RESUMEN
Self-replicability is a unique attribute observed in all living organisms, and the question of how the life was physically initiated could be equivalent to the question of how self-replicating informative polymers were formed in the abiotic material world. It has been suggested that the present DNA and proteins world was preceded by an RNA world in which genetic information of RNA molecules was replicated by the mutual catalytic function of RNA molecules. However, the important question of how the transition occurred from a material world to the very early pre-RNA world remains unsolved both experimentally and theoretically. We present an onset model of mutually catalytic self-replicative systems formed in an assembly of polynucleotides. A quantitative expression of the critical condition for the onset of growing fluctuation towards self-replication in this model is obtained by analytical and numerical calculations.
Asunto(s)
Polinucleótidos , ARN , Polinucleótidos/genética , ARN/genética , ADN/genéticaRESUMEN
In Serratia marcescens JNB5-1, prodigiosin was highly produced at 30°C, but it was noticeably repressed at ≥37°C. Our initial results demonstrated that both the production and the stability of the O-methyl transferase (PigF) and oxidoreductase (PigN) involved in the prodigiosin pathway in S. marcescens JNB5-1 sharply decreased at ≥37°C. Therefore, in this study, we improved mRNA stability and protein production using de novo polynucleotide fragments (PNFs) and the introduction of disulfide bonds, respectively, and observed their effects on prodigiosin production. Our results demonstrate that adding PNFs at the 3' untranslated regions of pigF and pigN significantly improved the mRNA half-lives of these genes, leading to an increase in the transcript and expression levels. Subsequently, the introduction of disulfide bonds in pigF improved the thermal stability, pH stability, and copper ion resistance of PigF. Finally, shake flask fermentation showed that the prodigiosin titer with the engineered S. marcescens was increased by 61.38% from 5.36 to 8.65 g/liter compared to the JNB5-1 strain at 30°C and, significantly, the prodigiosin yield increased 2.05-fold from 0.38 to 0.78 g/liter at 37°C. In this study, we revealed that the introduction of PNFs and disulfide bonds greatly improved the expression and stability of pigF and pigN, hence efficiently enhancing prodigiosin production with S. marcescens at 30 and 37°C. IMPORTANCE This study highlights a promising strategy to improve mRNA/enzyme stability and to increase production using de novo PNF libraries and the introduction of disulfide bonds into the protein. PNFs could increase the half-life of target gene mRNA and effectively prevent its degradation. Moreover, PNFs could increase the relative intensity of target genes without affecting the expression of other genes; as a result, it could alleviate the cellular burden compared to other regulatory elements such as promoters. In addition, we obtained a PigF variant with improved activity and stability by the introduction of disulfide bonds into PigF. Collectively, we demonstrate here a novel approach for improving mRNA/enzyme stability using PNFs, which results in enhanced prodigiosin production in S. marcescens at 30°C.
Asunto(s)
Proteínas Bacterianas/genética , Metiltransferasas/genética , Prodigiosina/biosíntesis , Serratia marcescens/genética , Serratia marcescens/metabolismo , Regiones no Traducidas 3' , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Disulfuros/química , Fermentación , Concentración de Iones de Hidrógeno , Metiltransferasas/química , Metiltransferasas/metabolismo , Simulación de Dinámica Molecular , Polinucleótidos/genética , Estabilidad Proteica , ARN Mensajero/genética , TemperaturaRESUMEN
Ribonucleoprotein (RNP) granules are membraneless organelles (MLOs), which majorly consist of RNA and RNA-binding proteins and are formed via liquid-liquid phase separation (LLPS). Experimental studies investigating the drivers of LLPS have shown that intrinsically disordered proteins (IDPs) and nucleic acids like RNA and other polynucleotides play a key role in modulating protein phase separation. There is currently a dearth of modelling techniques which allow one to delve deeper into how polynucleotides play the role of a modulator/promoter of LLPS in cells using computational methods. Here, we present a coarse-grained polynucleotide model developed to fill this gap, which together with our recently developed HPS model for protein LLPS, allows us to capture the factors driving protein-polynucleotide phase separation. We explore the capabilities of the modelling framework with the LAF-1 RGG system which has been well studied in experiments and also with the HPS model previously. Further taking advantage of the fact that the HPS model maintains sequence specificity we explore the role of charge patterning on controlling polynucleotide incorporation into condensates. With increased charge patterning we observe formation of structured or patterned condensates which suggests the possible roles of polynucleotides in not only shifting the phase boundaries but also introducing microscopic organization in MLOs.
