RESUMEN
Peptide and RNA epitope tags as tools for routine analysis of transgene expression and protein accumulation in transformed plant cell cultures was evaluated using three genes that encode very structurally and functionally different proteins. A T7 peptide was introduced at the amino- and carboxyl-termini of phosphinothricin-N-acetyl transferase and avidin and at the carboxyl-terminus of galactose oxidase. An RNA sequence that forms a higher order structure that is recognized by antibodies raised against the FLAG peptide was separately introduced into the 3' nontranslated region of these genes. Constructs were introduced into maize cell cultures using particle bombardment and transgene expression, protein accumulation, protein function and presence of the tags in RNA and/or protein as appropriate were evaluated in up to approximately 25 culture lines per construct. Results indicate that, while there will likely always be a need for some empirical evaluation of any tag-protein combination, introduction of the peptide tag at the amino-terminus was generally more successful than was incorporation at the carboxyl-terminus. RNA tags show promise for this purpose, but routine application will require development of a very sensitive immunoassay.
Asunto(s)
Epítopos/genética , Epítopos/inmunología , Expresión Génica/genética , Proteínas Recombinantes de Fusión/inmunología , Transgenes/genética , Zea mays/genética , Acetiltransferasas/genética , Acetiltransferasas/inmunología , Acetiltransferasas/metabolismo , Animales , Avidina/genética , Avidina/inmunología , Avidina/metabolismo , Biolística , Western Blotting , Pollos , Hongos , Galactosa Oxidasa/genética , Galactosa Oxidasa/inmunología , Galactosa Oxidasa/metabolismo , Genes Reporteros/genética , Vectores Genéticos/genética , Inmunoensayo , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Streptomyces , Transformación Genética , Zea mays/citología , Zea mays/metabolismoRESUMEN
OBJECTIVE: To increase the timeliness and sensitivity of a procedure that uses viral nucleic acid amplification followed by restriction fragment length polymorphism (RFLP) analysis for identifying strains of porcine reproductive and respiratory syndrome virus (PRRSV). SAMPLE POPULATION: 24 strains of PRRSV. PROCEDURE: A nested-set reverse transcriptase-polymerase chain reaction (RT-PCR) was developed and compared with a nonnested-set RT-PCR for sensitivity in amplifying known quantities of infective PRRSV. Once reaction conditions were optimized, the nested-set RT-PCR was tested for effectiveness with 24 strains of PRRSV isolated from swine. RESULTS: The nested-set RT-PCR was 100- to 1,000-fold more sensitive than the nonnested-set RT-PCR, detecting as little as 1 infective unit of PRRSV/ml of sample. It also was generally as sensitive as the combination of steps, namely virus isolation or propagation and nonnested-set RT-PCR, currently used routinely for amplifying PRRSV prior to RFLP analysis, and it was effective for amplifying all of the 24 strains of PRRSV tested. Using this RT-PCR, all tests were completed within 1.5 days (including RFLP analysis), compared with the > 7 days often required for the currently used method involving virus isolation and propagation. CONCLUSIONS: The nested-set RT-PCR was generally as sensitive as the combination of methods now used for PRRSV amplification prior to RFLP analysis, and it can markedly reduce the time required for testing. CLINICAL RELEVANCE: Presumptive identification of PRRSV strains can be provided in a more timely manner by use of a nested-set RT-PCR.
Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina/diagnóstico , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Animales , Líquido del Lavado Bronquioalveolar/virología , Células Cultivadas , Cartilla de ADN/química , ADN Viral/química , Electroforesis en Gel de Agar/veterinaria , Femenino , Sistemas de Lectura Abierta/genética , Polimorfismo de Longitud del Fragmento de Restricción , Síndrome Respiratorio y de la Reproducción Porcina/clasificación , Virus del Síndrome Respiratorio y Reproductivo Porcino/química , Virus del Síndrome Respiratorio y Reproductivo Porcino/clasificación , ARN Viral/sangre , Reproducción/fisiología , Enfermedades Respiratorias/diagnóstico , Enfermedades Respiratorias/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Sensibilidad y Especificidad , Organismos Libres de Patógenos EspecíficosRESUMEN
Type I repeat sequences are evolutionarily conserved sequence elements found in the replication origin and transcriptional promoter region of the rRNA genes (rDNA) in Tetrahymena thermophila. An abundant single-stranded DNA binding protein, ssA-TIBF, specifically interacts with the A-rich strand of the Type I repeat sequence. Quantitative binding competition experiments performed with purified ssA-TIBF demonstrate that the binding site for ssA-TIBF includes sequences both within the conserved 33 nt element and in a 3' flanking region: addition of the 3' flanking sequence to the Type I repeat oligonucleotide increases the binding affinity of ssA-TIBF by nearly 100-fold (apparent Kd = 3.0 x 10(-10) M). A mutation in the ssA-TIBF binding site previously shown to be the determinant of an rDNA replication defect in vivo results in a 25-fold decrease in ssA-TIBF binding affinity in vitro. ssA-TIBF also binds with high affinity to a copy of the Type I repeat sequence within the essential promoter region defined by in vitro transcription assays. The affinity of ssA-TIBF for the promoter repeat, which differs from other copies of the repeat at 8 out of 33 positions, is at least equal to its affinity for the Type I repeat sequences in the origin region. The biochemical properties of ssA-TIBF in vitro suggest that it could play a role in both replication and transcription of Tetrahymena rDNA in vivo.
Asunto(s)
ADN Protozoario/metabolismo , ADN Ribosómico/metabolismo , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Origen de Réplica , Tetrahymena thermophila/genética , Animales , Secuencia de Bases , Replicación del ADN , ADN Protozoario/genética , ADN Ribosómico/genética , ADN de Cadena Simple/metabolismo , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Unión Proteica , ARN Ribosómico/genética , Transcripción GenéticaRESUMEN
An origin of DNA replication has been mapped within the 5' non-transcribed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila. Mutations in 33 nt conserved AT-rich Type I repeat sequences located in the origin region cause defects in the replication and/or maintenance of amplified rDNA in vivo. Fe(II)EDTA cleavage footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by proteins in extracts of Tetrahymena cells. Two classes of proteins that bound the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays. One of these, ds-TIBF, bound preferentially to duplex DNA and exhibited only moderate specificity for Type I repeat sequences. In contrast, a single-stranded DNA-binding protein, ssA-TIBF, specifically recognized the A-rich strand of the Type I repeat sequence. Deletion of the 5' or 3' borders of the conserved sequence significantly reduced binding of ssA-TIBF. The binding properties of ssA-TIBF, coupled with genetic evidence that Type I sequences function as cis-acting rDNA replication control elements in vivo, suggest a possible role for ssA-TIBF in rDNA replication in Tetrahymena.