RESUMO
An approach for determination of cytotoxicity of chemical and biological drugs which combine the estimation of quality and quantity of cells after their treatment are proposed by the authors. The visual control of cellular minicultures permits one: 1) to register specific and pathologic alterations of cell morphology, 2) to trace their development in dynamics under the action of individual or complex drugs and 3) to choose the optimum time for the experiment fixation. The special staining of the treated cells and measurement of the optical density of absorbed histological dye permits one to make a conclusion about the changing of the cells quantity. A combined method of double estimation gives the opportunity to detect the artefacts taking place after staining the cells treated by some drugs and extracts of natural origin in high concentrations.
Assuntos
Antineoplásicos/toxicidade , Técnicas de Cultura de Células/métodos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Coloração e Rotulagem/métodos , Células Tumorais CultivadasRESUMO
Regulatory interaction of the L10 protein and translation operator of L10 mRNA was studied in vivo using a double-plasmid system. Feedback regulation of rplJ gene expression in Thermotoga maritima was proved, and the possibility of feedback cross-regulation was demonstrated in a heterologous system containing T. maritima and enterobacterial components.
Assuntos
Enterobacteriaceae/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Genes Bacterianos , Bactérias Anaeróbias Gram-Negativas/genética , Sequência de Bases , Retroalimentação , Dados de Sequência MolecularRESUMO
Comparative analysis of the structural organization of an untranslated sequence upstream from the rplJ gene in Thermotoga maritima revealed a potential binding site for the L10 ribosomal protein. The structure of the site detected is highly homologous to that of the 23S rRNA L10 target sequence. Structural organization of the potential mRNA L10 target site detected in T. Maritima is similar to that of mRNA targets of seven species of Enterobacteria and Synechocystis PCC 6803. Additional elements of structural homology between the mRNA and rRNA L10 targets in T. maritima are also shown. Location of the target site within the rplJ mRNA leader and ability of this region to form alternative conformations show that expression of the rplJ gene is autogenously controlled by the L10 ribosomal protein.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Genes Bacterianos , Bactérias Anaeróbias Gram-Negativas/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/genética , Proteína Ribossômica L10RESUMO
Construction of the recombinant plasmids, containing the antisense sequence of the E. coli rplJ gene under control of lac promoter has shown their effect on the level of the detector rplJ-lacZ gene expression.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , RNA Antissenso/genética , Óperon Lac/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genéticaAssuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Ribossômicas/genética , Salmonella typhimurium/genética , Homologia de Sequência do Ácido Nucleico , Genes Bacterianos , Dados de Sequência Molecular , Plasmídeos , Conformação Proteica , Proteína Ribossômica L10RESUMO
Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101 E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL' DNA portion and partially for rplJL' genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Klebsiella pneumoniae/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Salmonella typhimurium/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Dados de Sequência Molecular , Óperon , Plasmídeos , Proteínas Recombinantes/metabolismo , Proteína Ribossômica L10RESUMO
A recombinant phage mp9MW/rpoB containing the BglII-B fragment of the Escherichia coli rplJL-rpoBC gene cluster was constructed on the basis of filamentous phage M13. Stability of the phage was increased by insertion of a transcription terminator t beta' which blocked transcription of cloned genes from the Plac of the vector.
Assuntos
Colífagos/genética , Escherichia coli/genética , Genes Bacterianos , Recombinação Genética , Clonagem Molecular , Regulação Viral da Expressão Gênica , Genes Virais , PlasmídeosRESUMO
A mutation in lac-operator region of pUC19 plasmid causing an increase in beta-galactosidase activity was observed. The plasmid was used as a vector to provide high level of expression of the cloned E. coli rplL gene.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Plasmídeos/genética , Clonagem Molecular/métodos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/enzimologia , Vetores Genéticos , Óperon Lac , Mutação , Fenótipo , beta-Galactosidase/genéticaRESUMO
The recombinant plasmids pUC and pNM481 were constructed. The efficiency of P1ac promoter of the vector plasmid pUC and PL10. supporting the transcription of rplJL-rpoBC-operons of Escherichia coli was compared. It was found that the stronger promoter P1ac, due to convergent transcription directed by the promoter, makes possible the cloning on the multicopy plasmid pUC of the DNA fragment (the gene rplJ controlled by its own promoter PL10), coding for the synthesis of the regulatory ribosomal protein L10.
Assuntos
Escherichia coli/genética , Vetores Genéticos , Óperon , Regiões Promotoras Genéticas , Transcrição Gênica , Clonagem Molecular , Enzimas de Restrição do DNA , Desoxirribonuclease HindIII , Eletroforese em Gel de Ágar , PlasmídeosAssuntos
Escherichia coli/genética , Genes Reguladores , Proteínas Ribossômicas/genética , Transcrição Gênica , Enzimas de Restrição do DNA , Escherichia coli/metabolismo , Genes Bacterianos , Mutação , Óperon , Plasmídeos , Recombinação Genética , Proteína Ribossômica L10 , Proteínas Ribossômicas/metabolismoRESUMO
Recombinant pUC9 and pNM481 plasmids containing a 0.64 kbp PstI-fragment of the Escherichia coli rplJL operon have been constructed. The fragment was of high promoter efficiency when cloned onto pNM481.
Assuntos
Escherichia coli/genética , Óperon , Plasmídeos , Regiões Promotoras Genéticas , Clonagem MolecularRESUMO
Two BglII fragments of pJC703 cosmid were inserted into the plasmid PUC19. E. coli cells containing the recombinant PUC19/A plasmid acquired rifampicin resistance due to inserted rpoB gene but still expressed the Lac+ phenotype.
Assuntos
DNA Bacteriano/genética , DNA Recombinante , Escherichia coli/genética , Galactosidases/genética , Plasmídeos , beta-Galactosidase/genética , Mapeamento Cromossômico , Enzimas de Restrição do DNA , Escherichia coli/enzimologiaRESUMO
E. coli DNA fragment containing the rpoB gene with an rpoB3 rifampicin resistance dominant mutation (coding for the beta-subunit of RNA polymerase), genes rpI J and rpI L coding for the ribosomal proteins L7/L12 and L10, and promoters determining transcription of all these genes were cloned in M13mp8 and WB2348 filamentous phages. E. coli cells containing recombinant phages acquired resistance to rifampicin up to its 600 micrograms/ml concentration. When cloned into M13mp8 and WB2348 phages, the given fragment is oriented in such a way that the direction of the transcription initiated from its own promoter coincides with that initiated from the lac UV5 promoter. In both cases the recombinant phages have no stable rifampicin resistance which is coded by the fragment.
