RESUMEN
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.
Asunto(s)
Hemaglutininas Virales/genética , Orthomyxoviridae/metabolismo , Secuencia de Bases , Quimera , Clonación Molecular , ADN Recombinante/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mapeo RestrictivoRESUMEN
The mutation system has been suggested in an effort to test insertion and deletion mutants by changing the Lac-phenotype of bacterial colonies transformed by mutant DNA. This system also makes possible to determine heterozygotes and homozygotes among the mutants. The yield of mutants in shown to depend on the structure of the DNA heteroduplex region. The yield of deletion mutants is greater than that of insertion mutants. Heterozygotes prevail in mutant colonies (greater than 90%).
Asunto(s)
Reparación del ADN , Mutágenos , Mutación , Oligonucleótidos , Organofosfatos , Compuestos Organofosforados , Secuencia de Bases , ADN Bacteriano/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Plásmidos , Transformación BacterianaRESUMEN
A new approach is proposed to obtain the directed mutations in the gene under study. The technique is based on using alkylphosphotriester analogues of oligodeoxyribonucleotides as site-specific mutagens. The deletion C in lacZ' gene of bacteriophage M13mpB was obtained by cotransfection of Escherichia coli cells with a mix of DNA and phosphotriester analogues of oligonucleotides.
Asunto(s)
Mutación , Oligonucleótidos/genética , Secuencia de Bases , Colifagos/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Moldes Genéticos , Transfección , Transformación GenéticaRESUMEN
The high effectivity of using phosphotriester analogs of oligonucleotides for aimed mutagenesis in vitro and in vivo was shown. A general scheme, describing the mutagenic effects of phosphotriester analogs of oligonucleotides and their natural homologs, was derived by analysis of data on the structures of the obtained mutants. This scheme can serve as a foundation for selecting the structure of effective agents for aimed mutagenesis.
Asunto(s)
Mutágenos , Oligodesoxirribonucleótidos/toxicidad , Organofosfatos/toxicidad , Compuestos Organofosforados/toxicidad , Secuencia de Bases , Colifagos/genética , ADN Circular/genética , Modelos Genéticos , Pruebas de MutagenicidadRESUMEN
Nucleotide sequences of 10 mutant genes of human leukocyte interferon alpha 2 (IFN) with the use of 4 oligonucleotide primers containing ethyl substituents at phosphate groups were determined. To design primer sequences, an approach based on the local similarity profile of the IFN gene and M13mp7 vector DNA is described.
Asunto(s)
Secuencia de Bases , Interferón Tipo I/genética , Mutación , Homología de Secuencia de Ácido Nucleico , Humanos , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Clonación Molecular , Escherichia coli/genética , Galactosidasas/genética , Interferón Tipo I/genética , Biosíntesis de Proteínas , beta-Galactosidasa/genética , Regulación de la Expresión Génica , Humanos , Proteínas Recombinantes/genéticaRESUMEN
A two-step method of labelling DNA through aliphatic amino groups with various reporter molecules has been developed. The method is based on reaction of cytosine-residues of polynucleotide chain with 4-aminooxybutylamine; the latter's synthesis is described. DNA modified on this way with fluorescein isothiocyanate functions a probe in molecular hybridisation. The sensitivity of this method for fluorochrome-labelled DNA probes in the dot hybridisation procedure is about 200 pg per mm2.
Asunto(s)
Butilaminas , ADN/análisis , Colorantes Fluorescentes , Hibridación de Ácido Nucleico , Fenómenos Químicos , QuímicaRESUMEN
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.
Asunto(s)
Colifagos/genética , Mutación , Oligodesoxirribonucleótidos/genética , Organofosfatos , Compuestos Organofosforados , ADN Viral/genética , ADN Viral/metabolismo , Oligodesoxirribonucleótidos/síntesis químicaRESUMEN
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.
Asunto(s)
Mutación , Oligodesoxirribonucleótidos/genética , Secuencia de Bases , Oligodesoxirribonucleótidos/síntesis química , Organofosfatos/síntesis química , Moldes Genéticos , Transformación BacterianaRESUMEN
Alkyl triester analogues of oligodeoxynucleotides were synthesized: [Tp(CH3)]8T(Ac), [Tp(C2H5)]8T, [dGp(C2H5)]2G, [dAp(C2H5)]2A. Their binding to complementary polyribo-and polydeoxyribonucleotides was studied by UV-spectroscopy and gel-filtration. The DNA complexes of analogues were shown to be more thermostable than the RNA ones independent on the nucleic base nature and alkyl residue size of the analogue used. Mg-salt increased the thermostability of the oligonucleotide analogue--RNA complexes.