RESUMO
Plasmid-based promoter-probe vectors pPV4 and pPV5 have been constructed which are useful for comparing the relative efficiencies of bacterial promoters. The vectors utilize the beta-galactosidase (lacZ) gene of E. coli as an indicator gene. The latter was modified using synthetic DNA fragments. The promotor-probe system contains the ampicillin resistance gene and the origin of replication of plasmid pBR322. The plasmids pPV4 and pPV5 carry clustered unique restriction sites usable for promoter insertions, and SD sequence. A synthetic DNA fragment corresponding to transcription terminator was inserted downstream the lacZ gene. Presence of the terminator made it possible to clone strong promoters controlling transcription of the lacZ gene. To prevent any undesired promotor effect, the plasmid pPV5 has also second synthetic terminator upstream from the polylinker sequence. Using this promoter-probe system, relative efficiencies of a series of synthetic promoters, including PL promoter of phage lambda and its mutant, gene X promotor of phage fd and several model statistic promoters, have been compared.
Assuntos
Escherichia coli/genética , Galactosidases/genética , Genes Bacterianos , Vetores Genéticos , Regiões Promotoras Genéticas , beta-Galactosidase/genética , Resistência a Ampicilina/genética , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica , Dados de Sequência Molecular , Plasmídeos , Transcrição GênicaRESUMO
With the use of synthetic DNA fragments, a set of new plasmid vectors has been obtained. The vectors provided high level expression of peptides and small proteins in E. coli as fusions with fragments of beta-galactosidase of various length. These vectors were used to achieve expression of a synthetic gene for a functionally active fragment of bacteriorhodopsin. The yields of hybrid proteins consisting of beta-galactosidase and bacteriorhodopsin fragments were in the range of 5-30% from the total amount of cellular protein.
Assuntos
Escherichia coli/genética , Galactosidases/genética , Genes Sintéticos , Vetores Genéticos , Plasmídeos , beta-Galactosidase/genética , Bacteriorodopsinas/biossíntese , Bacteriorodopsinas/genética , Sequência de Bases , Enzimas de Restrição do DNA , DNA Bacteriano/genética , Regulação da Expressão Gênica , Genes BacterianosRESUMO
A useful and efficient approach to the synthesis of DNA duplexes of practically unlimited length has been developed. The proposed methodology is based on the use of temporary restriction sites for subcloning and assembling the segments of the desired DNA. It allows the utilization of chemically synthesized oligonucleotides of various length (from 10- to 100-mers) for the duplex construction. The application of this approach to the synthesis of a gene for the functionally active bacteriorhodopsin fragment is described.
Assuntos
DNA/síntese química , Genes Sintéticos , Engenharia Genética/métodos , Bacteriorodopsinas/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Enzimas de Restrição do DNA , DNA Bacteriano/síntese química , Escherichia coli/genética , Vetores Genéticos , Plasmídeos , Polinucleotídeos/síntese químicaRESUMO
A procedure has been designed for changing specific nucleotides in a DNA sequence with efficiency. The method involves the use of the specially constructed cloning vectors pBRS1, pHS1, and pHS2. These plasmids are derivatives of pBR322 in which the EcoRI-HindIII region has been replaced by synthetic duplexes carrying SmaI, HpaI and XhoI sites, in addition to EcoRI and HindIII sites. The DNA fragment to be mutagenized is cloned in pHS1 (or pBRS1, or pHS2) using restriction sites close to the SmaI and HpaI sites. The recombinant plasmid obtained is digested with one of these enzymes to produce double-stranded DNA with blunt ends. This linear DNA is a substrate for exonuclease III (or T4 DNA polymerase). The digestion under controlled conditions produces duplex with protruding single-stranded 5'-regions which include the site of the desired mutation. The synthesis of DNA by DNA-polymerase I (Klenow's fragment), primed in part by the synthetic oligonucleotide containing the desired mutation, leads to the linear heteroduplex. The closed circular heteroduplex is formed by ligation. After transformation into E. coli, DNA replication generates homoduplexes, some of which contain the mutation. Colony hybridization with the same 32P-labeled oligonucleotide is used to select mutants. The yield of the mutants is 10-20%. This technique can be extended to replicative form of M13 vectors. It can be also applied to any DNA sequence which has a unique site of restriction endonuclease generating blunt ends.
Assuntos
DNA/genética , Mutação , Oligodesoxirribonucleotídeos/genética , Oligonucleotídeos/genética , Clonagem Molecular , Replicação do DNA , Enzimas de Restrição do DNA/genética , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , Escherichia coli/genética , Ácidos Nucleicos Heteroduplexes/biossíntese , PlasmídeosRESUMO
A new modification of the oligonucleotide-mediated mutagenesis technique has been developed. The proposed methodology has been used to produce specific base changes in the double-stranded plasmid DNA. For this purpose, special cloning vectors have been constructed using the synthetic oligodeoxyribonucleotides. The developed method allows the production of mutant DNA from those of the wild-type with a yield of 10-20%.
