RESUMO
DNAs obtained from bone marrow cells of 31 children with neoplasms of mesenchymal origin were tested for the presence of proviral simian sarcoma associated virus by southern blot-hybridization. The lower limit of detection in this method was one provirus per 20-30 cells. Applying stringent conditions of hybridization, no proviral DNA could be detected in any of the samples tested, most of which were from children with leukemia or with bone marrow invading lymphosarcomas. Relaxed conditions of hybridization revealed a diffuse pattern of hybridizing fragments which was similar in all DNAs tested, including normal human DNA.
Assuntos
Medula Óssea/microbiologia , DNA Viral/isolamento & purificação , Leucemia/microbiologia , Retroviridae/genética , Vírus do Sarcoma do Macaco-Barrigudo/genética , Sarcoma/microbiologia , Linhagem Celular , Criança , DNA Viral/genética , Humanos , Peso Molecular , Hibridização de Ácido Nucleico , Rabdomiossarcoma/microbiologiaRESUMO
Polioviral genes coding for P1, the precursor for the structural proteins, and 3CD, the viral protease, were cloned in a Saccharomyces cerevisiae inducible expression system. N-antigenic empty capsids could be isolated from the yeast cell extract provided that pirodavir, a capsid-binding compound and capsid stabilizer, was added during the induction period and during purification. Purification was by immunoaffinity chromatography. The purified empty capsids had the same immunogenicity as poliovirus virions. The techniques described might be useful for the production of new non-infectious vaccines.
Assuntos
Antígenos Virais/biossíntese , Cisteína Endopeptidases/biossíntese , Genes Virais , Poliovirus/genética , Proteínas Virais , Proteínas Estruturais Virais/biossíntese , Proteases Virais 3C , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Capsídeo/biossíntese , Capsídeo/imunologia , Capsídeo/isolamento & purificação , Cromatografia de Afinidade , Clonagem Molecular , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/isolamento & purificação , Piperidinas , Poliovirus/imunologia , Vacina Antipólio de Vírus Inativado , Conservantes Farmacêuticos , Piridazinas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Proteínas Estruturais Virais/isolamento & purificaçãoRESUMO
Rabbit reticulocyte lysates were programmed with either RNA extracted from purified poliovirus or a mixture of mRNAs encoding the capsid precursor, P1, and proteinase 3CD. In both cases, 14S subunits were formed at 30 degrees C and empty capsids at 37 degrees C. Both the 14S subunits and empty capsids had the expected polypeptide composition and neutralization epitopes. It is concluded that the proteinase 3CD gene is the only viral genetic information needed for the correct processing of P1 and the formation of 14S subunits, and their assembly into antigenically correct empty capsids.
Assuntos
Poliovirus/genética , Biossíntese de Proteínas , Proteínas Virais , Proteases Virais 3C , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Capsídeo/genética , Capsídeo/imunologia , Sistema Livre de Células , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Poliovirus/imunologia , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , RNA Viral/metabolismo , CoelhosRESUMO
The sequence of the poliovirus genome encoding 3CD (a protease) was transferred to the yeast Saccharomyces cerevisiae on expression vectors with either a constitutive or an inducible promoter. Transformants could only be obtained with vectors carrying the inducible transcription unit. Extracts of induced cells were able to cleave cell-free synthesized P1, the precursor of the poliovirus capsid proteins, into VP0, VP3 and VP1. In yeast cells constitutively expressing P1, induction of 3CD expression resulted in only trace amounts of processed products. Processing could be improved considerably by simultaneous induction of both P1 and 3CD expression. Analysis of extracts of such induced cells revealed the presence of particles that resembled authentic subviral particles.
Assuntos
Capsídeo/biossíntese , Cisteína Endopeptidases/genética , Poliovirus/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Virais , Proteases Virais 3C , Sequência de Bases , Capsídeo/genética , Capsídeo/isolamento & purificação , Capsídeo/metabolismo , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/metabolismo , Indução Enzimática , Expressão Gênica , Dados de Sequência Molecular , Poliovirus/genética , Vacina Antipólio de Vírus Inativado/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/biossínteseRESUMO
A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per microg of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (> or =10(8) colonies per microg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.