RESUMEN
Antibody-induced conformational changes of proteins have been recently frequently suggested to explain a variety of observations. In spite of the fundamental importance of this phenomenon for both in vivo and in vitro antigen-antibody interactions, it is not generally accepted because of the lack of conclusive evidence. This report utilizes a novel approach to the study of antibody-induced antigenic conformational changes. Pairs of monoclonal antibodies (mAb) were used to induce and to assess conformational changes in potato virus X (PVX) protein. Blocking ELISA with native and glutaraldehyde treated virus was used to detect conformational changes. Double antibody sandwich (DAS) ELISA was designed to investigate possible inter-molecular spread of conformational changes. Detection of one way blocking in a blocking ELISA, with a pair of mAbs reacting to non-overlapping epitopes, suggested conformational change as the mechanism of blocking. The putative conformational change was confirmed when the one way blocking was prevented using conformationally restrained virus. Inter-molecular spread of the conformational change among the molecules of PVX protein was demonstrated in DAS-ELISA, when capture mAb inhibited binding of detecting mAb in the absence of steric hindrance. Unlike X-ray crystallography, the methodology utilized in this study indicates directly the significance of a changed conformation to antibody binding.
Asunto(s)
Anticuerpos Antivirales/fisiología , Antígenos Virales , Proteínas de la Cápside , Cápside , Virus de Plantas/inmunología , Animales , Anticuerpos Monoclonales , Unión Competitiva , Western Blotting , Cápside/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Formaldehído/farmacología , Glutaral/farmacología , Ratones , Ratones Endogámicos BALB C , Conformación Proteica , Solanum tuberosumRESUMEN
The frequency of transcription of the ribosomal protein and RNA polymerase gene segments of the rplKAJL-rpoBC gene cluster was measured for Escherichia coli K-12 strains carrying mutations in the genes for transcriptional termination factors. The results of our study suggest that Rho increases and that both NusA and the product of sfrB decrease termination frequency in the rplL-rpoB intercistronic region.
Asunto(s)
Proteínas Bacterianas/fisiología , ARN Polimerasas Dirigidas por ADN/genética , Genes Reguladores , Factor Rho/fisiología , Proteínas Ribosómicas/genética , Regiones Terminadoras Genéticas , Factores de Transcripción/fisiología , Escherichia coli/genética , Genes Bacterianos , ARN Bacteriano/genética , Transcripción GenéticaRESUMEN
A multiple-copy (plasmid) vector and a single-copy (lambda) vector were constructed for the in vitro formation of transcriptional fusions to lacZ. In both vectors the transcription of lacZ is dependent upon the attachment of a promoter upstream, but beta-galactosidase is independently translated from the hybrid mRNA. These vectors are based on the W205 trp-lac deletion, but most of the trpBA DNA has been removed. Promoters are fused to the 3' end of trpA rather than the HindIII site at the 5' end of trpB used in other vectors containing the W205 deletion. This modification avoids the polar effects encountered when either an untranslated sequence, or a sequence translated in a different reading frame is fused to trpB. Hence the level of beta-galactosidase synthesized by fusions in these new vectors accurately reflects the frequency of transcription from the attached promoter. A polyrestriction site linker precedes the lacZ gene in both vectors and allows the direct ligation of promoter containing DNA fragments produced by a large collection of restriction endonucleases.
Asunto(s)
Bacteriófago lambda/genética , Vectores Genéticos , Operón Lac , Plásmidos , Secuencia de Bases , Mapeo Cromosómico , Enzimas de Restricción del ADN , Escherichia coli/genética , Regiones Promotoras Genéticas , Transcripción Genética , beta-Galactosidasa/genéticaRESUMEN
The rate of synthesis of the beta and beta' subunits of RNA polymerase relative to the rate of synthesis of total protein was found to remain constant with increasing steady state growth rate. This is in contrast to the relative synthesis rates of ribosomal proteins which are known to increase with growth rate. Yet the ratio of the rate of transcription of the ribosomal protein (rplJL) and RNA polymerase (rpoBC) domains of the rplKAJLrpoBC gene cluster was found to be invariant. Fusions to lacZ were used to relate the rate of transcription of the rplKAJL genes to the rate of synthesis of total protein. No change was seen at growth rates above 0.8 doublings per hour. This indicates that the growth rate-dependent expression of these ribosomal proteins is regulated at the post-transcriptional level. However because both the relative rate of transcription of rpoBC and rate of synthesis of beta and beta' were found to remain invariant over this growth range it suggests the expression of these RNA polymerase subunits is regulated at the transcriptional level.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/biosíntesis , Escherichia coli/metabolismo , Proteínas Bacterianas/biosíntesis , División Celular , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Genes Bacterianos , Transcripción GenéticaRESUMEN
The pattern of transcription of the rplKAJLrpoBC gene cluster of Escherichia coli appears to be complex. At least four different promoters and a transcriptional attenuator have been identified. To compare the relative effect of each of the putative promoters and the attenuator on transcription of these genes, we fused these regulatory sites to lacZ. These transcriptional fusions were constructed on lambda transducing phages so a single copy of each could be stably integrated into the chromosome. The level of beta-galactosidase in a lysogen of each phage reflects the activity of the transcriptional regulatory site. We find that the promoters preceding rplK (rplKp) and rplJ (rplJp) are indeed the major promoters of this gene cluster. The minor promoter before rplL (rplLp) is much weaker and contributes little to the transcription of the downstream genes. Under these conditions, we find no evidence of a promoter (rpoBp) in the rplL-rpoB intercistronic region. The attenuator (atn) terminates ca. 70% of the transcripts initiated at the promoters preceding it. Although we cannot rule out that some transcripts from rplKp may read through into rplJLrpoBC, we find that rplJp alone is sufficient for high-level expression of these genes.