RESUMEN
Bacteriophage MS2 was employed for targeted delivery of an apoptosis-inducing agent, Tl+, into a tumor tissue. The targeted delivery was ensured by iRGD peptide, a ligand of integrins presumably located on the surface of endotheliocytes of the tumor tissue neovasculature and certain tumor cells. The synthesized peptide was conjugated to MS2 capsid proteins. Tl+ ions from TlNO3 penetrated the phage particles and tightly bound to phage RNA. Peptide-modified MS2 preparations filled with Tl+ caused cell death in two types of cultivated human breast cancer cells and effected necrosis of these tumor xenografts in mice. Neither peptide-conjugated bacteriophage MS2 without Tl+ nor the phage filled with Tl+ but without the peptide or the same phage with the non-conjugated peptide in solution produced such effects. The preparation exhibited no acute toxicity at a therapeutic dose.
RESUMEN
An Escherichia coli strain producing transposase of a repeated sequence of Bordetella pertussis chromosome (RSBP) was constructed. A defective MGE-helper plasmid method, which allowed the determination of transposase functional activity was developed. It was shown that transposase synthesized in E. coli cells ensures transposition of "defective" RSBP into the host chromosome. Overexpression of transposase was shown to markedly decrease the vital activity of E. coli cells under selective cultivation conditions. Reasons for a decrease in viability transposase-producing cells are discussed. Results showing the impact of transposase on replication of recombinant plasmids and E. coli cell division were obtained.
Asunto(s)
Bordetella pertussis/genética , Escherichia coli/enzimología , Secuencias Repetitivas Esparcidas , Transposasas/metabolismo , Cromosomas Bacterianos , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transposasas/genéticaRESUMEN
A computer-aided analysis of the repeating sequence of Bordetella pertussis chromosome (RSBP3) revealed 3 open reading frames, one of whose (ORF1) can code a protein whose structure and properties are similar to those of transposasas, i.e. enzymes in charges for the traveling of migrating genetic elements of pro- and eukaryote. Mutants of the RSBP3 insertion sequence with the affected and unaffected ORF1 sequence were constructed in order to substantiate the above assumption. Two independent experimental models (formation of inter-plasmid co-integrates and of co-integrates between plasmid and E. coli chromosome) were used to show that the RSBP3-stimulated formation of co-integrates is only true for plasmids containing RSBP3 with the unaffected ORF1 sequence. An activity of the Hpr protein (a component of the phosphoenolpyruvate-dependent phosphotransferase) was proven to influence the formation process of inter-plasmid co-integrates.
Asunto(s)
Bordetella pertussis/genética , Sistemas de Lectura Abierta , Transposasas/genética , Proteínas Bacterianas/fisiología , Bordetella pertussis/enzimología , Cromosomas Bacterianos , Computadores , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/fisiología , Plásmidos , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Strain chi6007 obtained from the parent E. coli strain chi5097 is a result of ptsH5 mutation, which allowed cells to grow without common components of the phosphoenolpyruvate-dependent phosphotransferase system. Segregants of strain chi6007 retaining the Pol+ gene responsible for inability to grow at 37 degrees C, but gaining rifampicin resistance (RifR) were used for cloning of cointegrate plasmids. Pre-integration complexes of HIV-1 were co-integrated with the pBR-322 plasmid and transformed strain chi6018. Sequencing showed that the pPIC91 hybrid plasmid contains full-length genome of HIV-1 with shortened 5-terminal LTR and full-length copy of pBR322. Elimination of the pPIC91 plasmid from chi6018 cells was followed by the appearance of auxotrophic insertion mutants. Sequencing of the insert region showed that chromosome DNA of the host cell includes integrated genomes of pBR-322 and HIV-1.
