RESUMEN
Previously characterized by us barley chloroplast psbA gene, which encodes one of the main Photosystem II components--D1 protein, has been inserted in a set of special plasmid vectors and its expression in vitro and in vivo has been investigated. Experiments on the in vitro expression in the rabbit reticulocyte lysate system revealed a major product with a molecular weight ca. 33.5 kD, which corresponds to the unprocessed D1 barley protein. A lower molecular weight protein (about 29 kD) was also observed. These results are in agreement with the existence of two potential translation start sites in the psbA gene in the same reading frame, the second one starting from Met37 residue. The results fully correlate with the earlier data on the in vitro expression of psbA genes of maize, pea, and tobacco. Experiments on the in vivo expression of psbA gene in E. coli cells with the above constructions also revealed proteins with m. w. about 33.5 and 29 kD. The yield of the target recombinant protein in some cases was about 25-30% of the total E. coli cellular protein. The correspondence of the bands to the desired products was proved by the immunoenzyme analysis with the use of polyclonal antibodies. The data obtained show for the first time the construction of E. coli strains producing recombinant D1 protein of cereals in a high level.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Hordeum/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Complejo de Proteína del Fotosistema II , Regiones Promotoras Genéticas , Proteínas ViralesRESUMEN
Mutagenesis of two previously cloned plant genes, maize storage protein cZ22B1 gene and barley Photosystem II protein D1 gene (psbA), was carried out. To improve the nutritional quality of zein, the DNA region corresponding to the protein sixth alpha-helix rod was substituted by a synthetic segment bearing three codon changes for Lys. Additional stabilization of this helix was achieved by three more codon changes for Glu. By means of oligonucleotide directed mutagenesis five different copies of psbA gene were obtained, bearing single codon change of Ser264 (wild type) for Gly, Ala, Cys, Asn, and Thr, respectively. These constructs can be used for studying functional topography of protein D1 and core region.
Asunto(s)
Mutagénesis Sitio-Dirigida , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón , Hordeum/genética , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Zea mays/genéticaRESUMEN
In order to determine structures of the barley photosystem II subunits, the following genes have been cloned: psbB, encoding 47 kDa chlorophyll-binding subunit; psbH, encoding 7.7 kDa phosphoprotein; psbE and psbF, encoding 9.3 and 4.4 kDa subunits of the cytochrome b559 apoprotein, respectively; and a fragment of psbC gene, encoding the 43 kDa chlorophyll-binding subunit. The nucleotide sequences of these genes and the deduced amino acid sequences of their products are highly homologous to the corresponding sequences for other plant species.
Asunto(s)
Cloroplastos , ADN/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema II , Secale/genética , Clorofila/metabolismo , Clonación Molecular , Grupo Citocromo b/genética , Complejos de Proteína Captadores de Luz , Operón , Péptidos/genética , Fosfoproteínas/genética , Plásmidos , Conformación Proteica , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoAsunto(s)
Bacillus thuringiensis/genética , Toxinas Bacterianas , Clonación Molecular/métodos , ADN Bacteriano/genética , ADN Viral/genética , ADN/genética , Endotoxinas , Escherichia coli/genética , Vectores Genéticos , Plásmidos , Fagos T/genética , Regiones Terminadoras Genéticas , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Hemolisinas , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN/métodos , Transcripción GenéticaAsunto(s)
Clorofila/genética , Cloroplastos/análisis , Grupo Citocromo b/genética , Grano Comestible/genética , Hordeum/genética , Complejo de Proteína del Fotosistema II , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clorofila/aislamiento & purificación , Cloroplastos/enzimología , Grupo Citocromo b/aislamiento & purificación , Hordeum/enzimología , Complejos de Proteína Captadores de Luz , Datos de Secuencia Molecular , Proteínas del Complejo del Centro de Reacción Fotosintética , Proteínas de Plantas/aislamiento & purificaciónAsunto(s)
Clorofila/genética , Cloroplastos/análisis , Grupo Citocromo b/genética , Grano Comestible/genética , Hordeum/genética , Familia de Multigenes , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clorofila/aislamiento & purificación , Cloroplastos/enzimología , Grupo Citocromo b/aislamiento & purificación , Complejo de Citocromo b6f , Hordeum/enzimología , Complejos de Proteína Captadores de Luz , Datos de Secuencia Molecular , Proteínas del Complejo del Centro de Reacción Fotosintética , Proteínas de Plantas/aislamiento & purificaciónRESUMEN
The psbD gene for the membrane polypeptide D2 has been isolated from barley chloroplast DNA and its sequence, along with the flanking regions, has been determined. The 3'-end of the psbD gene is overlapped with a 50 bp stretch of a second open reading frame which belongs to the psbC gene encoding the P6 protein of photosystem II.
