleotide phosphorylase (PNPase) – a major component of E. coli RNase E‐based degradosomes. The interacting arginine‐ and proline‐rich segments resemble the C‐terminally located degradosome scaffold region of E. coli RNase E. Our results indicate that RNase ES is a structurally shuffled RNase E homologue showing evolutionary conservation of functional RNase E‐like enzymatic activity, and suggest the existence of degradosome‐like complexes in Gram‐positive bacteria.
SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis
Authors: Braunstein Miriam , Espinosa Benjamin J. , Chan John , Belisle John T. , R. Jacobs William
Published: January 2004

Tuberculosis remains a severe worldwide health threat. A thorough understanding of Mycobacterium tuberculosis pathogenesis will facilitate the development of new treatments for tuberculosis. Numerous bacterial pathogens possess specialized protein secretion systems that are dedicated to the export of virulence factors. Mycobacterium tuberculosis is part of a developing group of pathogenic bacteria that share the uncommon property of possessing two secA genes (secA1 and secA2). In mycobacteria, SecA1 is the essential ‘housekeeping’ SecA protein whereas SecA2 is an accessory secretion factor. Here we demonstrate that SecA2 contributes to the pathogenesis of M. tuberculosis. A deletion of the secA2 gene in M. tuberculosis attenuates the virulence of the organism in mice. By comparing the profile of proteins secreted by wild‐type M. tuberculosis and the ΔsecA2 mutant, we identified superoxide dismutase A (SodA) as a protein dependent on SecA2 for secretion. SodA lacks a classical signal sequence for protein export. Our data suggests that SecA2‐dependent export is a new type of secretion pathway that is part of a virulence mechanism of M. tuberculosis to elude the oxidative attack of macrophages.
A new Schizosaccharomyces pombe base excision repair mutant, nth1, reveals overlapping pathways for repair of DNA base damage
Authors: Osman Fekret , Bjørås Magnar , Alseth Ingrun , Morland Ingrid , McCready S. , Seeberg Erling , Tsaneva Irina
Published: January 2004

Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or γ‐irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl‐N‐nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3‐methyladenine and 7‐methylguanine whereas methyl‐formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1‐like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears   to   be   their   lack   of   AP   lyase   activity.   The nth1 mutant   also   exhibited   elevated   frequencies   of spontaneous mitotic intrachromosomal recombination,   which   is   a   phenotype   shared   by   the   MMS‐hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.
Genetic and biochemical characterization of MbeA, the relaxase involved in plasmid ColE1 conjugative mobilization
Authors: Varsaki Athanasia , Lucas María , Afendra Amalia S. , Drainas Constantin , De La Cruz Fernando
Published: January 2004

MbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand‐transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5′‐(1469)CTGG/CTTA(1462)‐3′. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site‐directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand‐transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they  are  directly  implicated  in  catalysis  of  DNA‐cleavage and strand‐transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a    variant    of    the    common    relaxase    theme    with    a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases.
An archaeal XPF repair endonuclease dependent on a heterotrimeric PCNA
Authors: Roberts J. A. , Bell S. D. , White M. F.
Published: January 2004

Archaea share many similarities with eukarya in their information processing pathways and have proven to be a useful model for studies of DNA replication and transcription, but DNA repair pathways are not well understood in archaea. Nucleotide Excision Repair (NER) deals with many bulky DNA lesions and involves over 30 proteins in eukarya. Archaeal NER has not been characterized biochemically, but homologues of the human repair nucleases XPF and XPG have been identified by homology searches. Crenarchaeal XPF proteins have a simplified domain structure, consisting of the C‐terminal nuclease domain conserved   in   XPF   and   Mus81   but   lacking   the   N‐terminal ‘helicase’ domain that is found in eukaryal and euryarchaeal sequences. Unexpectedly, Sulfolobus XPF is only active in the presence of the sliding clamp PCNA, which is a heterotrimer in this organism. Interactions with two of the three subunits of PCNA are mediated via a C‐terminal interaction motif. The PCNA‐XPF complex acts as a structure‐specific nuclease on a similar range of DNA flap, bubble and junction substrates as the human protein, suggesting a fundamental conservation through billions of years of evolution.
Origins of metal ion selectivity in the DtxR/MntR family of metalloregulators
Authors: Guedon Emmanuel , Helmann John D.
Published: January 2004

