bluuebunny/crossref_metadata_2025_split huggingface dataset:
Costs and benefits of cold tolerance in transgenic Arabidopsis thaliana
Authors: JACKSON M. W. , STINCHCOMBE J. R. , KORVES T. M. , SCHMITT J. 
Published: December 2004

AbstractCold tolerance in plants is an ecologically important trait that has been under intensive study for basic and applied reasons. Determining the fitness benefits and costs of cold tolerance has previously been difficult because cold tolerance is normally an induced trait that is not expressed in warm environments. The recent creation of transgenic plants constitutively expressing cold tolerance genes enables the investigation of the fitness consequences of cold tolerance in multiple temperature environments. We studied three genes from the CBF (C‐repeat/dehydration responsive element binding factor) cold tolerance pathway, CBF1, 2 and 3, in Arabidopsis thaliana to test for benefits and costs of constitutive cold tolerance. We used multiple insertion lines for each transgene and grew the lines in cold and control conditions. Costs of cold tolerance, as determined by fruit number, varied by individual transgene. CBF2 and 3 overexpressers showed costs of cold tolerance, and no fitness benefits, in both environments. CBF1 overexpressing plants showed no fitness cost of cold tolerance in the control environment and showed a marginal fitness benefit in the cold environment. These results suggest that constitutive expression of traits that are normally induced in response to environmental stress will not always lead to costs in the absence of that stress, and that the ecological risks of CBF transgene escape should be assessed prior to their use in commercial agriculture.
How to track and assess genotyping errors in population genetics studies
Authors: BONIN A. , BELLEMAIN E. , BRONKEN EIDESEN P. , POMPANON F. , BROCHMANN C. , TABERLET P. 
Published: December 2004

AbstractGenotyping errors occur when the genotype determined after molecular analysis does not correspond to the real genotype of the individual under consideration. Virtually every genetic data set includes some erroneous genotypes, but genotyping errors remain a taboo subject in population genetics, even though they might greatly bias the final conclusions, especially for studies based on individual identification. Here, we consider four case studies representing a large variety of population genetics investigations differing in their sampling strategies (noninvasive or traditional), in the type of organism studied (plant or animal) and the molecular markers used [microsatellites or amplified fragment length polymorphisms (AFLPs)]. In these data sets, the estimated genotyping error rate ranges from 0.8% for microsatellite loci from bear tissues to 2.6% for AFLP loci from dwarf birch leaves. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs, but in both cases human factors were non‐negligible error generators. Therefore, tracking genotyping errors and identifying their causes are necessary to clean up the data sets and validate the final results according to the precision required. In addition, we propose the outline of a protocol designed to limit and quantify genotyping errors at each step of the genotyping process. In particular, we recommend (i) several efficient precautions to prevent contaminations and technical artefacts; (ii) systematic use of blind samples and automation; (iii) experience and rigor for laboratory work and scoring; and (iv) systematic reporting of the error rate in population genetics studies.
Mitochondrial gene diversity in the common vole Microtus arvalis shaped by historical divergence and local adaptations
Authors: FINK SABINE , EXCOFFIER LAURENT , HECKEL GERALD 
Published: December 2004

AbstractThe phylogeography of the common vole (Microtus arvalis) was examined by analysing mitochondrial DNA (mtDNA) sequence variation in 1044 base pairs (bp) of the cytochrome b (cytb) gene and in 322 bp of the control region (ctr) among 106 individuals from 58 locations. The geographical distribution of four previously recognized cytb evolutionary lineages in Europe was refined and a new lineage was found in southern Germany. All lineages were distributed allopatrically, except in one sample that was probably located in a contact zone. The occurrence of several lineages in the Alps is in keeping with their recent recolonization from distinct sources. The translation of 84 cytb DNA sequences produced 33 distinct proteins with relationships that differed from those of the DNA haplotypes, suggesting that the mtDNA lineages did not diverge in response to selection. In comparison with M. agrestis, a neutrality test detected no overall evidence for selection in the cytb gene, but a closer examination of a structural model showed that evolutionarily conserved and functionally important positions were often affected. A new phylogeographical test of random accumulation of nonsynonymous mutations generated significant results in three lineages. We therefore conclude that the molecular diversity of cytb in M. arvalis is overall the result of the demographic history of the populations, but that there have been several episodes of local adaptation to peculiar environments.
A novel mechanism controls anaerobic and catabolite regulation of the Escherichia coli tdc operon
Authors: Sawers Gary 
Published: December 2004

