bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantifying genotyping errors in noninvasive population genetics
Authors: BROQUET THOMAS , PETIT ERIC 
Published: December 2004

AbstractThe use of noninvasively collected samples greatly expands the range of ecological issues that may be investigated through population genetics. Furthermore, the difficulty of obtaining reliable genotypes with samples containing low quantities of amplifiable DNA may be overcome by designing optimal genotyping schemes. Such protocols are mainly determined by the rates of genotyping errors caused by false alleles and allelic dropouts. These errors may not be avoided through laboratory procedure and hence must be quantified. However, the definition of genotyping error rates remains elusive and various estimation methods have been reported in the literature. In this paper we proposed accurate codification for the frequencies of false alleles and allelic dropouts. We then reviewed other estimation methods employed in hair‐ or faeces‐based population genetics studies and modelled the bias associated with erroneous methods. It is emphasized that error rates may be substantially underestimated when using an erroneous approach. Genotyping error rates may be important determinants of the outcome of noninvasive studies and hence should be carefully computed and reported.
Major histocompatibility complex and microsatellite variation in two populations of wild gorillas
Authors: LUKAS D. , BRADLEY B. J. , NSUBUGA A. M. , DORAN‐SHEEHY D. , ROBBINS M. M. , VIGILANT L. 
Published: December 2004

AbstractIn comparison to their close relatives the chimpanzees and humans, very little is known concerning the amount and structure of genetic variation in gorillas. Two species of gorillas are recognized and while the western gorillas number in the tens of thousands, only several hundred representatives of the mountain gorilla subspecies of eastern gorillas survive. To analyse the possible effects of these different population sizes, this study compares the variation observed at microsatellite and major histocompatibility complex (MHC) loci in samples of wild western and mountain gorillas, collected using a sampling scheme that targeted multiple social groups within defined geographical areas. Noninvasive samples proved a viable source of DNA for sequence analysis of the second exon of the DRB loci of the MHC. Observed levels of variation at the MHC locus were similar between the two gorilla species and were comparable to those in other primates. Comparison of results from analysis of variation at multiple microsatellite loci found only a slight reduction in heterozygosity for the mountain gorillas despite the relatively smaller population size.
Generalized approach to the regulation and integration of gene expression
Authors: Oh Jeong‐Il , Kaplan Samuel 
Published: December 2004

The volume of electron flow through the cbb3 branch of the electron transport chain and the redox state of the quinone pool generate signals that regulate photosynthesis gene expression in Rhodobacter sphaeroides. An inhibitory signal is generated at the level of the catalytic subunit of the cbb3 cytochrome c oxidase and is transduced through the membrane‐localized PrrC polypeptide to the PrrBA two‐component activation system, which controls the expression of most of the photosynthesis genes in response to O2. The redox state of the quinone pool is monitored by the redox‐active AppA antirepressor protein, which determines the functional state of the PpsR repressor protein. The antirepressor/repressor system as well as a modulator of AppA function, TspO, together with FnrL and PrrA stringently control photopigment gene expression. These regulatory elements, together with spectral complex‐specific assembly factors, control the ultimate cellular levels and composition of the photosynthetic membrane.
Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila
Authors: Lüneberg Edeltraud , Mayer Barbara , Daryab Neda , Kooistra Oliver , Zähringer Ulrich , Rohde Manfred , Swanson John , Frosch Matthias 
Published: December 2004

We recently described the phase‐variable expression of a virulence‐associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild‐type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high‐copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non‐functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high‐copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA‐independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.
Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant
Authors: Minder Alexander C. , De Rudder Karel E. E. , Narberhaus Franz , Fischer Hans‐Martin , Hennecke Hauke , Geiger Otto 
Published: December 2004

Phosphatidycholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen‐fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N‐methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA‐deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild‐type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild‐type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.
A two‐component system that controls the expression of pneumococcal surface antigen A (PsaA) and regulates virulence and resistance to oxidative stress in Streptococcus pneumoniae
Authors: McCluskey J. , Hinds J. , Husain S. , Witney A. , Mitchell T. J. 
Published: December 2004