Corynebacterium diphtheriae
DtxR is an iron‐specific repressor of diphtheria toxin expression and iron homeostasis functions. A homologue, MntR, serves as a manganese‐specific repressor of Mn(II) uptake in
Bacillus subtilis
. When expressed in
B. subtilis
, DtxR regulates gene expression in response to either iron or manganese with comparable sensitivity. Replacement of two amino acids in the metal‐sensing site with the corresponding residues from MntR results in a DtxR mutant that is highly selective for Mn(II). However, iron responsiveness can be partially restored in a
fur
mutant in which iron uptake is derepressed and intracellular iron pools elevated. Conversely, if the putative metal‐binding residues in MntR are altered to those in DtxR, the resulting protein responds to both iron and manganese. These results suggest that the composition and geometry of the metal‐binding site plays a major role in defining the metal‐selectivity in this protein family. However, the broadened selectivity of DtxR when expressed in
B. subtilis
, and the effects of a
fur mutation, demonstrate that cellular milieu also influences metal responsiveness.
LuxO controls luxR expression in Vibrio harveyi: evidence for a common regulatory mechanism in Vibrio
Authors: Miyamoto Carol M. , Dunlap Paul V. , Ruby Edward G. , Meighen Edward A.
Published: January 2004

Quorum‐sensing control of luminescence in Vibrio harveyi, which involves an indirect autoinducer‐mediated phosphorelay signal transduction system, contrasts with the prototypical quorum‐sensing system of Vibrio fischeri, in which the autoinducer and the transcriptional activator LuxR directly activate lux operon expression. In V. harveyi, a regulator not homologous to V. fischeri LuxR and also designated LuxR (LuxRvh), binds specifically to the lux operon promoter region and activates the expression of luminescence. A direct connection has not been identified previously between V. harveyi LuxRvh and the autoinducer‐mediated phosphorelay system. Here, we demonstrate by mobility shift assays and measurement of luxRvh mRNA levels with luxO+ and luxO– cells that the central response regulator of the V. harveyi phosphorelay system (LuxO) represses the level of LuxRvh. Expression of a luxRvh‐bearing plasmid strongly stimulated luminescence of a luxO– mutant but had no effect on luminescence of wild‐type luxO+ cells, indicating tight regulation of luxRvh by LuxO. Furthermore, luxO null mutants of V. fischeri MJ‐1 and two autoinducer mutants, MJ‐211 (luxI–) and MJ‐215 (luxI–ainS–), emitted more light and exhibited more elevated levels of litR, a newly identified V. harveyi luxRvh homologue, than their luxO+ counterparts. These results suggest that activity of the autoinducer‐mediated phosphorelay system is coupled to LuxRvh/LitR control of luminescence through LuxO in V. harveyi and V. fischeri. The presence of homologues of V. harveyi LuxRvh, LuxO and other phosphorelay system proteins in various Vibrio species and the control of LuxRvh and its homologues by LuxO identified here in V. harveyi and V. fischeri and recently in Vibrio cholerae suggest that the luxO–luxRvh couple is a central feature of this quorum‐sensing system in members of the genus Vibrio.
virK,somAandrcsCare important for systemicSalmonella entericaserovar Typhimurium infection and cationic peptide resistance
Authors: Detweiler Corrella S. , Monack Denise M. , Brodsky Igor E. , Mathew Hanza , Falkow Stanley
Published: January 2004

