RESUMEN
The development of a short incubation model of scrapie (strain 263K), in golden hamsters has added impetus to the purification of the infectious agent. Our own attempts have been based on methods pioneered by Millson and developed by Prusiner. We present here results indicating that a purification factor of up to 10(4) with respect to protein may now be possible. Fractions from brain with high infectivity had a sedimentation range of 70-300S and contained an abundance of fibrils closely similar to the scrapie-associated fibrils (SAF) discovered by Merz et al.. Material of molecular weight (Mr) 26,000, which is probably protein, appears to be a major constituent of the fibrils. The association between infectivity and fibrils raises two possibilities: the fibrils are an infectious form of the scrapie agent or they are a pathological response to scrapie infection.
Asunto(s)
Scrapie/transmisión , Animales , Encéfalo/fisiopatología , Cricetinae , Peso Molecular , Proteínas/aislamiento & purificación , Scrapie/patología , OvinosRESUMEN
Most tra proteins encoded by the Escherichia coli F sex factor are incorporated into the minicell envelope. We have now assigned the tra proteins to cytoplasm (TraIp and 2b), inner membrane (TraEp, TraMp, and TraSp), and outer membrane (6e, TraAp, TraBp, TraJp, TraKp, TraLp, and TraTp). two proteins, TraDp and 6d, were associated with both inner and outer membranes. The proteins exported to the inner or outer membranes did not undergo proteolytic cleavage (processing) whereas beta-lactamase was processed normally.
Asunto(s)
Escherichia coli/metabolismo , Factor F , Genes , Proteínas de la Membrana/biosíntesis , Membrana Celular/metabolismo , Peso Molecular , Mutación , Biosíntesis de Proteínas , Transcripción GenéticaAsunto(s)
Colifagos/genética , Replicación del ADN , Proteínas Virales/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/inmunología , Reacciones Cruzadas , Prueba de Complementación Genética , Lisogenia , Peso Molecular , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
The purification of the Escherichia coli dnaB protein by affinity chromatography on nucleotides bound to agarose is described. The dnaB protein, which contains an associated ribonucleoside triphosphatase activity (Wickner, S., Wright, M., and Hurwitz, J. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 783-787) binds to immobilized ATP, ADP, and UDP, but not to AMP. The type of linkage of ATP to agarose influences the adsorption, elution, and purification of the enzyme. Optimal purification is achieved using ATP bound to agarose via its oxidized ribose moiety. By this means, the dnaB protein can be obtained at least 95% electrophoretically pure after only three purification steps. The enzyme can be eluted from immobilized nucleoside-5'-di- and -triphosphates by ATP, ADP, and pyrophosphate, but not by AMP or orthophosphate. ADP and pyrophosphate, as well as the substrate ATP in high concentration are at the same time inhibitors of the ribonucleoside triphosphatase. The dnaB complementing and ribonucleoside triphosphatase activities could not be separated from each other by affinity chromatography, supporting the finding of others that they both reside on the same protein complex, namely a dnaB multimer. The results indicate that the dnaB protein binds to immobilized nucleotides by means of its ribonucleoside triphosphatase, and that at least the pyrophosphate moiety is essential for adsorption as well as elution of the enzyme.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/análisis , Cromatografía de Afinidad/métodos , Peso Molecular , RibonucleótidosRESUMEN
An Escherichia coli K12 dnaB dnaC mutant was constructed by P1 transduction of the dnaC allele into a dnaB recipient stain. dnaB dnaC transductant were discriminated from dnaB mutants by their inability to grow at 40 degree C after lysogenization with phage P1bac. The dnaB dnaC mutant character was verified by 1. P1 transduction, and 2. by in vitro complementation with dnaB and dnaC wild type protein fractions. DNA synthesis was studied in strains containing dnaB, dnaB dnaC alleles in an otherwise uniform genetic background with the dnaB character either unsuppressed or suppressed by P1bac prophage. Degradation at 42 degree C of [3H]-thymidine pulse-labeled DNA in dnaB and dnaB dnaC mutants is suppressed by P1bac. However, unlike the dnaC mutant, the P1bac lysogen of the dnaB dnaC mutant exhibits an abrupt cessation of DNA synthesis and less residual cell divisions at 42 degree C indicating an inhibition of DNA chain elongation rather than a defect in DNA initiation. It is suggested that denaturation of the dnaB protein effects the dnaC function.
Asunto(s)
ADN/biosíntesis , Escherichia coli/metabolismo , Colifagos , Prueba de Complementación Genética , Calor , Lisogenia , Mutación , Fenotipo , Transducción GenéticaRESUMEN
The proximal hooks of plain and complex flagella produced by a strain of Pseudomonas rhodos have been analyzed by electron microscopy and optical diffraction and filtering. Plain flagellar hooks are cone-shaped, 70 nm long, and 13 to 21.5 nm wide, and consist of helically arranged subunits. Complex flagellar hooks are cylinders, 180 to 190 nm long, and 15 to 16 nm wide, and are composed of globular subunits. The structure comprises four small-scale helical rows of subunits intersecting bewteen 10 and 11 large-scale helices of pitch angle 80 degrees. The axial and lateral dimensions of the unit cell, which define the surface lattice, are 4.9 and 4.7 nm, respectively. In addition, a core structure, approximately 5 nm wide, has been demonstrated inside the hook cylinder. Complex flagellar hooks were isolated and purified by gradient centrifugation after acid degradation of the attached filaments. Isolated hook particles have an average sedimentation constant of 130S and consist of a protein of molecular weight 43,000. A model of the complex flagellar hook is presented, and its possible role in flagellar assembly and rotation is discussed.