Asunto(s)
Clorofila/genética , ADN/genética , Grano Comestible/genética , Hordeum/genética , Proteínas de Plantas/genética , Secuencia de Bases , Enzimas de Restricción del ADN , Complejos de Proteína Captadores de Luz , Datos de Secuencia Molecular , Proteínas del Complejo del Centro de Reacción Fotosintética , Complejo de Proteína del Fotosistema IIAsunto(s)
Clorofila/genética , Cloroplastos/metabolismo , Genes , Proteínas de Plantas/genética , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Hordeum/genética , Complejos de Proteína Captadores de Luz , Sustancias Macromoleculares , Datos de Secuencia Molecular , Proteínas del Complejo del Centro de Reacción Fotosintética , Complejo de Proteína del Fotosistema IIAsunto(s)
Clorofila/genética , Cloroplastos/metabolismo , Genes , Proteínas de Plantas/genética , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Hordeum/genética , Complejos de Proteína Captadores de Luz , Sustancias Macromoleculares , Datos de Secuencia Molecular , Proteínas del Complejo del Centro de Reacción Fotosintética , Complejo de Proteína del Fotosistema IIRESUMEN
A useful and efficient approach to the synthesis of DNA duplexes of practically unlimited length has been developed. The proposed methodology is based on the use of temporary restriction sites for subcloning and assembling the segments of the desired DNA. It allows the utilization of chemically synthesized oligonucleotides of various length (from 10- to 100-mers) for the duplex construction. The application of this approach to the synthesis of a gene for the functionally active bacteriorhodopsin fragment is described.
Asunto(s)
ADN/síntesis química , Genes Sintéticos , Ingeniería Genética/métodos , Bacteriorodopsinas/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Enzimas de Restricción del ADN , ADN Bacteriano/síntesis química , Escherichia coli/genética , Vectores Genéticos , Plásmidos , Polinucleótidos/síntesis químicaRESUMEN
A chemical-enzymatic synthesis of 271- and 286-bp DNA duplexes, each of which contains the entire sequence coding for human proinsulin has been accomplished. In addition to the coding sequence, the 271-bp fragment carries translation initiation and termination signals plus EcoRI-HindIII restriction enzyme sites for insertion into an appropriate plasmid vector. The 286-bp fragment also contains a Shine-Dalgarno (SD) sequence preceding an ATG codon. Employing the 286-bp polynucleotide, the 568-bp tandem proinsulin gene has been obtained. The synthesis of these DNA fragments involved preparation of 42 oligonucleotides by a rapid N-methylimidazolide phosphotriester method and enzymatic conversion of the oligonucleotides into the gene subfragments, which were cloned separately and fused to yield the desired DNAs coding for proinsulin. The proinsulin gene fragments were cloned in Escherichia coli and shown to have the correct sequences.
Asunto(s)
Genes Sintéticos , Proinsulina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/síntesis química , ADN Recombinante/análisis , Escherichia coli/genética , Genes , Genes Reguladores , Vectores Genéticos , Humanos , PlásmidosRESUMEN
A modified phosphotriester method has been employed for the efficient chemical synthesis of long-chain deoxyribooligonucleotides. During the course of this work, a general and rapid procedure was developed for the preparation of 24-62-mers in solution. Preparative reversed phase column chromatography on silanized silica gel was used to purify triester intermediates starting from 10-mers. The rapid synthesis of 32-mer and 42-mer on glass and silica gel supports using suitably protected 2-8-mer blocks as coupling units has been also accomplished. In particular, a convenient procedure for the solid-phase synthesis of oligonucleotide blocks bearing 3'-terminal phosphodiester groups is described.
Asunto(s)
Oligodesoxirribonucleótidos/síntesis química , Oligonucleótidos/síntesis química , Adenilil Imidodifosfato/análogos & derivados , Cromatografía Líquida de Alta Presión , Vidrio , Imidazoles , Indicadores y Reactivos , Dióxido de Silicio , Relación Estructura-ActividadRESUMEN
A rapid and convenient method for the synthesis of deoxyribooligonucleotides has been developed using the phosphotriester approach. The advantage of this methodology for work in solution was successfully demonstrated in synthesis of a number of DNA fragments up to 32-long. Adaptation of the presented method to solid-phase synthesis allows a pentadecamer to be assembled in 4-5 hours using dinucleotides as coupling units.