Asunto(s)
Escherichia coli/genética , Escherichia coli/virología , Genoma Viral , VIH-1/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Recombinante/genética , Escherichia coli/efectos de los fármacos , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Rifampin/farmacologíaAsunto(s)
Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Secuencia de Aminoácidos , Antivirales/farmacología , Secuencia de Bases , Línea Celular Tumoral , Genoma Viral , Hepacivirus/inmunología , Humanos , Conformación de Ácido Nucleico , Oligonucleótidos/farmacología , Plásmidos/genética , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Two fragments (1.9 and 1.8 kb) including, respectively, "structural" genes (A, B, and lysis polypeptide) and "replicase" gene, were obtained by reverse transcription PCR on the template of bacteriophage MS2 genome RNA. The fragments contained a common sequence which allowed the assembly of the entire functional genome sDNA. 1.8 kb fragment was subjected to two independent modifications: 1) so that the replicase gene could be replaced by other sequences no more than 1.5 kb in size and 2) it was inserted (on an artifician Tn-like structure) into E. coli chromosome. A system for replication of MS2-like corpuscles was thus created, containing a "shortened" genome and having extra genes. For extra genes, aminoglycoside-3'-phosphotransferase (kanamycin resistance) or a sequence of antiviral genetic program directed against hepatitis C virus were used.
Asunto(s)
Levivirus/genética , ARN Viral , Transducción Genética , Virión/genética , Secuencia de Bases , Clonación Molecular , Elementos Transponibles de ADN , ADN Viral/genética , Ácido Hipocloroso/farmacología , Levivirus/efectos de los fármacos , Datos de Secuencia Molecular , Virión/patogenicidadAsunto(s)
Hepacivirus/efectos de los fármacos , Vacunas de ADN/farmacología , Vacunas Virales/farmacología , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Hepacivirus/inmunología , ARN Mensajero/genética , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas de ADN/genética , Vacunas Virales/genéticaRESUMEN
Integration of a plasmid carrying the TnBP3 transposon of Bordetella pertussis into the chromosome of Escherichia coli and transpositions of the integrated structure within a chromosome in the wild-type and mutant cells ptsH devoid of the major Hpr protein of the phosphoenolpyruvate-dependent phosphotransferase system were studied. When transposed to a new chromosome site, the integrated structure was precisely (or almost precisely) excised from the metY gene sequence, which resulted in restoration of the Met+ phenotype. The integration and transposition events were only observed in the E. coli cells carrying the ptsH+ allele. The ptsH mutations inhibited integration and intramolecular transposition, which were restored after phenotypic or genetic suppression of the ptsH mutation. The intensity of the processes studied were suggested to depend on the integrity of a chain that ensures transferring of the phosphoryl residue by proteins of the phosphotransferase system in E. coli K12. The results obtained indicate that the ptsH mutants of E. coli can serve as the optimal host for cloning of fragments carrying repeated sequences of B. pertussis, which may apply to the repeated sequences of other microorganisms.
Asunto(s)
Proteínas Bacterianas , Bordetella pertussis/genética , Elementos Transponibles de ADN/genética , Escherichia coli/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Clonación Molecular/métodos , Regulación Bacteriana de la Expresión Génica , Mutación , Plásmidos/genética , Secuencias Repetidas en TándemRESUMEN
Transposition of Bordetella pertussis transposon in E. coli chromosome has been studied on a model of exclusion of donor multicopy pKK3 plasmid with coumermicin. TnBP3 induced the formation of co-integrates between the plasmid and chromosome. The structure of co-integrate was determined. Facts of exclusion of integrated structure and transposon transposition within integrated plasmid into new sites on a recipient chromosome were detected. Relationship between these processes and activity of bacterial cell recombination system has been determined.
Asunto(s)
Bordetella pertussis/genética , Cromosomas Bacterianos/genética , Elementos Transponibles de ADN , Escherichia coli/genética , Plásmidos , Secuencia de Bases , Cartilla de ADN , Farmacorresistencia Microbiana/genética , Marcadores Genéticos , Transducción GenéticaRESUMEN
B. pertussis genetically mobile element TnBp3 integrates the plasmid in E. coli chromosome. During culturing under nonselective conditions the majority of cells of some E. coli strains lose the kanamycin resistance marker, which indicates the instability of TnBp3 inheriting. The stability of inheriting the integrated structure is higher in E. coli cells with recB-21 recC-12 sbcB-2 mutations. The role of RecBC recombination system in extrusion of TnBp3 is discussed.
