Parthenogenetic flatworms have more symbionts than their coexisting, sexual conspecifics, but does this support the Red Queen?

The Red Queen hypothesis predicts that sexuality is favoured when virulent parasites adapt quickly to host genotypes. We studied a population of the flatworm Schmidtea polychroa in which obligate sexual and parthenogenetic individuals coexist. Infection rates by an amoeboid protozoan were consistently higher in parthenogens than in sexuals. Allozyme analysis showed that infection was genotype specific, with the second most common clone most infected. A laboratory measurement of fitness components failed to reveal high infection costs as required for the Red Queen. Although fertility was lower in more infected parthenogens, this effect can also be explained by the accumulation of mutations. We discuss these and other characteristics of our model system that may explain how a parasite with low virulence can show this pattern

Impact of growth matrix on pharmacodynamics of antimicrobial drugs for pig pneumonia pathogens

Abstract Background The most widely used measure of potency of antimicrobial drugs is Minimum Inhibitory Concentration (MIC). MIC is usually determined under standardised conditions in broths formulated to optimise bacterial growth on a species-by-species basis. This ensures comparability of data between laboratories. However, differences in values of MIC may arise between broths of differing chemical composition and for some drug classes major differences occur between broths and biological fluids such as serum and inflammatory exudate. Such differences must be taken into account, when breakpoint PK/PD indices are derived and used to predict dosages for clinical use. There is therefore interest in comparing MIC values in several broths and, in particular, in comparing broth values with those generated in serum. For the pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, MICs were determined for three drugs, florfenicol, oxytetracycline and marbofloxacin, in five broths [Mueller Hinton Broth (MHB), cation-adjusted Mueller Hinton Broth (CAMHB), Columbia Broth supplemented with NAD (CB), Brain Heart Infusion Broth (BHI) and Tryptic Soy Broth (TSB)] and in pig serum. Results For each drug, similar MIC values were obtained in all broths, with one exception, marbofloxacin having similar MICs for three broths and 4–5-fold higher MICs for two broths. In contrast, for both organisms, quantitative differences between broth and pig serum MICs were obtained after correction of MICs for drug binding to serum protein (fu serum MIC). Potency was greater (fu serum MIC lower) in serum than in broths for marbofloxacin and florfenicol for both organisms. For oxytetracycline fu serum:broth MIC ratios were 6.30:1 (P. multocida) and 0.35:1 (A. pleuropneumoniae), so that potency of this drug was reduced for the former species and increased for the latter species. The chemical composition of pig serum and broths was compared; major matrix differences in 14 constituents did not account for MIC differences. Bacterial growth rates were compared in broths and pig serum in the absence of drugs; it was concluded that broth/serum MIC differences might be due to differing growth rates in some but not all instances. Conclusions For all organisms and all drugs investigated in this study, it is suggested that broth MICs should be adjusted by an appropriate scaling factor when used to determine pharmacokinetic/pharmacodynamic breakpoints for dosage prediction

Comparison of standardised versus non-standardised methods for testing the in vitro potency of oxytetracycline against mannheimia haemolytica and pasteurella multocida

The in vitro pharmacodynamics of oxytetracycline was established for six isolates of each of the calf pneumonia pathogens Mannheimia haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and bacterial time-kill curves were determined in two matrices, Mueller Hinton broth (MHB) and calf serum. Geometric mean MIC ratios, serum:MHB, were 25.2:1 (M. haemolytica) and 27.4:1 (P. multocida). The degree of binding of oxytetracycline to serum protein was 52.4%. Differences between serum and broth MICs could not be accounted for by oxytetracycline binding to serum protein. In vitro time-kill data suggested a co-dependent killing action of oxytetracycline. The in vitro data indicate inhibition of the killing action of oxytetracycline by serum factor(s). The nature of the inhibition requires further study. The outcome of treatment with oxytetracycline of respiratory tract infections in calves caused by M. haemolytica and P. multocida may not be related solely to a direct killing action

RNA polymerase is poised for activation across the genome

Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter, followed by initiation of RNA synthesis and the transition to productive elongation. In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region. To determine the importance of polymerase stalling for transcription regulation, we carried out a genome-wide search for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. Our data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues

A Widespread Distribution of Genomic CeMyoD Binding Sites Revealed and Cross Validated by ChIP-Chip and ChIP-Seq Techniques

