RESUMO
A mixed infection of a single tick or host by Lyme disease spirochetes is common and a unique challenge for the diagnosis, treatment, and surveillance of Lyme disease. Here, we describe a novel protocol for differentiating Lyme strains on the basis of deep sequencing of the hypervariable outer surface protein C locus (ospC). Improving upon the traditional DNA-DNA hybridization method, the next-generation sequencing-based protocol is high throughput, quantitative, and able to detect new pathogen strains. We applied the method to more than one hundred infected Ixodes scapularis ticks collected from New York State, USA, in 2015 and 2016. An analysis of strain distributions within individual ticks suggests an overabundance of multiple infections by five or more strains, inhibitory interactions among coinfecting strains, and the presence of a new strain closely related to Borreliella bissettiae A supporting bioinformatics pipeline has been developed. The newly designed pair of universal ospC primers target intergenic sequences conserved among all known Lyme pathogens. The protocol could be used for culture-free identification and quantification of Lyme pathogens in wildlife and potentially in clinical specimens.
Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Borrelia burgdorferi/genética , Ixodes/microbiologia , Doença de Lyme/parasitologia , Animais , Carga Bacteriana , Borrelia/classificação , Borrelia/genética , Borrelia/isolamento & purificação , Borrelia burgdorferi/isolamento & purificação , Coinfecção/epidemiologia , Coinfecção/microbiologia , Coinfecção/parasitologia , DNA Bacteriano/genética , Feminino , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Doença de Lyme/epidemiologia , Doença de Lyme/microbiologia , Masculino , New York/epidemiologia , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor (F(+)) to a recipient (F(-)) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F(+) transconjugants and the recovery time between successive conjugation events of the F(+) donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10(-10) (cells/mL · min)(-1). We confirm a plasmid/chromosome ratio of 1:1 in homogenous F(+) populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome.
Assuntos
Conjugação Genética , Escherichia coli/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Simulação por Computador , Fatores de TempoRESUMO
The advent of cost-effective genotyping and sequencing methods have recently made it possible to ask questions that address the genetic basis of phenotypic diversity and how natural variants interact with the environment. We developed Camelot (CAusal Modelling with Expression Linkage for cOmplex Traits), a statistical method that integrates genotype, gene expression and phenotype data to automatically build models that both predict complex quantitative phenotypes and identify genes that actively influence these traits. Camelot integrates genotype and gene expression data, both generated under a reference condition, to predict the response to entirely different conditions. We systematically applied our algorithm to data generated from a collection of yeast segregants, using genotype and gene expression data generated under drug-free conditions to predict the response to 94 drugs and experimentally confirmed 14 novel gene-drug interactions. Our approach is robust, applicable to other phenotypes and species, and has potential for applications in personalized medicine, for example, in predicting how an individual will respond to a previously unseen drug.
Assuntos
Farmacorresistência Fúngica/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/genética , Saccharomyces cerevisiae/genética , Algoritmos , Antifúngicos/farmacologia , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica Múltipla/efeitos dos fármacos , Farmacorresistência Fúngica Múltipla/genética , Retroalimentação Fisiológica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Genótipo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
As opiates increase dopamine transmission, we measured the effects of morphine on dopamine-related genes using a real-time optic PCR assay that reliably detects small differences in mRNA in discrete brain regions. Tissue from dopaminoceptive and dopaminergic brain regions was collected from rats injected twice daily for 7 days with saline or increasing doses of morphine. Tissues were assayed for D1, D2 and D3 dopamine receptor mRNAs (D1R, D2R and D3R), as well as for mRNAs for tyrosine hydroxylase (TH) and the dopamine transporter (DAT). The neuron-associated mRNAs for SNAP-25 and synaptophysin, as well as the glial-associated mRNA for S100-beta and three 'housekeeping' mRNAs, were also measured. As reported previously by others, there was no alteration in D1R mRNA and a 25% decrease in D2R mRNA in the caudate-putamen, 2 h after the final morphine injection. Importantly, in the same RNA extracts, D3R mRNA showed significant increases of 85% in the caudate-putamen and 165% in the ventral midbrain, including the substantia nigra and ventral tegmental area. There were no other significant morphine effects. Mapping of brain regions in saline control rats agreed with previous studies, including showing the presence of low abundance TH mRNA and the absence of DAT mRNA in the caudate-putamen. The finding that chronic, intermittent injections of morphine caused an increase in D3R mRNA extends our understanding of the ability of D3R agonists to reduce the effects of morphine.