Asunto(s)
Bordetella pertussis/genética , Cromosomas Bacterianos , Elementos Transponibles de ADN , Escherichia coli/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Microbiana , Kanamicina/farmacología , Mutación , Plásmidos , Reacción en Cadena de la PolimerasaAsunto(s)
Antibacterianos/farmacología , Difteria/tratamiento farmacológico , Factor de Crecimiento Epidérmico/genética , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusión/farmacología , Animales , Antibacterianos/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Cobayas , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/efectos de los fármacos , Piel/microbiologíaRESUMEN
The DNA fragment, coding a part of the protein molecule--the precursor of the epidermal growth factor (pre-EGF76-208)--and containing the sequence of 133 N-end amino acid residues, was obtained with the use of gene engineering and molecular biological techniques. For this purpose a fraction of poly(A+) = RNA was isolated from the kidney of a newborn infant; on this fraction the "library" of cDNA fragments whose coding capacity corresponded to the required sequence of mRNA in the pre-EGF gene was obtained in the reaction of reverse transcription, conjugated with the polymerase chain reaction (PCR). After the determination of the nucleotide sequences in 11 fragments only 1 fragment which contained practically no mutations appearing in PCR as the result of amplifications was chosen. This fragment was used for the construction of a hybrid plasmid controlling the synthesis of hybrid fusion protein with the sequence of pre-EGF76-208. Fusion protein was synthesized with very low effectiveness, but the use of metallo-affinity chromatography permitted to isolate and purify it, its purity reaching 98%. Then its antitoxic activity was determined in the skin test on guinea pigs and found to be not less then 10(6) I.U./mg of protein.
Asunto(s)
Difteria/prevención & control , Factor de Crecimiento Epidérmico/inmunología , Fragmentos de Péptidos/inmunología , Precursores de Proteínas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Secuencia de Bases , Cromatografía de Afinidad , Cartilla de ADN , Toxoide Diftérico/inmunología , Evaluación Preclínica de Medicamentos , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/aislamiento & purificación , Escherichia coli/genética , Cobayas , Humanos , Recién Nacido , Riñón , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Ingeniería de Proteínas/métodos , Precursores de Proteínas/genética , Precursores de Proteínas/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Vacunas de ADN/inmunologíaRESUMEN
To study the role of cAMP in the virulence of S. typhimurium, cAMP-producing plasmid pTG 4 was transferred to cAMP-deficient S. typhimurium mutant. The transfer of the plasmid enhanced the virulence of the microorganisms due to the increased destruction of macrophages and the intensified multiplication of salmonellae in the spleen of mice.
Asunto(s)
AMP Cíclico/fisiología , Salmonella typhimurium/patogenicidad , Animales , Macrófagos/fisiología , Ratones , VirulenciaAsunto(s)
Escherichia coli/genética , Genes Bacterianos/efectos de los fármacos , Penicilina G/farmacología , Bacteriófago lambda/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Genes Bacterianos/efectos de la radiación , Genotipo , Rayos Ultravioleta , Activación Viral/efectos de los fármacosAsunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Genes Reguladores , Rec A Recombinasas/genética , Mapeo Cromosómico , ADN Bacteriano/genética , ADN de Cadena Simple/genética , Escherichia coli/efectos de los fármacos , Regulación de la Expresión Génica , Genes Bacterianos/efectos de los fármacos , Genes Reguladores/efectos de los fármacos , Conformación de Ácido Nucleico , Recombinación Genética , Transcripción Genética , Rayos UltravioletaRESUMEN
Penicillin has been found to have a bacteriostatic effect on logarithmically growing E. coli K-12 cells. The capacity for mitosis is restored due to the aggregation of cells. The bacteriostatic effect of this antibiotic is not linked with morphological changes in the cells.