SummaryRecent genomic‐based studies have identified 13 two‐component signal transduction systems (TCS) in Streptococcus pneumoniae. Bacterial TCSs are important for regulating expression of bacterial genes, including those which are important to the virulence of pathogenic bacteria. We have used virulence assays together with microarray analysis to investigate the importance of pneumococcal TCS04 in the virulence and gene regulation of this pathogen. Deletion mutants of the response regulator of TCS04, rr04, were examined in three independent pneumococcal strains representing three different pneumococcal serotypes. Analysis of the virulence of the three strains enabled us to identify a serotype‐specific attenuation of virulence due to deletion of rr04. Microarray comparison of the transcriptional profiles of the wild‐type strains with the rr04 mutants allowed us to determine which transcriptional changes were occurring in the rr04 mutants. Virulence‐associated changes were demonstrated in the attenuated strain with significant downregulation of a previously determined virulence locus, psaB, psaC and psaA.
The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo
Authors: Wu Shiping , Howard Susan T. , Lakey David L. , Kipnis Andre , Samten Buka , Safi Hassan , Gruppo Veronica , Wizel Benjamin , Shams Homayoun , Basaraba Randall J. , Orme Ian M. , Barnes Peter F. 
Published: December 2004

SummaryThe ability of Mycobacterium tuberculosis to grow in macrophages is central to its pathogenicity. We found previously that the widespread 210 strain of M. tuberculosis grew more rapidly than other strains in human macrophages. Because principal sigma factors influence virulence in some bacteria, we analysed mRNA expression of the principal sigma factor, sigA, in M. tuberculosis isolates during growth in human macrophages. Isolates of the 210 strain had higher sigA mRNA levels and higher intracellular growth rates, compared with other clinical strains and the laboratory strain H37Rv. SigA was also upregulated in the 210 isolate TB294 during growth in macrophages, compared with growth in broth. In contrast, H37Rv sigA mRNA levels did not change under these conditions. Overexpression of sigA enhanced growth of recombinant M. tuberculosis in macrophages and in lungs of mice after aerosol infection, whereas recombinant strains expressing antisense transcripts to sigA showed decreased growth in both models. In the presence of superoxide, sense sigA transformants showed greater resistance than vector controls, and the antisense sigA transformant did not grow. We conclude that M. tuberculosis sigA modulates the expression of genes that contribute to virulence, enhancing growth in human macrophages and during the early phases of pulmonary infection in vivo. This effect may be mediated in part by increased resistance to reactive oxygen intermediates.
Chromosomal fragmentation in dUTPase‐deficient mutants of Escherichia coli and its recombinational repair
Authors: Kouzminova Elena A. , Kuzminov Andrei 
Published: December 2004

SummaryRecent findings suggest that DNA nicks stimulate homologous recombination by being converted into double‐strand breaks, which are mended by RecA‐catalysed recombinational repair and are lethal if not repaired. Hyper‐rec mutants, in which DNA nicks become detectable, are synthetic‐lethal with recA inactivation, substantiating the idea. Escherichia coli dut mutants are the only known hyper‐recs in which presumed nicks in DNA do not cause inviability with recA, suggesting that nicks stimulate homologous recombination directly. Here, we show that dut recA mutants are synthetic‐lethal; specifically, dut mutants depend on the RecBC–RuvABC recombinational repair pathway that mends double‐strand DNA breaks. Although induced for SOS, dut mutants are not rescued by full SOS induction if RecA is not available, suggesting that recombinational rather than regulatory functions of RecA are needed for their viability. We also detected chromosomal fragmentation in dut rec mutants, indicating double‐strand DNA breaks. Both the synthetic lethality and chromosomal fragmentation of dut rec mutants are suppressed by preventing uracil excision via inactivation of uracil DNA‐glycosylase or by preventing dUTP production via inactivation of dCTP deaminase. We suggest that nicks become substrates for recombinational repair after being converted into double‐strand DNA breaks.
Translocation of Escherichia coli RNA polymerase against a protein roadblock in vivo highlights a passive sliding mechanism for transcript elongation
Authors: Mosrin‐Huaman Christine , Turnbough Charles L. , Rahmouni A. Rachid 
Published: December 2004