Salmonellamust express and deploy a type III secretion system located inSalmonellapathogenicity island 2 (SPI‐2) in order to survive in host phagocytic vacuoles and to cause systemic infection in mouse models of typhoid fever. A genome‐wide approach to screening forSalmonellagenes that are transcriptionally co‐regulatedin vitrowith SPI‐2 genes was used to identify bacterial loci that might function in a mouse model of systemic disease. Strains with mutations in three SPI‐2 co‐expressed genes were constructed and tested for their ability to cause disease in mice. We found thatvirK, a homologue of aShigellavirulence determinant, andrcsC, a sensor kinase, are important at late stages of infection. A secondSalmonellagene that has VirK homology,somA, is also important for systemic infection in mice. We have shown that expression of bothvirKandsomArequires the transcription factor PhoP, whereasrcsCdoes not. Additionally,rcsCexpression does not require the transcription factor OmpR, but expression of one of the known targets of RcsC, theyojN rcsBputative operon, does require OmpR.virK,somAandrcsCare expressed in tissue culture macrophages and conferSalmonellaresistance to the cationic peptide polymyxin B. We conclude thatvirK,somAandrcsCare important for late stages ofSalmonellaenteric fever, and that they probably contribute to the remodelling of the bacterial outer membrane in response to the host environment.
The role of DNA base excision repair in the pathogenesis of Salmonella enterica serovar Typhimurium
Authors: Suvarnapunya Akamol E. , Lagassé H. A. Daniel , Stein Murry A.
Published: January 2004

The intracellular pathogen, Salmonella enterica serovar Typhimurium, is able to proliferate in phagocytes, although reactive oxygen and nitrogen intermediates are lethal to most phagocytosed bacteria. To determine whether repair of oxidatively damaged DNA is involved in S. typhimurium intramacrophage proliferation, null mutants of the DNA base excision repair (BER) system were generated. These mutants were deficient in discrete enzymes (Δnth, Δnei, Δxth, Δnfo) or in the defined glycosylase (Δnth/nei) and endonuclease (Δxth/nfo) steps. In this study, S. typhimurium BER mutants are characterized for the first time. In vitro characterization of the Salmonella BER mutants revealed phenotypes that are mostly consistent with characterized Escherichia coli BER mutants. These strains were used to evaluate the role of BER in the context of Salmonella virulence. S. typhimurium Δxth and Δxth/nfo were significantly impaired for survival in both cultured and primary macrophages activated with interferon (IFN)‐γ. Survival of Δxth and Δxth/nfo was improved nearly to wild‐type levels in activated primary macrophages lacking both phagocyte oxidase and inducible nitric oxide synthase. In the murine typhoid fever model, Δnth/nei was fivefold attenuated and Δxth/nfo was 12‐fold attenuated compared with wild type. These data indicate that DNA oxidation is a mechanism that macrophages use to damage intracellular Salmonella, and suggest that BER‐mediated repair of this damage may be important in the establishment of Salmonella infection. We speculate that adaptation to a pathogenic lifestyle may influence the acquisition and retention of redundant BER enzymes.
DNA supercoiling contributes to disconnect σS accumulation from σS‐dependent transcription in Escherichia coli
Authors: Bordes Patricia , Conter Annie , Morales Violette , Bouvier Jean , Kolb Annie , Gutierrez Claude
Published: January 2004

The σS subunit of RNA polymerase is a key regulator of Escherichia coli transcription in stress conditions. σS accumulates in cells subjected to stresses such as an osmotic upshift or the entry into stationary phase. We show here that, at elevated osmolarity, σS accumulates long before the beginning of the σS‐dependent induction of osmEp, one of its target promoters. A combination of in vivo and in vitro evidence indicates that a high level of DNA negative supercoiling inhibits transcription by EσS. The variations in superhelical densities occurring as a function of growth conditions can modulate transcription of a subset of σS targets and thereby contribute to the temporal disconnection between the accumulation of σS and σS‐driven transcription. We propose that, in stress conditions leading to the accumulation of σS without lowering the growth rate, the level of DNA supercoiling acts as a checkpoint that delays the shift from the major (Eσ70) to the general stress (EσS) transcriptional machinery, retarding the induction of a subset of the σS regulon until the conditions become unfavourable enough to cause entry into stationary phase.
Multiplication of a restriction–modification gene complex
Authors: Sadykov Marat , Asami Yasuo , Niki Hironori , Handa Naofumi , Itaya Mitsuhiro , Tanokura Masaru , Kobayashi Ichizo
Published: January 2004