Asunto(s)
Proteínas/genética , Proteínas de Unión al ARN/genética , ARN/genética , Ribonucleoproteínas/genética , Simulación por Computador , Proteínas Intrínsecamente Desordenadas/genética , Extracción Líquido-Líquido , Modelos Moleculares , Orgánulos/genética , Polinucleótidos/química , Polinucleótidos/genética , Dominios Proteicos/genética , Proteínas/químicaRESUMEN
BACKGROUND: We hypothesize prebiotic evolution of self-replicating macro-molecules (Alberts, Molecular biology of the cell, 2015; Orgel, Crit Rev Biochem Mol Biol 39:99-123, 2004; Hud, Nat Commun 9:5171) favoured the constituent nucleotides and biophysical properties observed in the RNA and DNA of modern organisms. Assumed initial conditions are a shallow tide pool, containing a racemic mix of diverse nucleotide monomers (Barks et al., Chembiochem 11:1240-1243, 2010; Krishnamurthy, Nat Commun 9:5175, 2018; Hirao, Curr Opin Chem Biol 10:622-627), subject to day/night thermal fluctuations (Piccirilli et al., Nature 343:33-37, 1990). Self-replication, like Polymerase Chain Reactions, followed as higher daytime thermal energy "melted" inter-strand hydrogen bonds causing strand separation while solar UV radiation increased prebiotic nucleobase formation (Szathmary, Proc Biol Sci 245:91-99, 1991; Materese et al., Astrobiology 17:761-770, 2017; Bera et al., Astrobiology 17:771-785, 2017). Lower night energies allowed free monomers to form hydrogen bonds with their template counterparts leading to daughter strand synthesis (Hirao, Biotechniques 40:711, 2006). RESULTS: Evolutionary selection favoured increasing strand length to maximize auto-catalytic function in RNA and polymer stability in double stranded DNA (Krishnamurthy, Chemistry 24:16708-16715, 2018; Szathmary, Nat Rev Genet 4:995-1001, 2003). However, synthesis of the full daughter strand before daytime temperatures produced strand separation, longer polymer length required increased speed of self-replication. Computer simulations demonstrate optimal polynucleotide autocatalytic speed is achieved when the constituent nucleotides possess a left-right asymmetry that decreases the hydrogen bond kinetic barrier for the free nucleotide attachment to the template on one side and increases bond barrier on the other side preventing it from releasing prior to covalent bond formation. This phenomenon is similar to asymmetric kinetics observed during polymerization of the front and the back ends of linear cytoskeletal proteins such as actin and microtubules (Orgel, Nature 343:18-20, 1990; Henry, Curr Opin Chem Biol 7:727-733, 2003; Walker et al., J Cell Biol 108:931-937, 1989; Crevenna et al., J Biol Chem 288:12102-12113, 2013). Since rotation of the nucleotide would disrupt the asymmetry, the optimal nucleotides must form two or more hydrogen bonds with their counterpart on the template strand. All nucleotides in modern RNA and DNA have these predicted properties. Our models demonstrate these constraints on the properties of constituent monomers result in biophysical properties found in modern DNA and RNA including strand directionality, anti-parallel strand orientation, homochirality, quadruplet alphabet, and complementary base pairing. Furthermore, competition between RNA and DNA auto-replicators for 3 nucleotides in common permit states coexistence and possible cooperative interactions that could be incorporated into nascent living systems. CONCLUSION: Our findings demonstrate the molecular properties of DNA/RNA could have emerged from Darwinian competition among macromolecular replicators that selected nucleotide monomers that maximized the speed of autocatalysis.
