RESUMEN
The emerging role of single-nucleotide polymorphisms (SNPs) in clinical association and pharmacogenetic studies has created a need for high-throughput genotyping technologies. We describe a novel method for multiplexed genotyping of SNPs that employs PCR amplification on microspheres. Oligonucleotide PCR primers were designed for each polymorphic locus such that one of the primers contained a recognition site for BbvI (a type IIS restriction enzyme), followed by 11 nucleotides of locus-specific sequence, which reside immediately upstream of the polymorphic site. Following amplification, this configuration allows for any SNP to be exposed by BbvI digestion and interrogated via primer extension, four-color minisequencing. Primers containing 5' acrylamide groups were attached covalently to the solid support through copolymerization into acrylamide beads. Highly multiplexed solid-phase amplification using human genomic DNA was demonstrated with 57 beads in a single reaction. Multiplexed amplification and minisequencing reactions using bead sets representing eight polymorphic loci were carried out with genomic DNA from eight individuals. Sixty-three of 64 genotypes were accurately determined by this method when compared to genotypes determined by restriction-enzyme digestion of PCR products. This method provides an accurate, robust approach toward multiplexed genotyping that may facilitate the use of SNPs in such diverse applications as pharmacogenetics and genome-wide association studies for complex genetic diseases.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Ácidos Nucleicos Heterodúplex/análisis , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodos , ADN/análisis , ADN/genética , Amplificación de Genes , Genoma Humano , Genotipo , Humanos , Microesferas , Técnicas de Amplificación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
DNA double-strand breaks are created by ionizing radiation or during V(D)J recombination, the process that generates immunological diversity. Breaks are repaired by an end-joining reaction that requires DNA-PKCS, the catalytic subunit of DNA-dependent protein kinase. DNA-PKCS is a 460 kDa serine-threonine kinase that is activated by direct interaction with DNA. Here we report its structure at 22 A resolution, as determined by electron crystallography. The structure contains an open channel, similar to those seen in other double-stranded DNA-binding proteins, and an enclosed cavity with three openings large enough to accommodate single-stranded DNA, with one opening adjacent to the open channel. Based on these structural features, we performed biochemical experiments to examine the interactions of DNA-PKCS with different DNA molecules. Efficient kinase activation required DNA longer than 12 bp, the minimal length of the open channel. Competition experiments demonstrated that DNA-PKCS binds to double- and single-stranded DNA via separate but interacting sites. Addition of unpaired single strands to a double-stranded DNA fragment stimulated kinase activation. These results suggest that activation of the kinase involves interactions with both double- and single-stranded DNA, as suggested by the structure. A model for how the kinase is regulated by DNA is described.
Asunto(s)
ADN/metabolismo , Proteínas Serina-Treonina Quinasas/química , Sitios de Unión , Cristalografía , Reparación del ADN/genética , ADN de Cadena Simple/metabolismo , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/química , Activación Enzimática , Lípidos/química , Modelos MolecularesRESUMEN
SUMMARY: Transcription factors IIB (TFIIB) and IIE (TFIIE) bound to RNA polymerase II have been revealed by electron crystallography in projection at 15.7 A resolution. The results lead to simple hypotheses for the roles of these factors in the initiation of transcription. TFIIB is suggested to define the distance from TATA box to transcription start site by bringing TATA DNA in contact with polymerase at that distance from the active center of the enzyme. TFIIE is suggested to participate in a key conformational switch occurring at the active center upon polymerase-DNA interaction.
Asunto(s)
Cristalografía/métodos , ARN Polimerasa II/metabolismo , ARN Polimerasa II/ultraestructura , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Factores de Transcripción/ultraestructura , Sitios de Unión , ADN de Hongos/genética , ADN de Hongos/metabolismo , Escherichia coli/genética , Análisis de Fourier , Sustancias Macromoleculares , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/ultraestructura , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , TATA Box , Factor de Transcripción TFIIBRESUMEN
An improved method for the production, cleavage, and purification of fusion proteins and peptides is described. The unique aspect of this method is dependent on the use of a proteinase from tobacco etch virus (TEV). The proteinase used is a recombinant TEV proteinase produced with a polyhistidine tract positioned at the amino terminus. The proteinase recognizes a specific, extended cleavage site sequence. The peptide or protein of interest is purified as a fusion protein with a TEV proteinase cleavage site sequence located between it and an affinity carrier portion of the fusion. Incubation with the recombinant TEV proteinase mediates release of the peptide or protein of interest. Use of the recombinant TEV proteinase to cleave fusion proteins is an improvement over use of other proteinases for several reasons, including its high degree of specificity, its insensitivity to many proteinase inhibitors generally used in protein purification, and the ready separation of both the affinity tag and the proteinase from the cleaved product of interest.