Previous works have suggested that some gene complexes encoding a restriction (R) enzyme and a cognate modification (M) enzyme may behave as selfish mobile genetic elements. RM gene complexes, which destroy ‘non‐self’ elements marked by the absence of proper methylation, are often associated with mobile genetic elements and are involved in various genome rearrangements. Here, we found amplification of a restriction–modification gene complex. BamHI gene complex inserted into the Bacillus chromosome showed resistance to replacement by a homologous stretch of DNA. Some cells became transformed with the donor without losing BamHI. In most of these transformants, multiple copies of BamHI and the donor allele were arranged as tandem repeats. When a clone carrying one copy of each allele was propagated, extensive amplification of BamHI and the donor unit was observed in a manner dependent on restriction enzyme gene. This suggests that restriction cutting of the genome participates in the am­plification. Visualization by fluorescent in situ hybridization revealed that the amplification occurred in single cells in a burst‐like fashion that is reminiscent of induction of provirus replication. The multiplication ability in a bacterium with natural capacity for DNA release, uptake and transformation will be discussed in relation to spreading of RM gene ­complexes.
Binding of the Escherichia coli MelR protein to the melAB promoter: orientation of MelR subunits and investigation of MelR–DNA contacts
Authors: Grainger David C. , Belyaeva Tamara A. , Lee David J. , Hyde Eva I. , Busby Stephen J. W.
Published: January 2004

The  Escherichia  coli
 MelR  protein  is  a  melibiose‐triggered transcription factor, belonging to the AraC family, that activates transcription initiation at the melAB promoter. Activation is dependent on the binding of MelR to four 18 bp sites, centred at position −42.5 (site 2′), position −62.5 (site 2), position −100.5 (site 1) and position −120.5 (site 1′) relative to the melAB transcription start point. Activation also depends on the binding of CRP to a single site located between MelR binding site 1 and site 2. All members of the AraC family contain two helix–turn–helix (HTH) motifs that contact two segments of the DNA major groove at target sites on the same DNA face. In this work, we have studied the binding of MelR to different sites at the melAB promoter, focusing on the orientation of binding of the two MelR HTH motifs, and the juxtaposition of the different bound MelR subunits with respect to each other. To do this, MelR was engineered to contain a single cysteine residue adjacent to either one or the other HTH motif. The MelR derivatives were purified, and the cysteine residues were tagged with p‐bromoacetamidobenzyl‐EDTA‐Fe, an inorganic DNA cleavage reagent. Patterns of DNA cleavage after MelR binding were then used to determine the positions of the two HTH motifs at target sites. In order to simplify our analysis, we exploited an engineered derivative of the melAB promoter in which MelR binding to site 2 and site 2′, in the absence of CRP, is sufficient for transcription activation. To assist in the interpretation of our results, we also used a shortened derivative of MelR, MelR173, that is able to bind to site 2 but not to site 2′. Our results show that MelR binds as a direct repeat to site 2 and site 2′ with the C‐terminal HTH located towards the promoter‐proximal end of each site. The orientation in which MelR binds to site 2′ appears to be determined by MelR–MelR interactions rather than by MelR–DNA interactions. In complementary experiments, we used genetic analysis to investigate the importance of different residues in the two HTH motifs of MelR. Epistasis experiments provided evidence that supports the proposed orientation of binding of MelR at its target site.
Predicting mutation frequencies in stem–loop structures of derepressed genes: implications for evolution
Authors: Wright Barbara E. , Reschke Dennis K. , Schmidt Karen H. , Reimers Jacqueline M. , Knight William
Published: January 2004

This work provides evidence that, during transcription, the mutability (propensity to mutate) of a base in a DNA secondary structure depends both on the stability of the structure and on the extent to which the base is unpaired. Zuker's DNA folding computer program reveals the most probable stem–loop structures (SLSs) and negative energies of folding (–ΔG) for any given nucleotide sequence. We developed an interfacing program that calculates (i) the percentage of folds in which each base is unpaired during transcription; and (ii) the mutability index (MI) for each base, expressed as an absolute value and defined as ­follows: MI = (% total folds in which the base is unpaired) × (highest –ΔG of all folds in which it is unpaired). Thus, MIs predict the relative mutation or reversion frequencies of unpaired bases in SLSs. MIs for 16 mutable bases in auxotrophs, selected during starvation in derepressed genes, are compared with 70 background mutations in lacI and ebgR that were not derepressed during mutant selection. All the results are consistent with the location of known mutable bases in SLSs. Specific conclusions are: (i) Of 16 mutable bases in transcribing genes, 87% have higher MIs than the average base of the sequence analysed, compared with 50% for the 70 background mutations. (ii) In 15 of the mutable bases of transcribing genes, the correlation between MIs and relative mutation frequencies determined experimentally is good. There is no correlation for 35 mutable bases in the lacI gene. (iii) In derepressed auxotrophs, 100% of the codons containing the mutable bases are within one codon's length of a stem, compared with 53% for the background mutable bases in lacI. (iv) The data suggest that environmental stressors may cause as well      as     select     mutations     in     derepressed     genes. The implications of these results for evolution are discussed.
The regulation of EFG1 in white–opaque switching in Candida albicans involves overlapping promoters
Authors: Lachke Salil A. , Srikantha Thyagarajan , Soll David R.
Published: January 2004