SummaryCurrent models for transcription elongation infer that RNA polymerase (RNAP) moves along the template by a passive sliding mechanism that takes advantage of random lateral oscillations in which single basepair sliding movements interconvert the elongation complex between pre‐ and post‐translocated states. Such passive translocational equilibrium was tested in vivo by a systematic change in the templated NTP that is to be incorporated by RNAP, which is temporarily roadblocked by the lac repressor. Our results show that, under these conditions that hinder the forward movement of the polymerase, the elongation complex is able to extend its RNA chain one nucleotide further when the incoming NTP is a kinetically favoured substrate (i.e. low Km). The addition of an extra nucleotide destabilizes the repressor–operator roadblock leading to an increase in transcriptional readthrough. Similar results are obtained when the incoming NTPs are less kinetically favoured substrates (i.e. high Kms) by specifically increasing their intracellular concentrations. Altogether, these in vivo data are consistent with a passive sliding model in which RNAP forward translocation is favoured by NTP binding. They also suggest that fluctuations in the intracellular NTP pools may play a key role in gene regulation at the transcript elongation level.
Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites
Authors: Dodge Matthew A. , Waller Ross F. , Chow Larry M. C. , Zaman Muhammad M. , Cotton Leanne M. , McConville Malcolm J. , Wirth Dyann F. 
Published: December 2004

SummaryUpregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)‐tagged versions of LeMDR1 and the fate of the traceable‐fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 –/– and overexpressing cell lines. The LeMDR1‐GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)‐lysosome. Pulse–chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT‐lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug‐resistant Leishmania to vinblastine and rhodamine 123.
The H2‐sensing complex of Ralstonia eutropha: interaction between a regulatory [NiFe] hydrogenase and a histidine protein kinase
Authors: Buhrke Thorsten , Lenz Oliver , Porthun Antje , Friedrich Bärbel 
Published: December 2004

SummaryTwo [NiFe] hydrogenases enable the proteobacterium Ralstonia eutropha H16 to grow on molecular hydrogen as the sole energy source. A third [NiFe] hydrogenase (RH) acts as an H2 sensor in a multiple component signal transduction chain that controls hydrogenase gene transcription. The RH forms a dimeric heterodimer (HoxBC)2 in which HoxC contains the H2‐sensing active site and HoxB the electron‐transferring components including an organic, not yet identified redox cofactor. This oligomer forms a tight complex with the histidine protein kinase HoxJ. Both the sensor and the kinase were analysed by mutagenesis for functional domains that are instrumental in H2 signal transmission. A mutant deleted for a C‐terminal peptide of 55 amino acids in HoxB lost its H2‐sensing ability but still catalysed H2 oxidation. The mutant protein failed to form the dimeric heterodimer and a complex with HoxJ. The organic redox cofactor was no longer detectable in the truncated sensor. H2 sensing was also abolished by deletion of the PAS domain of HoxJ, indicating that this domain is involved in signal transduction. A truncated version of HoxJ consisting of only the input domain of the kinase was still capable of forming a complex with the RH. Mass determination of the purified HoxJ protein revealed that the kinase forms a homotetramer. The unique oligomeric structure of the H2‐sensing complex with respect to its regulatory function is discussed.
Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system
Authors: Livny Jonathan , Friedman David I. 
Published: December 2004

SummaryShiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the λ family, such as H‐19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H‐19B prophage encoding the TnpR resolvase gene downstream of the phage PR promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H‐19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non‐Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H‐19B, usually both prophages in the same cell are induced.
IsdA of Staphylococcus aureus is a broad spectrum, iron‐regulated adhesin
Authors: Clarke Simon R. , Wiltshire Michael D. , Foster Simon J. 
Published: December 2004