Asunto(s)
Replicación del ADN , ADN/biosíntesis , Polinucleótidos/biosíntesis , ARN/biosíntesis , ADN/genética , Cinética , Polinucleótidos/genética , ARN/genéticaRESUMEN
The mechanisms that underpin how insertions or deletions (indels) become fixed in DNA have primarily been ascribed to replication-related and/or double-strand break (DSB)-related processes. Here, we introduce a method to evaluate indels, orientating them relative to gene transcription. In so doing, we reveal a number of surprising findings: First, there is a transcriptional strand asymmetry in the distribution of mononucleotide repeat tracts in the reference human genome. Second, there is a strong transcriptional strand asymmetry of indels across 2,575 whole genome sequenced human cancers. We suggest that this is due to the activity of transcription-coupled nucleotide excision repair (TC-NER). Furthermore, TC-NER interacts with mismatch repair (MMR) under physiological conditions to produce strand bias. Finally, we show how insertions and deletions differ in their dependencies on these repair pathways. Our analytical approach reveals insights into the contribution of DNA repair towards indel mutagenesis in human cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Neoplasias/genética , Secuencias Repetitivas de Ácidos Nucleicos , Secuencias de Aminoácidos , Biología Computacional , Análisis Mutacional de ADN , Replicación del ADN , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Variación Genética , Genoma Humano , Genómica , Humanos , Mutación INDEL , Mutagénesis , Neoplasias/metabolismo , Polinucleótidos/genética , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
The KnotGenome server enables the topological analysis of chromosome model data using three-dimensional coordinate files of chromosomes as input. In particular, it detects prime and composite knots in single chromosomes, and links between chromosomes. The knotting complexity of the chromosome is presented in the form of a matrix diagram that reveals the knot type of the entire polynucleotide chain and of each of its subchains. Links are determined by means of the Gaussian linking integral and the HOMFLY-PT polynomial. Entangled chromosomes are presented graphically in an intuitive way. It is also possible to relax structure with short molecular dynamics runs before the analysis. KnotGenome is freely available at http://knotgenom.cent.uw.edu.pl/.
Asunto(s)
Cromosomas/ultraestructura , Biología Computacional/tendencias , Internet , Programas Informáticos , Algoritmos , Cromosomas/genética , Simulación de Dinámica Molecular , Polinucleótidos/química , Polinucleótidos/genética , Conformación ProteicaRESUMEN
Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c7dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide.
Asunto(s)
ADN/genética , G-Cuádruplex , Polinucleótidos/química , Polinucleótidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción Reversa/genética , Cesio/química , Cloruros/química , Cloruro de Litio/química , Cloruro de Potasio/química , Cloruro de Sodio/químicaRESUMEN
Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5' region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA).