EFG1
, which encodes a
trans
‐acting factor, is expressed as a more abundant 3.2 kb transcript in the white phase and as a less abundant 2.2 kb transcript in the opaque phase of the white–opaque transition in
Candida albicans
. To understand how alternative phase‐specific mRNAs are transcribed from the same gene locus, the 2320 bp upstream region of the gene was functionally characterized by analysing the ­activity of deletion derivatives in a luciferase‐based reporter system. The white phase‐specific promoter contained three discrete sequences involved in white phase‐specific activation, between −2022 and −1809 bp (AR1), between −1809 and −1727 bp (AR2) and between −922 and −840 bp (AR3). A higher resolution deletion and mutation analysis of AR2 revealed two regions between −1809 and −1787 bp and between −1764 and −1728 bp that are responsible for AR2 activation. Targeting of promoter constructs to the ectopic
ADE2
genomic site and the 3′ end of the
EFG1
genomic site revealed a positional requirement for white phase‐regulated activation specific for the AR2 region of the promoter. Gel mobility shift assays using AR2 revealed a white phase‐specific activation complex. No discrete activation sequences were identified in the overlapping promoter of the opaque phase‐specific
EFG1 transcript. The strength of opaque phase activation was directly proportional to the length of the promoter. Northern analysis excluded the possibility of an opaque phase‐specific repressor. These results demonstrate overlapping promoters for white and opaque phase‐specific expression of the gene for the transcription factor Efg1, with distinctly different mechanisms of phase‐specific activation.
Salmonella effectors translocated across the vacuolar membrane interact with the actin cytoskeleton
Authors: Miao Edward A. , Brittnacher Mitchell , Haraga Andrea , Jeng Robert L. , Welch Matthew D. , Miller Samuel I.
Published: January 2004

A family of nine Salmonella typhimurium type III secretion effectors with a conserved amino‐terminus have been defined. Three family members (SifA, SifB and SseJ) have previously been demonstrated to localize to the Salmonella‐containing vacuole and to Salmonella‐induced filaments. In contrast, we demonstrate that two other family members, SspH2 and SseI, co‐localized with the polymerizing actin cytoskeleton. These proteins also interacted with the mammalian actin cross‐linking protein filamin in the yeast two‐hybrid assay through their highly conserved amino‐terminal domains. This amino‐terminus was sufficient to direct localization to the polymerizing actin cytoskeleton, suggesting that the interaction with filamin is important for this subcellular localization. In addition, SspH2 co‐localized with vacuole‐associated actin polymerizations (VAP) induced by intracellular bacteria through the Salmonella pathogenicity island (SPI)‐2 type III secretion system (TTSS). SspH2 interacted with the actin‐binding protein profilin in the yeast two‐hybrid assay and by affinity chromatography. This interaction was highly specific to SspH2 and was mediated by its carboxy‐terminus. Furthermore, SspH2 inhibited the rate of actin polymerization in vitro, suggesting that it functions to reduce or remodel VAP. Strains with mutations in sspH2 and sseI retained the ability to form VAP. However, a third intracellular virulence factor, spvB, which ADP‐ribosylates actin, strongly inhibited VAP formation in HeLa cells, suggesting a more subtle effect for SspH2 and SseI on the actin cytoskeleton.
An outline of the petrology, structure and age of the Pompangeo Schist Complex of central Sulawesi, Indonesia
Authors: Parkinson Chris
Published: January 2004