Identifying transcription factor binding sites genome-wide using chromatin immunoprecipitation (ChIP)-based technology is becoming an increasingly important tool in addressing developmental questions. However, technical problems associated with factor abundance and suitable ChIP reagents are common obstacles to these studies in many biological systems. We have used two completely different, widely applicable methods to determine by ChIP the genome-wide binding sites of the master myogenic regulatory transcription factor HLH-1 (CeMyoD) in C. elegans embryos. The two approaches, ChIP-seq and ChIP-chip, yield strongly overlapping results revealing that HLH-1 preferentially binds to promoter regions of genes enriched for E-box sequences (CANNTG), known binding sites for this well-studied class of transcription factors. HLH-1 binding sites were enriched upstream of genes known to be expressed in muscle, consistent with its role as a direct transcriptional regulator. HLH-1 binding was also detected at numerous sites unassociated with muscle gene expression, as has been previously described for its mouse homolog MyoD. These binding sites may reflect several additional functions for HLH-1, including its interactions with one or more co-factors to activate (or repress) gene expression or a role in chromatin organization distinct from direct transcriptional regulation of target genes. Our results also provide a comparison of ChIP methodologies that can overcome limitations commonly encountered in these types of studies while highlighting the complications of assigning in vivo functions to identified target sites

Temporal Coordination of Gene Networks by Zelda in the Early Drosophila Embryo

In past years, much attention has focused on the gene networks that regulate early developmental processes, but less attention has been paid to how multiple networks and processes are temporally coordinated. Recently the discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-activated genes in Drosophila, hinted at a mechanism for how batteries of genes could be simultaneously activated. Here we use genome-wide binding and expression assays to identify Zld target genes in the early embryo with the goal of unraveling the gene circuitry regulated by Zld. We found that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis, and pattern formation. In the absence of Zld, many target genes failed to be activated, while others, particularly the patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic “hotspot” regions, which are bound by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots, CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo

Robust Target Gene Discovery through Transcriptome Perturbations and Genome-Wide Enhancer Predictions in Drosophila Uncovers a Regulatory Basis for Sensory Specification

CisTarget X is a novel computational method that accurately predicts Atonal governed regulatory networks in the retina of the fruit fly

NELF Potentiates Gene Transcription in the Drosophila Embryo

A hallmark of genes that are subject to developmental regulation of transcriptional elongation is association of the negative elongation factor NELF with the paused RNA polymerase complex. Here we use a combination of biochemical and genetic experiments to investigate the in vivo function of NELF in the Drosophila embryo. NELF associates with different gene promoter regions in correlation with the association of RNA polymerase II (Pol II) and the initial activation of gene expression during the early stages of embryogenesis. Genetic experiments reveal that maternally provided NELF is required for the activation, rather than the repression of reporter genes that emulate the expression of key developmental control genes. Furthermore, the relative requirement for NELF is dictated by attributes of the flanking cis-regulatory information. We propose that NELF-associated paused Pol II complexes provide a platform for high fidelity integration of the combinatorial spatial and temporal information that is central to the regulation of gene expression during animal development

Erroneous attribution of relevant transcription factor binding sites despite successful prediction of cis-regulatory modules

<p>Abstract</p> <p>Background</p> <p><it>Cis</it>-regulatory modules are bound by transcription factors to regulate gene expression. Characterizing these DNA sequences is central to understanding gene regulatory networks and gaining insight into mechanisms of transcriptional regulation, but genome-scale regulatory module discovery remains a challenge. One popular approach is to scan the genome for clusters of transcription factor binding sites, especially those conserved in related species. When such approaches are successful, it is typically assumed that the activity of the modules is mediated by the identified binding sites and their cognate transcription factors. However, the validity of this assumption is often not assessed.</p> <p>Results</p> <p>We successfully predicted five new <it>cis</it>-regulatory modules by combining binding site identification with sequence conservation and compared these to unsuccessful predictions from a related approach not utilizing sequence conservation. Despite greatly improved predictive success, the positive set had similar degrees of sequence and binding site conservation as the negative set. We explored the reasons for this by mutagenizing putative binding sites in three <it>cis</it>-regulatory modules. A large proportion of the tested sites had little or no demonstrable role in mediating regulatory element activity. Examination of loss-of-function mutants also showed that some transcription factors supposedly binding to the modules are not required for their function.</p> <p>Conclusions</p> <p>Our results raise important questions about interpreting regulatory module predictions obtained by finding clusters of conserved binding sites. Attribution of function to these sites and their cognate transcription factors may be incorrect even when modules are successfully identified. Our study underscores the importance of empirical validation of computational results even when these results are in line with expectation.</p