RESUMO
A new approach to create chimeric genes by directed exchange of oligonucleotide fragments was developed. By oligonucleotide-directed mutagenesis a few deletion mutants of the influenza virus hemagglutinin (HA) gene were obtained. These variants of HA gene contain unique restriction sites in DNA regions coding for the A and B epitopes of the HA molecule. The obtained special vectors may be used for cloning DNA fragments coding for new amino acid sequences in internal sites of the HA gene.
Assuntos
Hemaglutininas Virais/genética , Orthomyxoviridae/metabolismo , Sequência de Bases , Quimera , Clonagem Molecular , DNA Recombinante/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mapeamento por RestriçãoAssuntos
Regulação Viral da Expressão Gênica/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Imunização/métodos , Precursores de Proteínas/genética , Vacinas Sintéticas/genética , Vaccinia virus/genética , Proteínas do Envelope Viral/genética , Vacinas Virais/genética , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Regulação Viral da Expressão Gênica/imunologia , Genes Virais/genética , Antígenos de Superfície da Hepatite B/imunologia , Imunidade Celular/genética , Imunidade Celular/imunologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Precursores de Proteínas/imunologia , Coelhos , Vacinas Sintéticas/imunologia , Vaccinia virus/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologiaRESUMO
The full-length copy of the hemagglutinin gene of influenza virus was inserted into M13 phage DNA. The DNA sequence coding for the hydrophobic prepeptide was removed from the gene by oligonucleotide-directed mutagenesis. The possibilities of expression of the full-length and mutant genes in E. coli were investigated. The beta-galactosidase-hemagglutinin fusion proteins were isolated. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess of viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.
Assuntos
Escherichia coli/genética , Regulação da Expressão Gênica , Hemaglutininas Virais/genética , Vírus da Influenza A/genética , Bacteriófagos/genética , Clonagem Molecular , DNA/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Genes Virais , Plasmídeos , Radioimunoensaio , Mapeamento por Restrição , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/isolamento & purificaçãoRESUMO
Genes for leucocyte interferon and alpha-donor of galactosidase were fused by deletion mutagenesis or by site-directed mutagenesis. In both cases the fused protein was expressed. The protein having an antiviral activity of leucocyte interferon was easily detected in bacteria and solutions by the reaction of beta-galactosidase alpha-complementation and retained the antigenic determinants of interferon and beta-galactosidase. The use of fused proteins for optimization of gene expression and for the analysis of interferon structure-function relationship is discussed.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Galactosidases/genética , Interferon Tipo I/genética , Biossíntese de Proteínas , beta-Galactosidase/genética , Regulação da Expressão Gênica , Humanos , Proteínas Recombinantes/genéticaRESUMO
The possibility to use the E. coli intact DNA polymerase I in the oligonucleotide-directed site-specific mutagenesis of DNA has been studied. Optimal conditions of the extension activity of this enzyme were found. We have shown that the substitution of the Klenow fragment of the E. coli DNA polymerase by the intact DNA polymerase I did not decrease the efficiency and fidelity of the oligonucleotide-directed mutagenesis.
Assuntos
DNA Polimerase I/genética , DNA Bacteriano/biossíntese , Escherichia coli/enzimologia , Mutação , Oligonucleotídeos/genética , DNA Bacteriano/genética , Escherichia coli/genética , Cinética , Conformação de Ácido Nucleico , Conformação ProteicaRESUMO
A model system is developed to test oligonucleotide-directed mutations: T----C transition, T and C deletions (delta T and delta C), C insertion, double mutations (A----G, delta T), (T----C, A----G), and large oligonucleotide deletions (36 or 44 nucleotides). The system includes 9 variants of the phage M13 DNA carrying fragment of beta-galactosidase gene, and oligodeoxyribonucleotides partially noncomplementary to DNA sequence of this gene. Six variants are obtained by the site-localized mutagenesis, the other were described earlier. Induced mutations are easily tested by phenotype change of transformed bacteria (Lac+----Lac-); by formation or loss of the sites for BamHI and EcoRI restrictases; by DNA hybridization with 32P-labeled oligonucleotides; and by DNA sequencing by the Sanger method. The system is used to study the role of some factors, such as completeness of RF DNA synthesis, thermal stability of the oligonucleotide: DNA complex, quality of enzymes and substrates used in polymerase reaction, mutation type or the efficiency of mutagenesis. A number of unexpected mutations were observed in the course of oligonucleotide-directed mutagenesis. Lower yields of some mutants induced by oligonucleotides are shown to be due to the action of repair systems of bacteria.
Assuntos
Colífagos/genética , DNA Viral/genética , Galactosidases/genética , Genes Virais , Mutação , beta-Galactosidase/genética , Colífagos/enzimologia , Enzimas de Restrição do DNA , Escherichia coli/genética , Óperon Lac , Mutagênicos , Oligodesoxirribonucleotídeos/biossíntese , Oligodesoxirribonucleotídeos/genéticaRESUMO
17- and 20-mer oligodeoxyribonucleotides and their analogues, containing one to four phosphate groups esterified with ethyl alcohol in different positions of oligonucleotide chain, were synthesized by modified triester method. Ethylated di- and trinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. The structures of the oligonucleotides were confirmed by Maxam-Gilbert sequencing method. Oligonucleotides were not totally complementary to the N-terminal region of lac Z'gene (coding for N-terminal fragment of beta-galactosidase) of phage M13mpB DNA and induced the formation of the proposed deletion mutant DNA M13mp1 delta T. Phosphotriester analogues were more effective mutagens as compared to phosphodiester oligonucleotides due to their stability to nucleases. The use of E. coli DNA-polymerase I provided the increase in the mutant yields in case of the phosphotriester analogues. The stability of the analogues to 5'----3'----5'-endonuclease action, the specificity of oligonucleotide: DNA binding and the structure of mutant DNA were studied by the Sanger sequencing method.