Assuntos
Encéfalo/efeitos dos fármacos , Dopamina/metabolismo , Glicoproteínas de Membrana , Morfina/farmacologia , Neurônios/efeitos dos fármacos , Receptores de Dopamina D2/genética , Regulação para Cima/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Dependência de Morfina/genética , Dependência de Morfina/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D3 , Proteínas S100/genética , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Sinaptofisina/genética , Proteína 25 Associada a Sinaptossoma , Tirosina 3-Mono-Oxigenase/genética , Regulação para Cima/genética , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
Drugs abused by humans are thought to activate areas in the ventral striatum of the brain that engage the organism in important adaptive behaviors, such as eating. In support of this, we report here that striatal regions of sugar-dependent rats show alterations in dopamine and opioid mRNA levels similar to morphine-dependent rats. Specifically, after a chronic schedule of intermittent bingeing on a sucrose solution, mRNA levels for the D2 dopamine receptor, and the preproenkephalin and preprotachykinin genes were decreased in dopamine-receptive regions of the forebrain, while D3 dopamine receptor mRNA was increased. While morphine affects gene expression across the entire dopamine-receptive striatum, significant differences were detected in the effects of sugar on the nucleus accumbens and adjacent caudate-putamen. The effects of sugar on mRNA levels were of greater magnitude in the nucleus accumbens than in the caudate-putamen. These areas also showed clear differences in the interactions among the genes, especially between D3R and the other genes. This was revealed by a novel multivariate analysis method that identified cooperative interactions among genes, specifically in the nucleus accumbens but not the caudate-putamen. Finally, a role for these cooperative interactions in a load-sharing response to perturbations caused by sugar was supported by the finding of a different pattern of correlations between the genes in the two striatal regions. These findings support a major role for the nucleus accumbens in mediating the effects of naturally rewarding substances and extend an animal model for studying the common substrates of drug addiction and eating disorders.
Assuntos
Encéfalo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Morfina/farmacologia , Vias Neurais/metabolismo , Recompensa , Sacarose/farmacologia , Animais , Encéfalo/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Encefalinas/genética , Transtornos da Alimentação e da Ingestão de Alimentos/genética , Transtornos da Alimentação e da Ingestão de Alimentos/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica , Masculino , Vias Neurais/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Peptídeos Opioides/genética , Precursores de Proteínas/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/genética , Receptores de Dopamina D3 , Transtornos Relacionados ao Uso de Substâncias/genética , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Taquicininas/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
In order to discern aggregation in solution, we present a quantum mechanical analog of the photon statistics from fluorescent molecules diffusing through a focused beam. A generating functional is developed to fully describe the experimental physical system as well as the statistics. Histograms of the measured time delay between photon counts are fit by an analytical solution describing the static as well as diffusing regimes. To determine empirical fitting parameters, fluorescence correlation spectroscopy is used in parallel to the photon counting. For expedient analysis, we find that the distribution's deviation from a single Poisson shows a difference between two single fluor monomers or a double fluor aggregate of the same total intensities. Initial studies were performed on fixed-state aggregates limited to dimerization. However preliminary results on reactive species suggest that the method can be used to characterize any aggregating system.
Assuntos
Biofísica/métodos , Fótons , Espectrometria de Fluorescência/métodos , DNA/química , DNA de Cadeia Simples/química , Difusão , Dimerização , Modelos Estatísticos , Modelos Teóricos , Distribuição de Poisson , Fatores de TempoRESUMO
Inter- and intraspecies horizontal gene transfer enabled by bacterial secretion systems is a powerful mechanism for bacterial genome plasticity. The type IV secretion system of Escherichia coli, encoded by the F plasmid, enables cell-to-cell contact and subsequent DNA transfer known as conjugation. Conjugation is compromised by phage infection that specifically targets the secretion machinery. Hence, the use of phages to regulate the spread of genes, such as acquired antibiotic resistance or as general biosanitation agents, has gained interest. To predict the potential efficacy, the competition kinetics must first be understood. Using quantitative PCR to enumerate genomic loci in a resource-limited batch culture, we quantify the infection kinetics of the nonlytic phage M13 and its impact on conjugation in the absence of selection pressure (isogenic set). Modeling the resulting experimental data reveals the cellular growth rate to be reduced to 60% upon phage infection. We also find a maximum phage infection rate of 3×10(-11) mL phage(-1) min(-1) which is only 1 order of magnitude slower than the maximum conjugation rate (3×10(-10) mL cell(-1) min(-1)), suggesting phages must be in significant abundance to be effective antagonists to horizontal gene transfer. In the regime where the number of susceptible cells (F(+)) and phages are equal upon initial infection, we observe the spread of the conjugative plasmid throughout the cell population despite phage infection, but only at 10% of the uninfected rate. This has interesting evolutionary implications, as even in the absence of selection pressure, cells maintain the ability to conjugate despite phage vulnerability and the associated growth consequences.
