RESUMO
A synthetic gene for a proteinase inhibitor (eglin C) that was obtained by direct amplification with oligonucleotides without using DNA ligase and polynucleotide kinase of T4 phage was cloned into expression vectors. A high yield of the polypeptide (110-130 mg/l) was attained in E. coli strains.
Assuntos
DNA Ligases/metabolismo , Escherichia coli/genética , Genes Sintéticos , Serpinas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Amplificação de Genes , Vetores Genéticos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , ProteínasRESUMO
The gene of human mutant (serine-17) fibroblast interferon was isolated with the use of highly efficient oligonucleotide-directed mutagenesis. On the basis of the constructed expression plasmid pPR-IFN Ser17 a strain producing human mutant beta-interferon (VKPM V-4678) was developed. It was shown that the specific activity of the human mutant (serine-17) fibroblast interferon was 1 order of magnitude higher than that of the recombinant interferon which reaches the specific activity of natural fibroblast interferon.
Assuntos
Biotecnologia/métodos , Escherichia coli , Escherichia coli/metabolismo , Interferon beta/biossíntese , Mutagênese Sítio-Dirigida/genética , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Técnicas In Vitro , Interferon beta-1a , Interferon beta-1b , Interferon beta/genética , Interferon beta/isolamento & purificaçãoRESUMO
A procedure for extensive deletion mutagenesis of DNA using the uracil repair system is exemplified by reconstruction of the pBR322 replication regulatory region cloned into M13tg131. By means of an oligonucleotide primer the 116-nucleotide fragment was excised and four nucleotides were introduced to form a BglII restriction site. Use of the uracil repair selection provided a 30-fold increase in the deletion mutagenesis efficiency.
Assuntos
Deleção Cromossômica , Reparo do DNA , Uracila , Sequência de Bases , Elementos de DNA Transponíveis , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/metabolismo , Eletroforese em Gel de Ágar , Escherichia coli/enzimologia , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
Site-directed multiple mutagenesis to obtain hybrids of homologous proteins was carried out by means of the oligonucleotide-targeting digestion of ss DNA. The procedure is more convenient, rapid and simple than the step-by-step approach. To demonstrate different approaches to targeting digestion of ss DNA, NcoI, HinfI, FokI endonucleases were used for DNA cleavage. A hydride of the human and porcine IFN-alpha-2-genes has been constructed.
Assuntos
DNA de Cadeia Simples/genética , Mutagênese Sítio-Dirigida , Animais , Sequência de Bases , Enzimas de Restrição do DNA , DNA Bacteriano/genética , Eletroforese em Gel de Ágar , Escherichia coli/metabolismo , Humanos , Interferon Tipo I/genética , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , SuínosRESUMO
The mutant forms of human IFN-alpha 2 gene are obtained by oligonucleotide-directed mutagenesis with the use of uracil-repair system. To intensify the process the procedure of the uracil-containing DNA template preparation is modified. It was determined that when mutagenesis is performed in the uracil-repair system the yield of the process depends on the mutant DNA-strand in vitro synthesis efficiency. It is shown that the stability of the 5'-end primer-template complex and the level of the endogenic primers elongation are the basis factors, that determine induction mutations.
Assuntos
Reparo do DNA , Interferon Tipo I/biossíntese , Uracila/metabolismo , Animais , Sequência de Bases , Colífagos/genética , DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Genes Bacterianos , Genes Virais , Humanos , Interferon Tipo I/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes , Suínos , Moldes GenéticosRESUMO
In order to elucidate the function of the IS1 insA gene derivatives of plasmid pUC19::Tn9' with insertions of synthetic oligonucleotides were obtained. The latter are equal or multiple of 9 b.p. in length and are located in the Pst1 site within each of the two IS1 copies of the Tn9' transposon. The insertions of the nine base oligonucleotides code for the neutral amino acids and do not shift the reading frame. One of the mutant transposon obtained - Tn9'/X was studied on the ability to form simple insertions and plasmid cointegrates. For this purpose the pUC19 derivatives carrying the wild type and mutant transposon were mobilized by conjugative plasmid pRP3.1. It was found that the damage of the insA gene does not influence the ability of transposon to form simple insertions and plasmid cointegrates in both recA - and rec+ cells of E. coli. However, the frequency of the cointegrate formation in the subsequent transposition of the mutant transposon from pRP3.1::Tn9'/X to pBR322 was by 10-20 times lower in comparison to the wild type transposon. Instable (dissociating) Tn9'/X-mediated plasmid cointegrates formed by interaction pUC19::Tn9'/X and pRP3.1 were obtained. It was shown that in the E. coli recA-cells such cointegrates dissociate, as a rule, "correctly", i.e. they segregate mainly plasmids of types pUC19::Tn9'/X and pUC19::IS1/X. The data obtained correspond with the notion that the gene insA product is not essential for transposition, but is, possibly, involved in the formation of the IS1-generated deletions.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Escherichia coli , Fases de Leitura Aberta , Sequência de Bases , DNA Bacteriano/genética , Eletroforese em Gel de Ágar , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos , Proteínas Repressoras/genética , Mapeamento por RestriçãoRESUMO
The DNA fragment identical to the right shoulder of the inverted repeat from the par-region of ColE1 plasmid has been synthesized chemically. It is shown to participate in the plasmid multimers resolution and to define the stable inheritance of the plasmid pKS1 containing the fragment in Escherichia coli C600 cells as well as in the multirecombinogenic strain Escherichia coli JC8679. The efficiency of the fragments functioning in Escherichia coli JC8679 is not enough for resolution of all forms of oligomeric pKS1 DNA. The site for recombinase action is found to be located in the synthesized oligonucleotide. However, some extra sequences of DNA located within the region of inverted repeat are necessary for maximally efficient functioning of the recombinase, the enzyme participating in plasmid multimers resolution.