SummaryAs an important facet of host–pathogen interaction, Staphylococcus aureus has the ability to adhere to human extracellular matrix (ECM) components via a range of surface proteins. Here we have shown that IsdA has broad‐spectrum ligand‐binding activity, including fibrinogen and fibronectin. Mapping studies revealed a distinct domain responsible for ligand binding. This domain is present in a number of iron‐regulated proteins of S. aureus and in other Gram‐positive organisms. The isdA gene is only expressed in iron‐limited conditions under the control of Fur and not in standard laboratory media. Such conditions occur in serum in vitro and during infection. Whole cell binding and clumping assays revealed that when the bacteria are grown under iron‐limited conditions, IsdA constitutes a physiologically relevant adhesin to both fibrinogen and fibronectin. Thus for S. aureus, iron is an important marker for the host environment, to which the bacterium responds by differential regulation of at least one element of its adhesive strategy.
A putative G protein‐coupled receptor negatively controls sexual development in Aspergillus nidulans
Authors: Han Kap‐Hoon , Seo Jeong‐Ah , Yu Jae‐Hyuk 
Published: December 2004

SummaryG protein‐coupled receptors (GPCRs) are key components of heterotrimeric G protein‐mediated signalling pathways that detect environmental signals and confer rapid cellular responses. To broaden our understanding of signalling mechanisms in the filamentous fungus Aspergillus nidulans, intensive analyses of the Aspergillus nidulans genome have been carried out and nine genes (gprA∼gprI) that are predicted to encode seven transmembrane spanning GPCRs have been identified. Six of nine putative GPCRs have been disrupted and the gprD gene was found to play a central role in coordinating hyphal growth and sexual development. Deletion of gprD (ΔgprD) causes extremely restricted hyphal growth, delayed conidial germination and uncontrolled activation of sexual development resulting in a small colony covered by sexual fruiting bodies. Genetic studies indicate that GprD may not signal through the FadA (Gα)‐protein kinase A (PKA) pathway. Elimination of sexual development rescues both growth and developmental abnormalities caused by ΔgprD, suggesting that the primary role of GprD is to negatively regulate sexual development. This is supported by the fact that environmental conditions inhibiting sexual development suppress growth defects of the ΔgprD mutant. We propose that the GprD‐mediated signalling cascade negatively regulates sexual development, which is required for proper proliferation of A. nidulans.
Induction of the sufA operon encoding Fe‐S assembly proteins by superoxide generators and hydrogen peroxide: involvement of OxyR, IHF and an unidentified oxidant‐responsive factor
Authors: Lee Joon‐Hee , Yeo Won‐Sik , Roe Jung‐Hye 
Published: December 2004

SummaryA promoter (sufAp), inducible by various oxidants, directs transcription of the sufABCDSE operon encoding an alternative Fe‐S cluster assembly system in Escherichia coli. Superoxide generators and H2O2 induced expression of sufA–lacZ even in ΔsoxRS and ΔoxyR mutants, suggesting participation of an additional regulator(s) in oxidant induction of the sufA operon. Through deletion and linker scanning mutagenesis, we found three cis‐acting oxidant‐responsive elements (OREs). ORE‐I lies between −236 and −197 nucleotides from the transcription start site, overlapping extensively with the OxyR binding site reported previously. ORE‐II (−156 to −127) was found to be the site of IHF action. ORE‐III (−56 to −35) had no predictable binding sites for known regulators. Gel mobility shift assays with a 50 bp DNA probe containing ORE‐III revealed the presence of an ORE‐III‐specific factor that binds only when cells are treated with oxidants. S1 mapping analysis revealed that phenazine methosulphate (PMS) and H2O2 induced sufA expression by more than 40‐fold. In a ΔoxyR mutant, sufA was still induced more than 10‐fold. Fur, a ferric uptake regulator that negatively regulates this operon in response to iron availability, did not mediate the oxidant induction. Deletion of the suf operon caused cells to be more sensitive to superoxide‐generating agents without affecting sensitivity to H2O2. From these results, we propose that the oxidant induction of the sufA operon is mediated through OxyR, IHF, plus an unidentified oxidant‐responsive factor, and that the suf gene products are needed to defend cells against oxidative stress caused by superoxide generators.