Variably dismembered and metamorphosed accretionary complexes constitute the basement of much of the Indonesian island of Sulawesi. The most extensive of these is the Pompangeo Schist Complex, which crops out over ∼ 5000 km2 in central Sulawesi, and is predominantly composed of interbanded phyllitic marble, calcareous phyllite, graphitic schist and quartzite; rocks of terrigenous to shallow marine origin. Along the eastern margin of the complex, schists are interthrust with unmetamorphosed Jurassic sandstone, which may represent parental material of the complex. The schists are unconformably overlain by pelagic sediments with an Albian–Cenomanian biostratigraphy. Synmetamorphic progressive deformation of the Pompangeo Schist Complex has resulted in repeated isoclinal folding and a strong transposition foliation striking north‐northwest/south‐southeast and dipping west, subparallel to the compositional banding of the complex; microstructural fabrics indicate a top‐to‐east sense of shear. On a regional scale the Pompangeo Schist Complex is lithostratigraphically coherent and an east‐to‐west metamorphic field gradient is recognizable, which, if continuous, represents a relatively low thermal gradient of ∼ 15 °C/km. K–Ar dating yielded ages of ca 111 Ma. Correlative metamorphic rocks appear to underlie the entire Neogene magmatic province, since they occur sporadically throughout western Sulawesi, including the Bantimala region of the South Arm. The Pompangeo schist metamorphism cannot be correlated with arc magmatism in western Sulawesi, which is of Neogene age. The Pompangeo and Bantimala schists, as well as other accretionary complexes in western Sulawesi, were probably generated in the same subduction system that was responsible for the extensive Mesozoic continental arc in central Kalimantan, at the eastern margin of Sundaland.
K–Ar ages of the basaltic rocks from Far East Russia: Constraints on the tectono‐magmatism associated with the Japan Sea opening
Authors: Okamura Satoshi , Martynov Yuri A. , Furuyama Katsuhiko , Nagao Keisuke
Published: January 2004

K–Ar ages of the Cenozoic basaltic rocks from the Far East region of Russia (comprising Sikhote‐Alin and Sakhalin) are determined to obtain constraints on the tectono‐magmatic evolution of the Eurasian margin by comparison with the Japanese Islands, Northeast China, and the formation of the back‐arc basin. In the early Tertiary stage (54–26 Ma), the northwestward subduction of the Pacific Plate produced the active continental margin volcanism of Sikhote‐Alin and Sakhalin, whereas the rift‐type volcanism of Northeast China, inland part of the continent began to develop under a northeast–southwest‐trending deep fault system. In the early Neogene (24–17 Ma), a large number of subduction‐related volcanic rocks were erupted in connection with the Japan Sea opening. After an inactive interval of the volcanism ∼ 20–13 Ma ago, the late Neogene (12–5 Ma) volcanism of Sikhote‐Alin and Sakhalin became distinct from those of the preceding stages and indicated within‐plate geochemical features similar to those of Northeast China, in contrast to the Japan Arc which produces island arc volcanism. During the Japan Sea opening, the northeastern Eurasian margin detached and became a continental island arc system, and an integral part of continental eastern Asia comprising Sikhote‐Alin, Sakhalin and Northeast China, and the Japan Arc with a back‐arc basin. The convergence between the Eurasian Plate, the Pacific Plate and the Indian Plate may have contributed to the Cenozoic tectono‐magmatism of the northeastern Eurasian continent.
Evolution of Cretaceous active continental margins and their correlation with other global events
Authors: Filatova N. I.
Published: January 2004

