RESUMEN
We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab phages from libraries, and for selecting antigen binders by panning are presented. The latter protocol includes a procedure for trypsin elution of bound phage.
Asunto(s)
Bacteriófagos/genética , Biblioteca de Genes , Fragmentos Fab de Inmunoglobulinas/genética , Animales , Humanos , RatonesRESUMEN
Two genes, hom and thrB, involved in threonine biosynthesis in Lactococcus lactis MG1614, were cloned and sequenced. These genes, which encode homoserine dehydrogenase and homoserine kinase, were initially identified by the homology of their gene products with known homoserine dehydrogenases and homoserine kinases from other organisms. The identification was supported by construction of a mutant containing a deletion in hom and thrB that was unable to grow in a defined medium lacking threonine. Transcriptional analysis showed that the two genes were located in a bicistronic operon with the order 5' hom-thrB 3' and that transcription started 66 bp upstream of the translational start codon of the hom gene. A putative -10 promoter region (TATAAT) was located 6 bp upstream of the transcriptional start point, but no putative -35 region was identified. A DNA fragment covering 155 bp upstream of the hom translational start site was functional in pAK80, an L. lactis promoter probe vector. In addition, transcriptional studies showed no threonine-dependent regulation of hom-thrB transcription.
Asunto(s)
Homoserina Deshidrogenasa/genética , Lactococcus lactis/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Treonina/biosíntesis , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Secuencia de Bases , Clonación Molecular , Medios de Cultivo , ADN Bacteriano , Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/enzimología , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Operón , Regiones Promotoras Genéticas , Treonina/genética , Treonina/metabolismo , Transcripción GenéticaAsunto(s)
Vectores Genéticos , Fragmentos Fab de Inmunoglobulinas/genética , Animales , Secuencia de Bases , Clonación Molecular , Colifagos/genética , Cartilla de ADN/genética , ADN Complementario/genética , Escherichia coli/genética , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ingeniería de Proteínas , TripsinaAsunto(s)
Biblioteca de Genes , Genes de Inmunoglobulinas , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Secuencia de Aminoácidos , Animales , Bacteriófagos , Secuencia de Bases , Clonación Molecular/métodos , Cartilla de ADN , Humanos , Regiones Constantes de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/biosíntesis , Indicadores y Reactivos , Linfocitos/inmunología , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Bazo/inmunologíaRESUMEN
We have examined the termination region sequence of the rrnG operon and have observed its properties in vivo using a fusion plasmid test system. Transcription of rrnG terminator fragments was also studied in vitro. We found that termination of rrnG transcription is a complex process controlled by a tandem Rho-independent and Rho-dependent terminator arrangement which we designate rrnG-tt'. Together, these two elements were 98% efficient at terminating transcription initiated at the rrnG-P2 promoter. When the two elements were separated, however, we found that the Rho-independent structure was only 59% efficient while the Rho-dependent fragment alone could account for total transcriptional termination of the tandem arrangement. The rrnG termination region was resistant to rrn antitermination and, therefore, possesses some means of stopping antiterminated transcription. The distal rrnG sequence contains several additional noteworthy features; the rrnGt' fragment contains a REP (repetitive extragenic palindromic) sequence and homology with a small unidentified reading frame following rrnE. This sequence is followed by witA, which is homologous to a citrate transport gene, citB. Finally, our sequence, obtained from plasmid pLC23-30, contains a Tn1000 insertion that is absent from the E. coli chromosome. This insertion lies 975 bp beyond the 5S gene and is not involved in the termination events examined in this study.