Asunto(s)
ADN de Cadena Simple/genética , Repeticiones de Microsatélite , Polinucleótidos/genética , Cartilla de ADN , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Microbial rhodopsins are remarkable for the diversity of their functional mechanisms based on the same protein scaffold. A class of rhodopsins from cryptophyte algae show close sequence homology with haloarchaeal rhodopsin proton pumps rather than with previously known channelrhodopsins from chlorophyte (green) algae. In particular, both aspartate residues that occupy the positions of the chromophore Schiff base proton acceptor and donor, a hallmark of rhodopsin proton pumps, are conserved in these cryptophyte proteins. We expressed the corresponding polynucleotides in human embryonic kidney (HEK293) cells and studied electrogenic properties of the encoded proteins with whole-cell patch-clamp recording. Despite their lack of residues characteristic of the chlorophyte cation channels, these proteins are cation-conducting channelrhodopsins that carry out light-gated passive transport of Na(+) and H(+). These findings show that channel function in rhodopsins has evolved via multiple routes.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Criptófitas , Rodopsinas Sensoriales/metabolismo , Secuencia de Aminoácidos , Proteínas de Transporte de Catión/genética , Cationes Monovalentes/metabolismo , Chlorophyta , Evolución Molecular , Células HEK293 , Humanos , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Luz , Técnicas de Placa-Clamp , Polinucleótidos/genética , Polinucleótidos/metabolismo , Protones , Rodopsinas Sensoriales/genética , Sodio/metabolismoRESUMEN
Apurinic/apyrimidinic (AP) sites are some of the most frequent lesions in genomic DNA. It is widely accepted that, irrespective of their origin, AP sites are further processed by the base excision repair (BER) machinery, being the central intermediate of this process. Under special conditions, proteins, which recognize AP sites, are able to form covalent adducts with DNA. By combination of the cross-linking technique with mass-spectrometry analysis, Ku antigen (Ku)--the central player in nonhomologous end joining (NHEJ), the pathway of double-strand break (DSB) repair--was identified as a protein reactive to AP sites. Moreover, Ku was shown to be a 5'-dRP/AP lyase that acts near DSBs in NHEJ. The recent studies have demonstrated involvement of Ku in the different stages of BER. Here, Ku roles in NHEJ and BER pathways of DNA repair are overviewed.
Asunto(s)
Antígenos Nucleares/genética , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Animales , Antígenos Nucleares/química , Ácido Apurínico/química , Ácido Apurínico/genética , Dominio Catalítico/genética , Aductos de ADN/genética , Proteína Quinasa Activada por ADN/química , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/química , Autoantígeno Ku , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Polinucleótidos/química , Polinucleótidos/genética , Multimerización de Proteína/genéticaRESUMEN
Previously, the mitochondrial control region of 214 Belgian dogs was sequenced. Analysis of this data indicated length heteroplasmy of the polyT stretch in the polyC-polyT-polyC stretch from positions 16661 to 16674. Nine polyC-polyT-polyC haplotype combinations were observed, consisting of seven major haplotypes (highest signal intensity) combined with minor haplotypes (lower signal intensity) one T shorter than the major haplotype in all but three dogs. The longer the polyT stretch, the smaller was the difference in signal intensity between the major and minor haplotype peaks. Additional sequencing, cloning, and PCR trap experiments were performed to further study the intra-individual variation of this mitochondrial DNA (mtDNA) region. Cloning experiments demonstrated that the proportion of clones displaying the minor haplotypes also increased with the length of the polyT stretch. Clone amplification showed that in vitro polymerase errors might contribute to the length heteroplasmy of polyT stretches with at least 10 Ts. Although major and minor polyC-polyT-polyC haplotypes did not differ intra-individually within and between tissues in this study, interpretation of polyT stretch variation should be handled with care in forensic casework.
Asunto(s)
ADN Mitocondrial/genética , Perros/genética , Genoma , Polinucleótidos/genética , Análisis de Secuencia de ADN , Animales , Variación Genética , HaplotiposRESUMEN
The influence of local chromatin context on gene expression can be explored by integrating a transcription reporter at different locations in the genome as a sensor. Here we provide a detailed protocol for analyzing thousands of reporters integrated in parallel (TRIP) at a genome-wide level. TRIP is based on tagging each reporter with a unique barcode, which is used for independent reporter expression analysis and integration site mapping. Compared with previous methods for studying position effects, TRIP offers a 100-1,000-fold higher throughput in a faster and less-labor-intensive manner. The entire experimental protocol takes â¼42 d to complete, with high-throughput sequencing and data analysis requiring an additional â¼11 d. TRIP was developed by using transcription reporters in mouse embryonic stem (mES) cells, but because of its flexibility the method can be used to probe the influence of chromatin context on a variety of molecular processes in any transfectable cell line.
