RESUMO
We constructed a novel bacterial genome detection system using zinc finger protein (ZF) fused with firefly luciferase (ZF-luciferase). Taking advantage of the direct recognition of double-stranded DNA (dsDNA) by ZF, we previously constructed bacterial genome detection systems that did not require dehybridization processes. To detect polymerase chain reaction (PCR) products rapidly and with a high sensitivity, we constructed two kinds of ZF-luciferase, Sp1-fused luciferase (Sp1-luciferase), and Zif268-fused luciferase (Zif268-luciferase). ZF-luciferase not only maintains luciferase activity but also shows dsDNA-binding ability and specificity. Furthermore, we succeeded in the detection of 10 copies of the genome of Legionella pneumophila and Escherichia coli O157. ZF-luciferase would be a useful tool for highly sensitive detection of pathogenic bacterial genome.
Assuntos
Escherichia coli/isolamento & purificação , Legionella pneumophila/isolamento & purificação , Luciferases de Vaga-Lume/metabolismo , Dedos de Zinco/genética , Sítios de Ligação , DNA Bacteriano/análise , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Escherichia coli/genética , Legionella pneumophila/genética , Luciferases de Vaga-Lume/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismoRESUMO
A novel method of rapid and specific detection of polymerase chain reaction (PCR) products from bacterial genomes using Zn finger proteins was developed. Zn finger proteins are DNA-binding proteins that can sequence specifically recognize PCR products. Since Zn finger proteins can directly detect PCR products without undergoing dehybridization, unlike probe DNA, and can double check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to detect Legionella pneumophila using Sp1. It was found that a 49 bp L. pneumophila-specific region containing the Sp1 recognition site is located on the flhA gene of the L. pneumophila genome. We succeeded in specifically detecting PCR products amplified from L. pneumophila in the presence of other bacterial genomes by ELISA, and demonstrated that Sp1 enables the discrimination of L. pneumophila-specific PCR products from others. By fluorescence depolarization measurement, these specific PCR products could be detected within 1 min. These results indicate that the rapid and simple detection of PCR products specific to L. pneumophila using a Zn finger protein was achieved. This methodology can be applied to the detection of other bacteria using various Zn finger proteins that have already been reported.
Assuntos
Bactérias/isolamento & purificação , Proteínas de Ligação a DNA/química , Genoma Bacteriano , Reação em Cadeia da Polimerase/métodos , Dedos de Zinco , Bactérias/genética , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Legionella pneumophila/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Fator de Transcrição Sp1/química , Fator de Transcrição Sp1/metabolismoRESUMO
We have detected PCR products from Salmonella spp. and Influenza A virus using Zn finger protein Zif268 and Sp1, respectively. Previously, we demonstrated a novel method of rapid and specific detection of PCR products from Legionella pneumophila genome using Zn finger protein Sp1. In principle, this methodology might be applied to the detection of most bacteria and viruses using various Zn finger proteins. Here, to demonstrate the wider applicability of our method, we detected PCR products from Salmonella spp. and the Influenza A virus. BLAST data indicated the Zif268 and Sp1 recognition sequence were located on the gyrB gene of Salmonella spp. and the nucleoprotein gene of Influenza A virus, respectively. The PCR products from the oligonucleotide corresponding to the gyrB gene of Salmonella spp. or the nucleoprotein gene of the Influenza A virus could be specifically detected by ELISA or fluorescence depolarization measurement using Zif268 or Sp1. These results indicate the wide applicability of our novel methodology.
Assuntos
DNA Bacteriano/análise , DNA Viral/análise , Vírus da Influenza A/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Salmonella/isolamento & purificação , DNA Girase/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA/genética , Fator de Transcrição Sp1/metabolismo , Proteínas do Core Viral/genéticaRESUMO
A ring-opening reaction of cyclopropanes with five-membered heteroaromatics having a leaving group at C(2) was found to provide heteroaromatic-fused pyrrolidines in one step. This reaction was successfully applied to the synthesis of the protein kinase C-beta inhibitor JTT-010, which possesses a dihydropyrrolo[1,2-a]indole core.
Assuntos
Ciclopropanos/química , Proteína Quinase C/antagonistas & inibidores , Pirrolidinas/síntese química , Inibidores Enzimáticos/síntese química , Compostos Heterocíclicos/síntese química , Indanos/síntese química , Proteína Quinase C beta , Pirróis/síntese químicaRESUMO
An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268-luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF-luciferase fusion protein. By means of the automatic analyzer with ZF-luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0×10 to 1.0×10(6) copies.
Assuntos
Proteínas de Bactérias/genética , Inocuidade dos Alimentos/métodos , Luciferases/metabolismo , Reação em Cadeia da Polimerase , Dedos de Zinco/genética , Automação , DNA Viral/análise , DNA Viral/isolamento & purificação , Escherichia coli O157/genética , Humanos , Luciferases/genética , Norovirus/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Salmonella/genéticaRESUMO
A novel detection system of PCR products from bacterial genomes using Zinc finger proteins was developed. Zinc finger proteins are DNA-binding proteins that can bind to dsDNA with high affinity and specificity. Since Zinc finger proteins can directly detect PCR products and can double-check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to construct the detection system for three pathogen, Legionella pneumophila, Salmonella spp. and Influenza A virus using well-characterized Zinc finger proteins. As a result, we succeeded in detecting the PCR products from Legionella pneumophila, Salmonella spp. and Influenza A virus using Sp1 and Zif268. Therefore, this methodology can be applied to the detection of most pathogen using various Zinc finger proteins.
Assuntos
Bactérias/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase/métodos , Vírus/isolamento & purificação , Dedos de Zinco , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Legionella pneumophila/genética , Legionella pneumophila/isolamento & purificação , Salmonella/genética , Salmonella/isolamento & purificação , Fator de Transcrição Sp1/metabolismoRESUMO
The novel example of a vinylic hydrogen more reactive than a benzylic hydrogen was found by treatment of a twisted styrene derivative with a strong base followed by D(2)O quenching. In this paper, the full details of the examples of the highly activated vinyl hydrogens in twisted styrene derivatives are described, with a discussion on the correlation between the reactivity of the vinyl hydrogens and the magnitude of the twist. The highly reactive vinyl hydrogens could be rationalized by considering the novel orbital interaction between the pi(*) orbital of the benzene ring and the pi(*) orbital of the vinylic C-H bond in the twisted styrene derivatives.