The scenario of the Earth active continental margin evolution is reconstructed here for Early Cretaceous–Senonian times when pulsatory alternated magmatic and orogenic processes occurred. The correlation of these continental marginal manifestations with some global events was undertaken also. The first, Neocomian stage (starting from the Jurassic) was distinguished by the combined development of continental marginal volcanic belts (with extensional conditions in back‐arc regions) and peri‐oceanic island arcs. In the second, Aptian–early Middle Albian stage the mid‐Cretaceous global orogeny took place which resulted in almost complete extinction of subduction zones within the continent–ocean transition as well as in obduction (with metamorphism) of island arc, back‐arc, and oceanic terranes together with microcontinents over continents. This mid‐Cretaceous (120–100 Ma) hypercollision was caused by a sharp increase in the spreading rate and origination of new mid‐oceanic ridges accompanied the breakup of Pangea. Those events provoked an increase in the rate of convergence of oceanic plates and continents, strong compressional stress into peri‐oceanic domains, and fast growth of the continental edges due to fold–thrust terrane rim development. During the third, late Albian–Late Cretaceous stage (100–85 Ma), some decrease of the rapid oceanic crust growth led to the accelerated subduction of oceanic plates under continents. The new convergent plate boundaries with global systems of continental marginal magmatic belts with a very large volume of volcanics were developed. The peculiar events of the Cretaceous history at active continental margins coincided with simultaneous extraordinary global events: fast growth of the oceanic crust, peak of intraplate magmatism, and prolonged lack of the Earth magnetic reversals, and can be considered to be indicators of mantle superplume activity and whole‐mantle convection development during 120–80 Ma.
A petrogenetic grid for eclogite and related facies under high‐pressure metamorphism
Authors: Oh C. W. , Liou J. G.
Published: January 2004

The petrogenetic grid between the eclogite and other high‐pressure/temperature (P/T) metamorphic facies in a basaltic system is constructed by considering barroisite as one of the important phases in high‐P/T metamorphism and by using previous petrological data combined with Schreinemakers' analysis and slope calculation. In the constructed petrogenetic grid, the eclogite facies is bounded by the blueschist and epidote–amphibolite facies with negative‐slope reactions at lower temperatures (450–550 °C) and by the epidote–amphibolite, amphibolite and granulite facies with positive‐slope reactions at higher temperatures (> 550–600 °C). The eclogite facies does not contact the greenschist facies, and the lowest P condition for the eclogite facies exists at the boundary between the eclogite and epidote–amphibolite facies. The temperature range of the epidote–amphibolite facies increases with increasing pressure until 8–11 kbar and then decreases up to 13–15 kbar. Compared to boundaries of other facies, boundaries of the eclogite facies may have wider P–T ranges. The boundary between the blueschist and eclogite facies occurs over a large temperature range from 450 to 620 ± 30 °C, and the transitions between the eclogite and amphibolite or high‐pressure granulite facies occur over a pressure range in excess of 6–10 kbar.
Introduction to geodynamics for high‐ and ultrahigh‐pressure metamorphism
Authors: Liou J. G. , Aruyama S. M , Ong B. C
Published: January 2004

Since the first workshop on ultrahigh‐pressure (UHP) metamorphism at Stanford in 1994 and the special issue of The Island Arc `Ultrahigh‐Pressure Metamorphism and Tectonics' published in December 1995, many symposia and special sessions specifically with regard to the UHP metamorphic terrane have been held. While we are still wondering how exhumation of UHP rocks from mantle depths to the surface takes place, the finding of possible records from the mantle transition zone at 300–400 km depths is astonishing. The study of the UHP regime has expanded to include input from mineral physics, experimental geochemistry and kinetics in addition to the petrochemical and tectonic study of a variety of HP–UHP rocks. It was with this theme that the second workshop for the task group III‐6 `Ultrahigh‐Pressure Metamorphism and Geodynamics in Collision‐type Orogenic Belts' of the International Lithosphere Program was held during the International Geological Congress in Beijing, 1996. The Symposium 8–9 `Dynamic Metamorphic Rocks and High‐ and Ultrahigh‐Pressure Metamorphism' (Cong Bolin & J. G. Liou conveners) had more than 25 presentations in two oral sessions and 70 papers in one poster session. This second special issue of The Island Arc includes nine papers from this symposium and a few related contributions to the geodynamics of HP–UHP metamorphism and tectonics. It is our hope that The Island Arc will continue to publish a special issue on this increasingly recognized subject that is essential to our understanding of continental collision, mantle dynamics and geochemical + fluid cycles.
Differential exhumation of tectonic units and ultrahigh‐pressure metamorphic rocks in the Dabie Mountains, China
Authors: Dong Shuwen , Chen Jiangfeng , Huang Dezhi
Published: January 2004