Asunto(s)
Péptidos/metabolismo , Virus de Plantas/enzimología , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía de Afinidad , Endopeptidasas/aislamiento & purificación , Endopeptidasas/metabolismo , Escherichia coli/metabolismo , Glutatión/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Datos de Secuencia Molecular , Níquel , Plásmidos , Sefarosa , Proteínas Virales/aislamiento & purificaciónRESUMEN
Regulation of gene expression in eukaryotes relies on intricate protein-protein interactions. Transcription of the galactose genes in yeast has been a productive model for this type of interaction. The positive activator in this system, GAL4, has a bifunctional C-terminus. It contains both a prototypic acidic activation domain and a region that binds the negative regulator, GAL80. We have taken advantage of this colocalization of functions to subject the region to a constrained mutagenesis analysis: one function was maintained, while the other one was altered. This analysis and the experiments it suggested have led us to two conclusions: first, the acidic amino acids are not, as commonly thought, required for activation; second, this region is not unstructured or alpha helical, but its function may require a beta sheet.
Asunto(s)
Proteínas Fúngicas/química , Regulación Fúngica de la Expresión Génica , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/química , Secuencia de Aminoácidos , Aniones , Proteínas de Unión al ADN/química , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The most common class of activation domains, the so-called acidic activators, has been proposed either to adopt an amphipathic alpha-helical structure or to exist as unstructured "acid blobs." However, genetic analysis of an acidic activation domain in the yeast GAL4 protein has suggested that the structure of the activation region is a beta sheet. To distinguish between these models, we conducted a biophysical analysis of peptides corresponding to the yeast GAL4 and GCN4 acidic activation domains. Circular dichroism spectroscopy shows that the peptides are not alpha helical, but that they can undergo a transition to a structure that is almost 100% beta sheet in character in slightly acidic solution. We also show that the artificial acidic activator AH has structural properties that are markedly different from the natural GAL4 and GCN4 domains and does not adopt a beta-rich structure at reduced pH.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Fúngicas/ultraestructura , Proteínas Quinasas/ultraestructura , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/ultraestructura , Secuencia de Aminoácidos , Aniones , Secuencia de Bases , Dicroismo Circular , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Péptidos/química , Proteínas Quinasas/química , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Factores de Transcripción/químicaRESUMEN
Transcription of galactose-inducible genes in yeast is regulated by interaction between the activator protein GAL4 and the negative regulatory protein GAL80. It has been suggested that GAL80 binds to and represses GAL4 under uninduced conditions and dissociates from GAL4 on induction. However, the possibility that GAL80 remains associated with GAL4 after induction has not been ruled out. Experiments to discriminate between these two models were performed and revealed that GAL80 stays bound after induction.
Asunto(s)
Proteínas Fúngicas/metabolismo , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Alelos , Secuencia de Aminoácidos , Proteínas de Unión al ADN , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Galactosa/farmacología , Cinética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Transformación Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The carboxy-terminal 28 amino acids of the Saccharomyces cerevisiae transcriptional activator protein GAL4 execute two functions--transcriptional activation and interaction with the negative regulatory protein, GAL80. Here we demonstrate that these two functions are separable by single amino acid changes within this region. We determined the sequences of four GAL4C-mutations, and characterized the abilities of the encoded GAL4C proteins to activate transcription of the galactose/melibiose regulon in the presence of GAL80 and superrepressible GAL80S alleles. One of the GAL4C mutations can be compensated by a specific GAL80S mutation, resulting in a wild-type phenotype. These results support the idea that while the GAL4 activation function tolerates at least minor alterations in the GAL4 carboxyl terminus, the GAL80-interactive function is highly sequence-specific and sensitive even to single amino acid alterations. They also argue that the GAL80S mutations affect the affinity of GAL80 for GAL4, and not the ability of GAL80 to bind inducer.