Assuntos
Colífagos/genética , Mutação , Oligodesoxirribonucleotídeos/genética , Organofosfatos , Compostos Organofosforados , DNA Viral/genética , DNA Viral/metabolismo , Oligodesoxirribonucleotídeos/síntese químicaRESUMO
20-mer oligodeoxyribonucleotides d-ACGACGG (R') CCAG (R'') TGATCCGTA, where R' = R'' = H (20), F' = Et, R'' = H (20-Et), or R' = R'' = Et (20-Et2) were synthesized by modified triester method. Ethylated dinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. Structures of oligonucleotides were confirmed by Maxam - Gilbert method. Mutagenesis induced by oligonucleotides was studied on DNA of M13mpB phage. Oligonucleotides were not totally complementary to this DNA in the region of 4-11 codons of Z'-gene. They all were shown to direct the formation of the designed deletion mutants, phosphotriester analogues (20-Et) and (20-Et2) being more effective mutagens. The specificity of oligonucleotides: DNA binding and mutant DNA structure were shown by Sanger method.
Assuntos
Mutação , Oligodesoxirribonucleotídeos/genética , Sequência de Bases , Oligodesoxirribonucleotídeos/síntese química , Organofosfatos/síntese química , Moldes Genéticos , Transformação BacterianaRESUMO
Specific modification of promoter regions of DNA has been studied. Plasmid pK56B1 DNA has been used as a model to test RNA-polymerase binding with DNA under various conditions. RNA-polymerase is shown to form specific complexes with DNA which are stable in solutions with a moderate ionic strength (0.1-0.2 M NaCl), under pH 5-8 in the presence of 0.5 M O-methylhydroxylamine of O-delta-aminooxybutylhydroxylamine. Escherichia coli JM103 cells have been transfected with DNAs treated with 0.5 M O-methylhydroxylamine at 37 degrees C, pH 5.2. The inactivation effects of the mutagen on single-stranded DNA of bacteriophage M13 m p1, double-stranded form of this bacteriophage (replicative form-RF) and on the complex of RNA-polymerase with RF DNA have been compared. The obtained data confirmed the specificity of reagent action with DNA sites binding with the enzyme. Selectivity of promoters modification has been confirmed also by the analysis of M13 m p1 DNA mutations induced in lacZ' gene by delta-aminooxybutylhydroxylamine effect on the DNA complex with DNA-polymerase.
Assuntos
DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Sítios de Ligação , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , PlasmídeosRESUMO
An efficient method to obtain the mutant genes for human leucocyte alpha 2-interferon (IFN) has been elaborated. The technique includes the following main stages: cloning of interferon gene in M13mp8 DNA; isolation of double-stranded hybrid DNA complex, containing IFN gene as a single-stranded fragment; selective modification of a single-stranded hybrid DNA by sodium bisulphite; the repair of hybrid DNA by DNA polymerase I from Escherichia coli, transformation of Escherichia coli JN103 cells by double-stranded circular DNA, containing the selectively modified gene IFN. The technique is based on the protection of bacteriophage M13 genome from mutagen induced damage by means of converting phage DNA into the double-stranded structure leaving the single-stranded fragment to be mutagenized prone to mutagen action. This is achieved by reannealing of single-stranded M13mpB DNA hydrolyzed by restriction endonuclease BamHI. The technique preserves the infectiousness of vector DNA under the conditions permitting modification of up to 10% cytosine residues in IFN gene. Every clone resulting from transformation of Escherichia coli by modified DNA carried mutations in IFN gene, identified by sequencing after Sanger.
Assuntos
Clonagem Molecular , Genes , Interferon Tipo I/genética , Mutação , Sequência de Bases , DNA de Cadeia Simples/genética , Humanos , Interferon Tipo I/análiseAssuntos
Deleção Cromossômica , Elementos de DNA Transponíveis/efeitos dos fármacos , Galactosidases/genética , Mutagênicos/farmacologia , Mutação , Oligonucleotídeos/farmacologia , Translocação Genética/efeitos dos fármacos , beta-Galactosidase/genética , Sequência de Bases/efeitos dos fármacos , Colífagos/genética , DNA Viral/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Genes Virais/efeitos dos fármacos , Ácidos Nucleicos Heteroduplexes/genéticaRESUMO
The inactivation and the mutagenesis of lambda phage Cl 857 virR by O-methylhydroxylamine (OMHA) and O-delta-aminooxybuthylhydroxylamine (delta-HA) were studied. The inactivation of OMHA-treated phage was shown to be stronger in E. coli polA cells defective in DNA-polymerase I as compared to wild-type host E. coli W3350. In contrast delta-HA caused similar phage inactivation in these two strains. Wave-type kinetics of the inactivation and the mutagenesis of phage by OMHA and delta-HA was observed. delta-HA appeared to be a more effective mutagen than OMHA: it induced higher mutant yield at a given level of inactivation.