The tdc operon is subject to CRP‐controlled catabolite repression. Expression of the operon is also induced anaerobically, although this regulation does not rely on direct control by either FNR or ArcA. Recently, the anaerobic expression of the tdc operon was found to be fortuitously induced in the presence of glucose by a heterologous gene isolated from the Gram‐positive anaerobe Clostridium butyricum. The gene, termed tcbC, encoded a histone‐like protein of 14.5 kDa. Using tdc–lacZ fusions, it was shown that TcbC did not activate tdc expression by functionally replacing any of the operon regulators. In vitro transcription analyses with RNA polymerase and CRP revealed that faithful CRP‐dependent transcription initiation occurred only on supercoiled templates. No specific, CRP‐dependent transcription initiation was observed on relaxed or linear DNA templates. Surprisingly, purified His‐tagged TcbC activated transcription from a relaxed, circular template, but not from supercoiled or linear templates. Examination of the CRP binding site of the tdc promoter revealed that it was located 43.5 bp upstream of the transcription initiation site. Repositioning of the CRP site at −41.5 bp abolished activation by the TcbC protein and allowed CRP‐dependent transcription to occur on linear, relaxed and supercoiled templates. TcbC bound DNA non‐specifically; however, in topoisomerase I relaxation assays, it was demonstrated that TcbC imposed torsional constraints on negatively supercoiled DNA, which influenced the ability of the enzyme to relax the topoisomers. Taken together, these results strongly suggest that TcbC activates transcription of tdc by altering the local topological status of the tdc promoter and that, in the wild‐type tdc promoter, the CRP binding site is misaligned to allow transcription to occur only under optimal conditions. Indeed, in vivo transcription analyses revealed that repositioning of the CRP binding site to −41.5 bp resulted in high‐level, CRP‐dependent transcription, even under catabolite‐repressing conditions, and that transcription was no longer influenced by TcbC. Remarkably, however, anaerobic regulation of the mutant promoter was retained. This indicates that the other tdc regulators, TdcA and TdcR, govern anaerobic transcription activation by CRP.
Transfection studies to explore essential folate metabolism and antifolate drug synergy in the human malaria parasite Plasmodium falciparum
Authors: Wang Ping , Wang Qi , Aspinall Tanya V. , Sims Paul F. G. , Hyde John E. 
Published: December 2004

SummaryFolate metabolism in Plasmodium falciparum is the target of important antimalarial agents. The biosynthetic pathway converts GTP to polyglutamated derivatives of tetrahydrofolate (THF), essential cofactors for DNA synthesis. Tetrahydrofolate can also be acquired by salvage mechanisms. Using a transfection system adapted to studying this pathway, we investigated modulation of dihydropteroate synthase (DHPS) activity on parasite phenotypes. Dihydropteroate synthase incorporates p‐aminobenzoate (pABA) into dihydropteroate, the precursor of dihydrofolate. We were unable to obtain viable parasites where the dhps gene had been truncated. However, parasites where the protein was full‐length but mutated at two key residues and having < 10% of normal activity were viable in folate‐supplemented medium. Metabolic labelling showed that these parasites could still convert pABA to polyglutamated folates, albeit at a very low level, but they could not survive on pABA supplementation alone. This degree of disablement in DHPS also abolished the synergy of the antifolate combination pyrimethamine/sulfadoxine. These data indicate that DHPS activity above a low but critical level is essential regardless of the availability of salvageable folate and formally prove the role of this enzyme in antifolate drug synergy and folate biosynthesis in vivo. However, we found no evidence of a significant role for DHPS in folate salvage. Moreover, when biosynthesis was compromised by the absence of a fully functional DHPS, the parasite was able to compensate by increasing flux through the salvage pathway.
Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase σ subunit
Authors: Grainger David C. , Webster Christine L. , Belyaeva Tamara A. , Hyde Eva I. , Busby Stephen J. W. 
Published: December 2004

SummaryActivation of transcription initiation at the Escherichia coli melAB promoter is dependent on MelR, a transcription factor belonging to the AraC family. MelR binds to 18 bp target sites using two helix–turn–helix (HTH) motifs that are both located in its C‐terminal domain. The melAB promoter contains four target sites for MelR. Previously, we showed that occupation of two of these sites, centred at positions −42.5 and −62.5 upstream of the melAB transcription start point, is sufficient for activation. We showed that MelR binds as a direct repeat to these sites, and we proposed a model to describe how the two HTH motifs are positioned. Here, we have used suppression genetics to confirm this model and to show that MelR residue 273, which is in HTH 2, interacts with basepair 13 of each target site. As our model for DNA‐bound MelR suggests that HTH 2 must be adjacent to the melAB promoter −35 element, we searched this part of MelR for amino acid side‐chains that might be able to interact with σ. We describe genetic evidence to show that MelR residue 261 is close to residues 596 and 599 of the RNA polymerase σ70 subunit, and that they can interact. Similarly, MelR residue 265 is shown to be able to interact with residue 596 of σ70. In the final part of the work, we describe experiments in which the MelR binding site at position −42.5 was improved. We show that this is detrimental to MelR‐dependent transcription activation because bound MelR is mispositioned so that it is unable to make ‘correct’ interactions with σ.
Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes
Authors: Zenewicz Lauren A. , Skinner Jason A. , Goldfine Howard , Shen Hao 
Published: December 2004

SummaryVirulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways. We investigated whether L. monocytogenes modulates T‐cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells. In vitro culture of murine splenocytes with L. monocytogenes resulted in a specific and dose‐dependent upregulation of Fas ligand (FasL). Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non‐pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s). Examination of L. monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine‐preferring phospholipase C (PC‐PLC). Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC‐PLC synergized with LLO for the induction of FasL expression. FasL‐expressing cells induced by L. monocytogenes were capable of killing Fas‐expressing target cells. Furthermore, L. monocytogenes infection results in upregulation of FasL on T cells in mice. These results describe a novel function for LLO and PC‐PLC and suggest that L. monocytogenes may use these virulence factors to modulate the host immune response.
MicroReview: The bacterial Sm‐like protein Hfq: a key player in RNA transactions
Authors: Valentin‐Hansen Poul , Eriksen Maiken , Udesen Christina 
Published: December 2004

SummaryThe conserved RNA‐binding protein Hfq, originally discovered in Escherichia coli as a host factor for Qβ replicase, has emerged as a pleiotropic regulator that modulates the stability or the translation of an increasing number of mRNAs. During the past 5 years, Hfq‐mediated control has been an area of increasing focus because the protein has been linked to the action of many versatile RNA‐based regulators that use basepairing interactions to regulate the expression of target mRNAs. The recent findings that Hfq assists in bimolecular RNA–RNA interactions and is similar structurally and functionally to eukaryotic Sm proteins have further fuelled interest in this important post‐transcriptional regulator. Here, we summarize the history of Hfq and highlight results that have led to an important gain in insight into the physiology, biochemistry and evolution of Hfq and its homologues.
MreB, the cell shape‐determining bacterial actin homologue, co‐ordinates cell wall morphogenesis in Caulobacter crescentus
Authors: Figge Rainer M. , Divakaruni Arun V. , Gober James W. 
Published: December 2004

SummaryThe bacterial actin homologue, MreB, is required for the maintenance of a rod‐shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon‐shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid‐cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid‐cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin‐binding protein 2 (PBP2) also formed band‐like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co‐immunoprecipitated with several other penicillin‐binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2–peptidoglycan biosynthesis complex.
Re‐replication from non‐sequesterable origins generates three‐nucleoid cells which divide asymmetrically
Authors: Bach Trond , Skarstad Kirsten 
Published: December 2004

SummaryIn rapidly growing Escherichia coli cells replication cycles overlap and initiation occurs at multiple replication origins (oriCs). All origins within a cell are initiated essentially in synchrony and only once per cell cycle. Immediate re‐initiation of new origins is avoided by sequestration, a mechanism dependent on the SeqA protein and Dam methylation of GATC sites in oriC. Here, GATC sites in oriC were changed to GTTC. This reduced the sequestration to essentially the level found in SeqA‐less cells. The mutant origins underwent re‐initiation, showing that the GATC sites in oriC are required for sequestration. Each re‐initiation eventually gave rise to a cell containing an extra nucleoid. The three‐nucleoid cells displayed one asymmetrically placed FtsZ‐ring and divided into a two‐nucleoid cell and a one‐nucleoid cell. The three nucleoid‐cells thus divided into three daughters by two consecutive divisions. The results show that extra rounds of replication cause extra daughter cells to be formed prematurely. The fairly normal mutant growth rate and size distribution show, however, that premature rounds of replication, chromosome segregation, and cell division are flexibly accommodated by the existing cell cycle controls.
SCP1, a 356 023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2)
Authors: Bentley S. D. , Brown S. , Murphy L. D. , Harris D. E. , Quail M. A. , Parkhill J. , Barrell B. G. , McCormick J. R. , Santamaria R. I. , Losick R. , Yamasaki M. , Kinashi H. , Chen C. W. , Chandra G. , Jakimowicz D. , Kieser H. M. , Kieser T. , Chater K. F. 
Published: December 2004