Asunto(s)
Cromatina/metabolismo , Efectos de la Posición Cromosómica/genética , Genes Reporteros/genética , Animales , Células Cultivadas , Elementos Transponibles de ADN , Vectores Genéticos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ratones , Polinucleótidos/genéticaRESUMEN
Although lipoproteins of mycoplasmas are thought to play a crucial role in interactions with their hosts, very few have had their biochemical function defined. The gene encoding the lipoprotein MslA in Mycoplasma gallisepticum has recently been shown to be required for virulence, but the biochemical function of this gene is not known. Although this gene has no significant sequence similarity to any gene of known function, it is located within an operon in M. gallisepticum that contains a homolog of a gene previously shown to be a nonspecific exonuclease. We mutagenized both genes to facilitate expression in Escherichia coli and then examined the functions of the recombinant proteins. The capacity of MslA to bind polynucleotides was examined, and we found that the protein bound single- and double-stranded DNA, as well as single-stranded RNA, with a predicted binding site of greater than 1 nucleotide but less than or equal to 5 nucleotides in length. Recombinant MslA cleaved into two fragments in vitro, both of which were able to bind oligonucleotides. These findings suggest that the role of MslA may be to act in concert with the lipoprotein nuclease to generate nucleotides for transport into the mycoplasma cell, as the remaining genes in the operon are predicted to encode an ABC transporter.
Asunto(s)
Proteínas Portadoras/genética , Lipoproteínas/genética , Mycoplasma gallisepticum/genética , Mycoplasma gallisepticum/patogenicidad , Polinucleótidos/genética , Polinucleótidos/metabolismo , Factores de Virulencia/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Infecciones por Mycoplasma/genética , Infecciones por Mycoplasma/metabolismo , Infecciones por Mycoplasma/microbiología , Mycoplasma gallisepticum/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
In this paper, we revisit and adapt to viral evolution an approach based on the theory of branching process advanced by Demetrius et al. (Bull. Math. Biol. 46:239-262, 1985), in their study of polynucleotide evolution. By taking into account beneficial effects, we obtain a non-trivial multivariate generalization of their single-type branching process model. Perturbative techniques allows us to obtain analytical asymptotic expressions for the main global parameters of the model, which lead to the following rigorous results: (i) a new criterion for "no sure extinction", (ii) a generalization and proof, for this particular class of models, of the lethal mutagenesis criterion proposed by Bull et al. (J. Virol. 18:2930-2939, 2007), (iii) a new proposal for the notion of relaxation time with a quantitative prescription for its evaluation, (iv) the quantitative description of the evolution of the expected values in four distinct "stages": extinction threshold, lethal mutagenesis, stationary "equilibrium", and transient. Finally, based on these quantitative results, we are able to draw some qualitative conclusions.
Asunto(s)
Evolución Molecular , Modelos Genéticos , Polinucleótidos/genética , Replicación Viral/genética , Procesos EstocásticosRESUMEN
A polynucleotide probe, call a polymeric sequence probe (PSP), was used to detect influenza A (Influenza A/WSN/33) NA (Neuraminidase) viral RNA in Madin-Darby canine kidney (MDCK) cells. The PSP is a single-stranded DNA molecule with ~2,000 tandem repeat fluorescence binding sites and target binding sites that can bind with multiple fluorescence complementary oligos and target viral RNA using a fluorescence in situ hybridization (FISH) process. A single viral RNA labeled by PSP can be directly observed in MDCK cells. The simple FISH protocol enables the observation and quantitative analysis of the infectious process and drug effects with ultrahigh sensitivity and spatial resolution.