A model involving buoyancy, wedging and thermal doming is postulated to explain the differential exhumation of ultrahigh‐pressure (UHP) metamorphic rocks in the Dabie Mountains, China, with an emphasis on the exhumation of the UHP rocks from the base of the crust to the upper crust by opposite wedging of the North China Block (NCB). The Yangtze Block was subducted northward under the NCB and Northern Dabie microblock, forming UHP metamorphic rocks in the Triassic (240–220 Ma). After delamination of the subduction wedge, the UHP rocks were exhumed rapidly to the base of the crust by buoyancy (220–200 Ma). Subsequently, when the left‐lateral Tan–Lu transform fault began to be activated, continuous north–south compression and uplifting of the orogen forced the NCB to be subducted southward under the Dabie Orogen (`opposite subduction'). Opposite subduction and wedging of the North China continental crust is responsible for the rapid exhumation of the UHP and South Dabie Block units during the Early Jurassic, at ca 200–180 Ma at a rate of ∼ 3.0 mm/year. The UHP eclogite suffered retrograde metamorphism to greenschist facies. Rapid exhumation of the North Dabie Block (NDB) occurred during 135–120 Ma because of thermal doming and granitoid formation during extension of continental margin of the Eurasia. Amphibolite facies rocks from NDB suffered retrograde metamorphism to greenschist facies. Different unit(s) and terrane(s) were welded together by granites and the wedging ceased. Since 120–110 Ma, slow uplift of the entire Dabie terrane is caused by gravitational equilibrium.
Metamorphic zoning and thermal structure of the Dabie ultrahigh‐ pressure–high‐pressure terrane, central China
Authors: Tabata H. , Maruyama S. , Shi Z.
Published: January 2004

The ultrahigh‐ and high‐pressure (UHP–HP) metamorphic belt of the Dabie Mountains, central China, formed by the Triassic continental subduction and collision, is divided into four metamorphic zones; from south to north, the greenschist facies zone, epidote amphibolite to amphibolite facies zone, quartz eclogite zone, and coesite eclogite zone, based on metabasite mineral assemblages. Most of the coesite‐bearing eclogites consist mainly of garnet and omphacite with homogeneous compositions and have partially undergone hydration reactions to form clinopyroxene + plagioclase + calcic amphibole symplectites during amphibolite facies overprinting. However, the least altered eclogites sometimes contain garnet and omphacite that preserve compositional zoning patterns which may have originated during their growth at peak temperature conditions of ∼ 750 °C, suggesting a short duration of UHP metamorphic conditions and/or consequent rapid cooling during exhumation. Systematic investigation on peak metamorphic temperatures of coesite eclogite have revealed that, contrary to the general trend of metamorphic grade in the southern Dabie unit, the coesite eclogite zone shows rather flat thermal structure (T = 600 ± 50 °C) with the highest temperature reaching up to 850 °C and no northward increase in metamorphic temperature, which is opposed to the previous interpretations. This feature, along with the preservation of compositional zonation, implies complicated differential movement of each eclogite mass during UHP metamorphism and the return from the deeper subduction zone at mantle depths to the surface.
Chrome‐rich clinopyroxene in orthopyroxenite from Maowu, Dabie Mountains, central China: A second record and its implications for petrogenesis
Authors: Liu Xiaochun , Zhou Huyun , Ma Zhesheng , Chang Lihua
Published: January 2004

A second occurrence of chrome‐rich clinopyroxene has been discovered as inclusions in orthopyroxene in orthopyroxenite from Maowu, the Dabie Mountains, Central China. The average formula for chrome‐rich clinopyroxene can be expressed as (Na0.39Ca0.54)0.93(Mg0.57Fe2+0.06Fe3+0.01Cr0.24Al0.15)1.03Si2.02O6, with a maximum amount of kosmochlor component of 28.52 mol%. The unit cell parameters obtained from a single‐crystal are a = 9.614 Å, b = 8.800 Å, c = 5.240 Å, β = 106.59°, space group C2/c. The indices of refraction are α = 1.697, β = 1.704, γ = 1.726. Chrome‐rich clinopyroxene, which coexists with chromite, chromian rutile and chromian pyrope, crystallized at a temperature of 1025 °C and very high pressure, and therefore represents a mantle relic. Together with the appearance of low‐pressure inclusion mineral assemblage and the estimation of physical–chemical conditions for matrix minerals, the Maowu eclogite–ultramafic complex is considered to be formed during ultrahigh‐pressure metamorphism from the mantle‐derived protolith.