SummaryThe sequencing of the entire genetic complement of Streptomyces coelicolor A3(2) has been completed with the determination of the 365 023 bp sequence of the linear plasmid SCP1. Remarkably, the functional distribution of SCP1 genes somewhat resembles that of the chromosome: predicted gene products/functions include ECF sigma factors, antibiotic biosynthesis, a gamma‐butyrolactone signalling system, members of the actinomycete‐specific Wbl class of regulatory proteins and 14 secreted proteins. Some of these genes are among the 18 that contain a TTA codon, making them targets for the developmentally important tRNA encoded by the bldA gene. RNA analysis and gene fusions showed that one of the TTA‐containing genes is part of a large bldA‐dependent operon, the gene products of which include three proteins isolated from the spore surface by detergent washing (SapC, D and E), and several probable metabolic enzymes. SCP1 shows much evidence of recombinational interactions with other replicons and transposable elements during its history. For example, it has two sets of partitioning genes (which may explain why an integrated copy of SCP1 partially suppressed the defective partitioning of a parAB‐deleted chromosome during sporulation). SCP1 carries a cluster of probable transfer determinants and genes encoding likely DNA polymerase III subunits, but it lacks an obvious candidate gene for the terminal protein associated with its ends. This may be related to atypical features of its end sequences.
The large resolvase TnpX is the only transposon‐encoded protein required for transposition of the Tn4451/3family of integrative mobilizable elements
Authors: Lyras Dena , Adams Vicki , Lucet Isabelle , Rood Julian I. 
Published: December 2004

SummaryChloramphenicol resistance inClostridium perfringensandClostridium difficileis often encoded bycatPgenes located within the 6.3 kb integrative mobilizable elements Tn4451and Tn4453respectively. This family of transposons is capable of being mobilized into a recipient cell in the presence of another conjugative element. Transposition is mediated by the large resolvase TnpX, which excises the element to produce a circular molecule that is the integrative intermediate. In this study,in vivodeletion analysis of the transposon‐encodedtnpVandtnpYgenes showed that they are not essential for excision or integration of this group of elements. Similar studies ontnpWsuggested either that this gene is not essential for these functions or that TnpW does not function when providedin trans. Development and use of anin vivoinsertion assay showed that TnpX is the only transposon‐encoded protein required for the integration reaction. Subsequently, a TnpXLEH6protein was purified and shown to catalyse excisionin vitroin the absence of any other protein and preferentially to excise a supercoiled DNA substrate. In summary, these studies have shown that TnpX is the only transposon protein requiredin vivoandin vitrofor the excision process and that, like excision, integration also occurs by a serine recombinase‐mediated site‐specific recombination mechanism.
Post‐transcriptional regulation of bacterial motility by aconitase proteins
Authors: Tang Yue , Guest John R. , Artymiuk Peter J. , Read Robert C. , Green Jeffrey 
Published: December 2004

Summary Escherichia coli and Bacillus subtilis aconitases can act as iron and oxidative stress‐responsive post‐transcriptional regulators. Here, it is shown that a Salmonella enterica serovar Typhimurium LT2 acnB mutant exhibits impaired binding to the surface of J774 macrophage‐like cells. Proteomic analyses were used to investigate further the binding defect of the acnB mutant. These revealed that the levels of the flagellum protein FliC were much lower for the acnB mutant. This strain was correspondingly less motile and possessed fewer flagella than either the parental strain or the acnA and acnAB mutants. The acnB lesion did not alter fliC transcription, nor did apo‐AcnB select the fliC transcript from a library of S. enterica transcripts; thus, the effect of AcnB on FliC is indirect. Evidence is presented to show that apo‐AcnB regulates FliC synthesis via interaction with the ftsH transcript to decrease the intracellular levels of FtsH. The lower levels of FtsH protease activity then influence σ32, DnaK and, ultimately, FliC production.
Upregulation of the Hsp104 chaperone at physiological temperature during recovery from thermal insult
Authors: Seppä Laura , Hänninen Anna‐Liisa , Makarow Marja 
Published: December 2004

SummaryThermal insult at 50°C causes protein denaturation in yeast, but the cells survive if preconditioned at 37°C. Survival depends on refolding of heat‐denatured proteins. Refolding of cytoplasmic proteins requires Hsp104, the expression of which increases several‐fold upon shift of the cells from physiological temperature 24°C to 37°C. We describe here a novel type of regulation of Hsp104, designated delayed upregulation (DUR). When Saccharomyces cerevisiae cells grown at 24°C, preconditioned at 37°C and treated briefly at 50°C were shifted back to 24°C, Hsp104 expression was negligible for 1 h, but increased then to a three to nine times higher level than that detected after growth at 24°C, returning to normal after 5 h. A heat shock element (HSE) of the upstream sequence of HSP104 was necessary and sufficient for DUR, whereas stress response elements (STRE) were dispensable. Destruction of HSE plus all three STREs abolished Hsp104 expression, resulting in cell death after thermal insult. Deletion of MSN2/4, encoding transcription factors driving STRE‐dependent gene expression, decreased DUR. Deletion of HOG1, encoding a heat‐responsive and osmosensitive mitogen‐activated protein kinase implicated to be functionally connected to Msn2/4p, abolished DUR. We suggest that DUR was regulated via HSE, required Hog1p and involved Msn2/4p‐regulated gene products.