Asunto(s)
Virus de la Influenza A/química , Virus de la Influenza A/genética , Polinucleótidos/genética , Sondas ARN/química , Sondas ARN/genética , ARN Viral/análisis , ARN Viral/genética , Animales , Perros , Hibridación Fluorescente in Situ , Células de Riñón Canino Madin DarbyRESUMEN
Cationic polyrotaxanes, obtained by temperature activated threading of cationic cyclodextrin derivatives onto water-soluble cationic polymers (ionenes), form metastable nanometric polyplexes with pDNA and combinations of siRNA with pDNA. Because of their low toxicity, the polyrotaxane polyplexes constitute a very interesting system for the transfection of polynucleotides into mammalian cells. The complexation of Cy3-labeled siRNA within the polyplexes was demonstrated by fluorescence correlation spectroscopy. The uptake of the polyplexes (red) was imaged by confocal fluorescence microscopy using the A549 cell line as a model (blue: nuclei, green: membranes). The results prove the potential of polyrotaxanes for further investigations involving knocking down genes of therapeutic interest.
Asunto(s)
Ciclodextrinas/química , Portadores de Fármacos/química , Polinucleótidos/administración & dosificación , Rotaxanos/química , Cationes , Línea Celular , Ciclodextrinas/síntesis química , ADN/administración & dosificación , ADN/genética , Portadores de Fármacos/síntesis química , Endocitosis , Silenciador del Gen , Humanos , Luciferasas/genética , Espectroscopía de Resonancia Magnética , Microscopía Confocal , Microscopía Fluorescente , Tamaño de la Partícula , Plásmidos/administración & dosificación , Plásmidos/genética , Polinucleótidos/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Rotaxanos/síntesis química , Espectrometría de FluorescenciaRESUMEN
SuperSAGE is a variant of the Serial Analysis of Gene Expression (SAGE) technology, based on counting transcripts by sequencing analysis of short sequence tags. In SuperSAGE, 26 bp tags are extracted from cDNA using the Type III restriction endonuclease EcoP15I. The use of a longer tag size in SuperSAGE allows a secure tag-to-gene annotation in any eukaryotic organism. We have succeeded in combining SuperSAGE and high-throughput sequencing technology (Now- or Next-Generation Sequencing, NGS) in an approach we call High-throughput SuperSAGE (HT-SuperSAGE). This approach allows deep transcriptome analysis and multiplexing, while reducing time, cost, and effort for the analysis. In this chapter, we present the detailed HT-SuperSAGE protocol for both the Illumina Genome Analyzer and also the AppliedBiosystems SOLiD sequencer.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Animales , Secuencia de Bases , Tampones (Química) , Cartilla de ADN/genética , ADN Complementario/síntesis química , ADN Complementario/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Polinucleótidos/genética , Transcripción Reversa , Transcripción GenéticaRESUMEN
Biochemical and molecular biomarkers tools are utilized as early warning signatures of contaminant exposure to target and non-target organisms. The objective of this study was to investigate the sublethal effects of iron chloride to the larvae of the lace coral Pocillopora damicornis by measuring a suit of oxidative-stress biomarkers. The larvae were exposed to a range of sublethal concentrations of iron chloride (0.01, 0.1, 1, 10, and 100 ppm) for seven days. With reference to oxidative stress biomarkers, the no-observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) of iron chloride were observed to be 0.01 and 100 ppm respectively. At the end of the seventh day the antioxidant status of the larvae was evaluated by the levels of glutathione (GSH), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione-S-transferase (GST), in both experimental and control groups. For the quantification of cellular oxidative damage, lipid peroxidation (LPO) activity was determined in the same and the extent of DNA damage was assessed by the expression of DNA apurinic/apyrimidinic (AP) sites. Iron chloride exhibited a concentration-dependent inhibition of GSH and GPX and induction of GR, GST, LPO, and DNA-AP sites in the P. damicornis larvae when compared to the control group. The oxidative stress biomarkers of the larvae exposed to 0.1, 1, and 10 ppm of iron chloride did not show any significant overall differences when compared to the control group. However the activities of LPO, GSH, GPX, GR, GST and DNA-AP in the larval group exposed to 100 ppm of iron chloride exhibited statistically significant (P=0.002, 0.003, 0.002, 0.002, 0.005 and 0.007) differences when compared to the control group. The research results indicated that iron chloride in concentrations at the 100 ppm level caused oxidative stress in the P. damicornis larvae.
