RESUMO
We describe a new molecular technique for the analysis of microbial species and complex microbial populations based on the separation of PCR-amplified 16S rDNA fragments by denaturing high-performance liquid chromatography (DHPLC). Using marine bacterial samples, we determined the optimum conditions for the analysis of bacterial species and the examination of complex bacterial assemblages obtained from different environments. The incorporation of a 40-bp GC clamp into the amplification primer was essential to effectively discriminate genetic differences in DHPLC-primers with a 20-, 10-, or 0-bp GC clamp length were less efficient. A 64.5 degrees C column temperature in DHPLC allowed optimal separation of species in a complex bacterial population. PCR-DHPLC analysis of bacterial assemblages demonstrated profiles with distinguishable peaks, which constituted the different populations and their degree of abundance. Fraction collection and DNA sequencing from profile peaks enabled bacterial identification. PCR-DHPLC analysis can also provide opportunities for describing bacterial communities, cloning bacteria, and monitoring bacterial populations in environments of interest.
Assuntos
Bactérias/genética , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Cromatografia Líquida de Alta Pressão/métodos , Água do Mar/microbiologia , Sequência de Bases , Primers do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/genética , DNA Ribossômico/isolamento & purificação , Japão , Reação em Cadeia da Polimerase/métodosRESUMO
Electronic microarray technology is a potential alternative in bacterial detection and identification. However, conditions for bacterial detection by electronic microarray need optimization. Using the NanoChip electronic microarray, we investigated eight marine bacterial species. Based on the 16S rDNA sequences of these species, we constructed primers, reporter probes, and species-specific capture probes. We carried out two separate analyses for longer (533 bp) and shorter (350 and 200 bp) amplified products (amplicons). To detect simultaneously the hybridization signals for the 350- and 200-bp amplicons, we designed a common reporter probe from an overlapping sequence within both fragments. We developed methods to optimize detection of hybridization signals for processing the DNA chips. A matrix analysis was performed for different bacterial species and complementary capture probes on electronic microarrays. Results showed that, when using the longer amplicon, not all bacterial targets hybridized with the complementary capture probes, which was characterized by the presence of false-positive signals. However, with the shorter amplicons, all bacterial species were correctly and completely detected using the constructed complementary capture probes.
Assuntos
Bactérias/genética , Bactérias/isolamento & purificação , Contagem de Colônia Microbiana/métodos , Monitoramento Ambiental/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Ribossômico 16S/genética , Microbiologia da Água , Sondas de DNA/genética , Eletrônica Médica/instrumentação , Eletrônica Médica/métodos , Monitoramento Ambiental/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/análiseRESUMO
Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with jasmonic acid (JA) for 1/2h yielded a cDNA of a gene tentatively named RERJ1 that is upregulated in response to exogenous JA. Northern blot analysis indicated that the RERJ1 mRNA levels peaked at 1/2-1h after the addition of jasmonic acid and then decreased gradually. RERJ1 encodes a transcriptional regulator with a basic helix-loop-helix motif. The phenotypes of transgenic rice plants overexpressing sense or antisense RERJ1 mRNA demonstrated that RERJ1 is involved in the growth inhibition of rice shoots caused by JA. Other biological functions of RERJ1 are discussed from an evolutionary standpoint.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Sequências Hélice-Alça-Hélice/fisiologia , Oryza/fisiologia , Brotos de Planta/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Cultivadas , Proteínas de Ligação a DNA/química , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/fisiologia , Dados de Sequência Molecular , Oryza/efeitos dos fármacos , Proteínas de Plantas , Brotos de Planta/efeitos dos fármacos , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
In suspension-cultured rice ( Oryza sativaL.) cells, jasmonic acid (JA) functions as a signal transducer in elicitor N-acetylchitoheptaose-induced phytoalexin production. Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with JA (10(-4) M) for 2 h yielded a cDNA for a gene that responded to exogenous JA by an increase in mRNA level. Nucleotide sequence analysis indicated that the cDNA encodes an homologue of the yeast Old Yellow Enzyme. The deduced amino acid sequence was very similar to the sequences of 12-oxophytodienoic acid reductases (OPR) 1 and 2 from Arabidopsis thaliana(AtOPR1 and AtOPR2) and OPR1 from tomato ( Lycopersicon esculentum) (LeOPR1). The cDNA-encoded protein purified from recombinant Escherichia coli cells as a hexahistidine-tagged fusion protein exhibited OPR activity similar to that of AtOPR1, AtOPR2, and LeOPR1, which catalyze reduction of (-)- cis-12-oxophytodienoic acid (OPDA) preferentially over (+)- cis-OPDA, a natural precursor of JA. Thus the rice enzyme was termed OsOPR1. The physiological roles of OsOPR1 are discussed. This is the first report of the cloning of an OPR gene from a monocot plant.
Assuntos
Ciclopentanos/farmacologia , Oryza/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/genética , Reguladores de Crescimento de Plantas/farmacologia , Células Cultivadas , Clonagem Molecular , Cicloeximida/farmacologia , DNA Complementar/química , DNA Complementar/genética , Ácidos Graxos Insaturados/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Oryza/citologia , Oryza/efeitos dos fármacos , Oxirredutases/metabolismo , Oxilipinas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
Two cDNA clones for jasmonic acid (JA)-responsive genes, RRJ1 and RRJ2, were isolated by differential screening from suspension-cultured rice cells treated with JA for 2 h. The putative RRJ1 protein is completely identical to that of a putative rice cystathionine gamma-lyase, while the putative RRJ2 protein is highly similar in sequence to a rice pyruvate decarboxylase, PDC1.