Asunto(s)
Escherichia coli/genética , Operón , ARN Bacteriano/genética , ARN Ribosómico/genética , Regiones Terminadoras Genéticas , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
We have used a plasmid antitermination test system to examine the response of an Escherichia coli rRNA operon antiterminator to transcription through Rho-dependent and Rho-independent terminator-containing fragments. We also monitored transcription through multiple copies of a terminator to explore the mechanism of rrn antitermination. Four principal observations were made about antitermination and transcriptional terminators. (1) The rrn antiterminator mediated efficient transcription through Rho-dependent terminators. (2) Under the influence of the rrn antiterminator, RNA polymerase transcribed through two and three copies of the Rho-dependent 16 S----terminator with nearly the same efficiency as through one. (3) The antiterminator had less effect on fragments containing Rho-independent terminators; the rpoC t fragment and three fragments derived from the rrnB terminator region stopped antiterminated transcription. Four other Rho-independent terminator fragments were weakly antiterminated in our test system. (4) Surprisingly, the strength of these terminator fragments was not strongly related to properties such as the -delta G or number of trailing uridine residues of their canonical Rho-independent structures, but appears to be related to additional downstream terminators. We have drawn the following conclusions from these experiments. First, that ribosomal antitermination primarily reverses Rho-dependent termination by modifying the RNA polymerase elongation complex. Transcription through a 1700 nucleotide, multiple terminator sequence showed that the antiterminator caused persistent changes in the transcription process. Second, that fragments derived from the Rho-independent rrnB and rpoBC terminator regions can effectively stop antiterminated transcription. Third, that efficient in vivo termination may often involve regions with complex multiple terminators.
Asunto(s)
Escherichia coli/genética , Genes Reguladores , Operón , Regiones Terminadoras Genéticas , Transcripción Genética , Clonación Molecular , ARN Polimerasas Dirigidas por ADN/metabolismo , Factor Rho/metabolismoRESUMEN
Transcriptional regulation of the deoP2 promoter by the cyclic AMP/cyclic AMP receptor protein complex (cAMP/CRP) and the CytR repressor requires two high-affinity CRP targets located around -41 and -93 bp preceding the start site for transcription. Here we report the structure of cddP, another CRP/CytR-regulated promoter. In common with what was found in deo, the cdd promoter also contains multiple CRP targets. Thus, using the DNasel footprinting procedure, tandem CRP binding sites were identified around -41 and -93. These findings support a general model for CytR binding and CytR regulation, in which (i) CytR and the CRP/cAMP complex bind to similar or identical targets, (ii) two or more targets are necessary for proper binding of CytR to a promoter region, and (iii) CytR represses transcription by antagonizing cAMP/CRP activation.
Asunto(s)
Citidina Desaminasa/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Nucleósido Desaminasas/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Genes , Datos de Secuencia Molecular , Mapeo Nucleótido , Plásmidos , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
We describe the construction of low copy number operon-fusion vectors, and use one of these vectors for the cloning and transcriptional analysis of the terminator region after the deo operon of Escherichia coli K-12. The new vectors are miniderivatives of plasmid R1 containing the parB stability locus of this plasmid and the lac genes as a selectable marker. Since the copy number of the vectors is only one per genome-equivalent at temperatures below 37 degrees C this system is ideally suited for isolation and characterization of transcriptional and translational signals from E. coli. Our results show that a very strong terminator (deot), which resembles Rho-independent terminators, is located 60 bp downstream from the fourth structural gene of the deo operon. This confirms that deoD is the last gene in the operon. In addition, we have identified a new promoter just after the deot terminator and a short DNA sequence that is able to reduce lacZ expression by 85% when inserted between the deoP2 promoter and the lac genes.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos , Genes Reguladores , Genes , Vectores Genéticos , Operón , Regiones Terminadoras Genéticas , Secuencia de Bases , Clonación Molecular , N-Glicosil Hidrolasas/genética , Plásmidos , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
The sequences required for full repression of the Escherichia coli deoP1 and P2 promoters by the deoR repressor (DeoR) have been analyzed in vivo. Using recombinant techniques, we have constructed a set of deo-lacZ fusions which contain different parts of the sequences involved in the regulation of deo expression on low copy number fusion vectors. Since these vectors are present in only one copy per chromosome at temperatures below 37 degrees C, this vector system allows very accurate studies of gene control signals. Our results show that three DeoR operator sites exist in the deoP1-P2 regulatory region. Two of these loci overlap the initiation sites for deoP1 (O1) and deoP2 (O2) transcription located 599 bp apart, whereas the third site (OE) is present approximately 270 bp upstream of P1. DeoR repression of both P1 and P2 transcription is weak on promoter fragments which only contain one operator site (O1 or O2). Enhanced repression by deoR is observed on promoter fragments containing two operator sites. However, all three sites are needed for full repression. These findings are discussed with respect to upstream and downstream control regions of eukaryotic genes.