Asunto(s)
Antozoos/efectos de los fármacos , Cloruros/toxicidad , Daño del ADN , Compuestos de Hierro/toxicidad , Estrés Oxidativo/efectos de los fármacos , Animales , Antozoos/genética , Antozoos/metabolismo , Antioxidantes/metabolismo , Ácido Apurínico/genética , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Larva/efectos de los fármacos , Larva/genética , Larva/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Nivel sin Efectos Adversos Observados , Polinucleótidos/genética , Factores de TiempoRESUMEN
Chloroplast DNA sequences are of great interest for population genetics and phylogenetic studies. However, only a small set of markers are commonly used. Most of them have been designed for amplification in a large range of Angiosperms and are located in the Large Single Copy (LSC). Here we developed a new set of 100 primer pairs optimized for amplification in Monocotyledons. Primer pairs amplify coding (exon) and non-coding regions (intron and intergenic spacer). They span the different chloroplast regions: 72 are located in the LSC, 13 in the Small Single Copy (SSC) and 15 in the Inverted Repeat region (IR). Amplification and sequencing were tested in 13 species of Monocotyledons: Dioscorea abyssinica, D. praehensilis, D. rotundata, D. dumetorum, D. bulbifera, Trichopus sempervirens (Dioscoreaceae), Phoenix canariensis, P. dactylifera, Astrocaryum scopatum, A. murumuru, Ceroxylon echinulatum (Arecaceae), Digitaria excilis and Pennisetum glaucum (Poaceae). The diversity found in Dioscorea, Digitaria and Pennisetum mainly corresponded to Single Nucleotide Polymorphism (SNP) while the diversity found in Arecaceae also comprises Variable Number Tandem Repeat (VNTR). We observed that the most variable loci (rps15-ycf1, rpl32-ccsA, ndhF-rpl32, ndhG-ndhI and ccsA) are located in the SSC. Through the analysis of the genetic structure of a wild-cultivated species complex in Dioscorea, we demonstrated that this new set of primers is of great interest for population genetics and we anticipate that it will also be useful for phylogeny and bar-coding studies.
Asunto(s)
Cartilla de ADN/metabolismo , ADN de Cloroplastos/genética , Genética de Población/métodos , Magnoliopsida/genética , Filogenia , Secuencia de Bases , Repeticiones de Minisatélite/genética , Datos de Secuencia Molecular , Polimorfismo Genético , Polinucleótidos/genética , Especificidad de la EspecieRESUMEN
Numerous DNA chemistries for improving oligodeoxynucleotide (ODN)-based RNA targeting have been explored. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODN's are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH(3) modification on the nucleic acid structure and RNase H1 enzyme activity, we have investigated two DNA/RNA hybrids containing either a R(P) or S(P) BH(3) modification by nuclear magnetic resonance (NMR) spectroscopy. T(M) studies show that the stabilities of R(P) and S(P) modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to that of an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. To characterize such similar structures, we used a large number of NMR restraints (including dipolar couplings and backbone torsion angles) to determine structural features that were important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total R(x) values of <6%) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In the case of the S(P) modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the S(P) BH(3) group in the major groove while the R(P) BH(3) group points away from the DNA. However, this creates a potential clash of the R(P) BH(3) groups with important RNase H1 residues in a complex, while the S(P) BH(3) groups could be tolerated. We therefore predict that on the basis of our NMR structures a fully R(P) BH(3) DNA/RNA hybrid would not be a substrate for RNase H1.