Assuntos
Bacteriófago M13/fisiologia , Conjugação Genética , Escherichia coli/genética , Escherichia coli/virologia , Algoritmos , Simulação por Computador , Cinética , Modelos GenéticosRESUMO
BACKGROUND: PCR in principle can detect a single target molecule in a reaction mixture. Contaminating bacterial DNA in reagents creates a practical limit on the use of PCR to detect dilute bacterial DNA in environmental or public health samples. The most pernicious source of contamination is microbial DNA in DNA polymerase preparations. Importantly, all commercial Taq polymerase preparations inevitably contain contaminating microbial DNA. Removal of DNA from an enzyme preparation is problematical. METHODOLOGY/PRINCIPAL FINDINGS: This report demonstrates that the background of contaminating DNA detected by quantitative PCR with broad host range primers can be decreased greater than 10-fold through the simple expedient of Taq enzyme dilution, without altering detection of target microbes in samples. The general method is: For any thermostable polymerase used for high-sensitivity detection, do a dilution series of the polymerase crossed with a dilution series of DNA or bacteria that work well with the test primers. For further work use the concentration of polymerase that gave the least signal in its negative control (H(2)O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria compared to higher concentrations of Taq polymerase. CONCLUSIONS/SIGNIFICANCE: It is clear from the studies shown in this report that a straightforward procedure of optimizing the Taq polymerase concentration achieved "treatment-free" attenuation of interference by contaminating bacterial DNA in Taq polymerase preparations. This procedure should facilitate detection and quantification with broad host range primers of a small number of bona fide bacteria (as few as one) in a sample.
Assuntos
Taq Polimerase/química , Técnicas de Tipagem Bacteriana/métodos , Sequência de Bases , Clonagem Molecular , DNA/análise , DNA/isolamento & purificação , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Pseudomonas fluorescens/genética , RNA Ribossômico 16S/química , Homologia de Sequência do Ácido Nucleico , Taq Polimerase/metabolismo , beta-Lactamases/metabolismoRESUMO
The algebra of target theory for damage by radiation was laid out by Atwood and Norman in 1949. Their equations provide a widely embraced framework for distinguishing single-hit and multi-hit mechanisms of damage. The present work asks whether in vitro damage to DNA duplexes by different agents affects amplification by the polymerase chain reaction (PCR) in a single-hit manner. Real-time monitoring of fluorescent PCR product (qPCR) was used to measure the fraction of DNA (S) surviving doses (D) of three damaging agents: gamma irradiation, DNase I, and UV radiation. The log fraction surviving was compared to the best-fit straight line predicted for a random single-hit model (lnS=kD). Human DNA targets for analysis were segments of multiple (nested) DNA lengths from the nuclear and the mitochondrial genomes within 10% of 150, 250, 350, 450, 650, 1000 and 2000 bases. For gamma irradiation, the results were consistent with a single-hit model for all segment sizes. In the case of DNase I, the shortest segment (150 bp), for both genomic and mitochondrial DNA, experienced more damage at low concentrations of DNase than the random single-hit model predicted. Conversely, in the case of UV, all segments of the nuclear target gene were less damaged at low doses and more damaged at high doses than predicted by the one hit model. These deviations from the predictions of a random single-hit model were interpreted as evidence for concerted activity in the case of DNase and of a multi-hit, sequence-dependent mechanism in the case of UV, perhaps due to the accumulation of lesions that slowed but did not entirely block Taq polymerase elongation.
Assuntos
Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , DNA/efeitos da radiação , Desoxirribonuclease I/metabolismo , Raios gama , Humanos , Modelos Estatísticos , Reação em Cadeia da Polimerase/métodos , Raios UltravioletaRESUMO
It is shown that laminar thermal convection can drive a chain reaction of DNA replication. The convection is triggered by a constant horizontal temperature gradient, moving molecules along stationary paths between hot and cold regions. This implements the temperature cycling for the classical polymerase chain reaction (PCR). The amplification is shown to be exponential and reaches 100,000-fold gains within 25 min. Besides direct applications, the mechanism might have implications for the molecular evolution of life.
Assuntos
Replicação do DNA , DNA Viral/química , Bacteriófago lambda/genética , DNA Viral/biossíntese , DNA Viral/síntese química , Temperatura Alta , Reação em Cadeia da PolimeraseRESUMO
DNA beacons are short single-stranded chains which can form closed hairpin shapes through complementary base pairing at their ends. Contrary to the common polymer theory assumption that only their loop length matters, experiments show that their closing kinetics depend on the loop composition. We have modeled the closing kinetics and in so doing have obtained stacking enthalpies and entropies for single-stranded nucleic acids. The resulting change of persistence length with temperature effects the dynamics. With a Monte Carlo study, we answer another polymer question of how the closing time scales with chain length, finding tau approximately N(2.44+/-0.02). There is a significant crossover for shorter chains, bringing the effective exponent into good agreement with experiment.