Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Escherichia coli , Proteínas de Transporte de Monossacarídeos , Peptídeos/química , Proteínas/química , Proteínas Recombinantes de Fusão/química , Proteínas Repressoras , Fatores de Transcrição , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biotinilação , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/química , Proteínas de Transporte/genética , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/enzimologia , Vetores Genéticos , Indicadores e Reagentes , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Plasmídeos , Proteínas/análise , Proteínas/isolamento & purificação , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
We have purified a novel DNA polymerase from Thermus thermophilus. This was enabled by use of general gap filling assays to monitor polymerase activity and cross-reactive monoclonal antibodies against the alpha catalytic subunit of E. coli DNA polymerase III holoenzyme to distinguish a novel polymerase from the well characterized DNA polymerase I-like Thermus thermophilus DNA polymerase. Two proteins migrating with the polymerase after three chromatographic steps were isolated and subjected to partial amino acid sequencing. The amino termini of both were homologous to the two products of the E. coli dnaX gene, the gamma and tau subunits of the DNA polymerase III holoenzyme. Using this information and sequences conserved among dnaX-like genes, we isolated a gene fragment by PCR and used it as a probe to isolate the full length Thermus thermophilus dnaX gene. The deduced amino acid sequence is highly homologous to the DnaX proteins of other bacteria. Examination of the sequence permitted identification of a frameshift site similar to the one used in E. coli to direct the synthesis of the shorter gamma DnaX-gene product. Based on this information, we conclude that a conventional replicase exists in extreme thermophilic eubacteria. The general biological and practical technological implications of this finding are discussed.
Assuntos
Coenzimas/isolamento & purificação , DNA Polimerase III/isolamento & purificação , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Anticorpos Antibacterianos , Anticorpos Monoclonais , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Coenzimas/genética , Reações Cruzadas , DNA Polimerase III/análise , DNA Polimerase III/genética , Mudança da Fase de Leitura do Gene Ribossômico , Genes Bacterianos/genética , Dados de Sequência Molecular , Análise de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Thermus thermophilus/genética , Thermus thermophilus/imunologiaRESUMO
We report a quick, easy and inexpensive fluorometric assay that measures the activity of replication enzymes using Pico-GreenTM. The systems tested include replication of the natural template M13 Gori by E. coli DNA polymerase III holoenzyme and the replication of a synthetic homopolymer by human immunodeficiency virus reverse transcriptase. A direct comparison of the fluorometric assay with the conventional isotopic assay shows that the fluorometric assay accurately reflects the extent of replication. By performing the assay reactions directly in 96-well plates and using a fluorescence plate reader to determine the extent of reaction, the time required to measure replication activities is significantly shortened.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Fluorometria , HumanosAssuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Vetores Genéticos , Proteínas de Transporte de Monossacarídeos , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Repressoras/genética , Seleção Genética , Sequência de Aminoácidos , Bacteriófago M13/genética , Sequência de Bases , Proteínas de Transporte/genética , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Ligação Genética , Repressores Lac , Ligantes , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Ligação Proteica , Análise de Sequência de DNAAssuntos
DNA Polimerase III/isolamento & purificação , Escherichia coli/enzimologia , Técnicas Bacteriológicas , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , DNA Polimerase III/química , DNA Polimerase III/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/crescimento & desenvolvimento , Indicadores e Reagentes , Cinética , Substâncias Macromoleculares , Peso MolecularRESUMO
We have constructed a large library of random peptides fused to the C terminus of the lac repressor. The DNA binding activity of the repressor protein physically links the peptides to the plasmid encoding them by binding to lac operator sequences on the plasmid. This linkage allows efficient enrichment for specific peptide ligands in the random population of peptides by affinity purification of the peptide-repressor-plasmid complexes with an immobilized receptor. After transformation of Escherichia coli with recovered plasmids, the library can be amplified for additional rounds of affinity enrichment or specific plasmids can be sequenced to determine the primary structure of the peptides. We used a monoclonal antibody specific for the peptide dynorphin B as a model receptor to screen a random dodecamer library. After only two rounds of enrichment, the majority of the plasmids in the selected population encoded fusion peptides that bound specifically to the antibody. These peptides contain a consensus sequence similar to a segment of dynorphin B (RQFKVV). This technique should be useful to find peptide ligands for a variety of biological receptors.
Assuntos
Peptídeos/genética , Receptores de Superfície Celular/metabolismo , Proteínas Repressoras/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Clonagem Molecular/métodos , Dinorfinas/química , Dinorfinas/genética , Biblioteca Gênica , Ligantes , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Peptídeos/química , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/químicaRESUMO
Developmental mutants of Myxococcus xanthus have been previously described which appear to be defective in required cell-cell interactions. These mutants fall into four phenotypic classes, Asg, Bsg, Csg, and Dsg, each of which is unable to differentiate into spores but can be rescued by extracellular complementation by wild-type cells or by mutants of a different class. We report the identification of one of the loci in which mutations result in a Bsg phenotype. The cloned locus was contained on a 12-kilobase EcoRI fragment and then localized by subcloning and a combination of in vitro and transposon mutagenesis. All mutations in this locus behave as a single complementation group, which we designate bsgA (formerly ssbA). Each of the bsgA mutations results in a nonsporulating phenotype, which can be rescued by extracellular complementation. Furthermore, we report that the bsgA mutants have a distinctive interaction with wild-type cells when vegetatively growing, swarming colonies converge.
Assuntos
Clonagem Molecular , Genes Bacterianos , Myxococcales/genética , Alelos , Elementos de DNA Transponíveis , Mutação , Myxococcales/fisiologia , Plasmídeos , Esporos Bacterianos/fisiologia , Transdução GenéticaRESUMO
The ssbA mutants of Myxococcus xanthus behave as if they are unable to produce a cell-to-cell signal required for normal development. They are unable to form fruiting bodies or spores on developmental medium. They do sporulate, however, if allowed to develop in mixtures with wild-type cells. Fusions of developmentally induced promoters of M. xanthus to the Escherichia coli lacZ gene were used to characterize the effect of the ssbA mutations on developmental gene expression. Each of the five independent fusions tested was found to be dependent upon the ssbA+ allele for full expression. The ssbA mutants were able to express each of these fusions if the mutants were allowed to develop in mixtures with wild-type (Lac-) cells. These results cannot be explained on the basis of genetic exchange. The data are consistent with regulation of gene expression mediated by cell-to-cell interactions.