RESUMO
AIMS: A shift in primary carbon metabolism is the fastest response to oxidative stress. Induced within seconds, it precedes transcriptional regulation, and produces reducing equivalents in form of NADPH within the pentose phosphate pathway (PPP). RESULTS: Here, we provide evidence for a regulatory signaling function of this metabolic transition in yeast. Several PPP-deficiencies caused abnormal accumulation of intermediate metabolites during the stress response. These PPP-deficient strains had strong growth deficits on media containing oxidants, but we observed that part of their oxidant-phenotypes were not attributable to the production of NADPH equivalents. This pointed to a second, yet unknown role of the PPP in the antioxidant response. Comparing transcriptome profiles obtained by RNA sequencing, we found gene expression profiles that resembled oxidative conditions when PPP activity was increased. Vice versa, deletion of PPP enzymes disturbed and delayed mRNA and protein expression during the antioxidant response. INNOVATION: Thus, the transient activation of the PPP is a metabolic signal required for balancing and timing gene expression upon an oxidative burst. CONCLUSION: Consequently, dynamic rearrangements in central carbon metabolism seem to be of major importance for eukaryotic redox sensing, and represent a novel class of dynamic gene expression regulators.
Assuntos
Antioxidantes/metabolismo , Regulação da Expressão Gênica , Via de Pentose Fosfato , Transcrição Gênica , Transporte de Elétrons , Perfilação da Expressão Gênica , Glicólise , Mutação , NADP/metabolismo , OxirreduçãoRESUMO
The functional complexity of the human transcriptome is not yet fully elucidated. We report a high-throughput sequence of the human transcriptome from a human embryonic kidney and a B cell line. We used shotgun sequencing of transcripts to generate randomly distributed reads. Of these, 50% mapped to unique genomic locations, of which 80% corresponded to known exons. We found that 66% of the polyadenylated transcriptome mapped to known genes and 34% to nonannotated genomic regions. On the basis of known transcripts, RNA-Seq can detect 25% more genes than can microarrays. A global survey of messenger RNA splicing events identified 94,241 splice junctions (4096 of which were previously unidentified) and showed that exon skipping is the most prevalent form of alternative splicing.
Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica , Genoma Humano , Sítios de Splice de RNA , RNA Mensageiro/genética , Análise de Sequência de RNA , Linhagem Celular , Linhagem Celular Tumoral , Biologia Computacional , DNA Complementar , DNA Intergênico , Éxons , Humanos , Íntrons , Análise de Sequência com Séries de Oligonucleotídeos , RNA Polimerase II/metabolismoRESUMO
In this article, we describe a genotyping approach applicable to both individual and multiplexed single nucleotide polymorphism (SNP) analysis, based on a ligation detection reaction (LDR) performed directly on genomic DNA. During the ligation, the biallelic state of the SNP locus is converted into a bimarker state of ligated detector oligonucleotides. The state of the markers is then determined by a 5'-nuclease assay (TaqMan) with universal fluorescent probes. The LDR-TaqMan method was successfully applied for the genotyping of 30 SNP loci of Arabidopsis thaliana. The technology is cost-effective, needs no locus-specific optimization, requires minimal manipulations, and has very good potential for automation.
Assuntos
Análise Mutacional de DNA/métodos , DNA/genética , Genômica , Polimorfismo de Nucleotídeo Único/genética , Alelos , Arabidopsis/genética , Sequência de Bases , Sítios de Ligação , Análise Mutacional de DNA/economia , DNA de Plantas/genética , Genoma de Planta , Genótipo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
We describe here a method for the synthesis of oligonucleotides with block structure (padlock probes, primers for multiplex polymerase chain reaction (PCR), and ligation-independent cloning), based on the ligation of presynthesized parts by T4 DNA ligase. The advantages of this approach are: (i) suitability of the technology for any producer-from synthesis company to laboratory, (ii) high quality and adjustable scale of synthesis, and (iii) possibility of including any modified bases inexpensively in the common part of the oligonucleotide. Clear difference of sizes of products and substrates makes the synthesis amenable to automation. For large series of padlock probes, the price per one primer approaches the price of the locus-specific parts. We demonstrate the application of this method to two different tasks: preparative-scale production of padlock probes and small-scale synthesis of PCR primers.
Assuntos
Conformação de Ácido Nucleico , Oligonucleotídeos/biossíntese , Oligonucleotídeos/química , Sequência de Bases , Primers do DNA/biossíntese , Primers do DNA/química , Primers do DNA/genética , Oligonucleotídeos/genética , Reação em Cadeia da PolimeraseRESUMO
We describe a novel and handy method for generating a population of templates for sequencing. The method is based on the random insertion of antibiotic resistance gene in plasmid DNA digested by DNase I. The advantages of this approach are the small quantity of DNA necessary for mutagenesis and the complete independence from the restriction map of the plasmid. DNase I digestion provides a random distribution of the insertions, while antibiotic selection provides low background. We also present a convenient PCR-based procedure for the analysis and ordering of obtained insertion mutants.
