Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/isolamento & purificação , Anticorpos Monoclonais , Chlorella/virologia , Cromatografia de Afinidade/métodos , Desoxirribonucleases de Sítio Específico do Tipo II/imunologia , Guanidina/farmacologia , Imunoadsorventes/química , Phycodnaviridae/enzimologia , Sefarose/análogos & derivados , Sefarose/química , Ureia/farmacologiaRESUMO
In order to make high-copy-number multimers of DNA fragments in a tandem unit, two different gene amplification vectors (pSK9 and pBBS1) were developed. Two identical class-IIS restriction enzyme sites (BspMI for pSK9 and BbsI for pBBSI) were inversely oriented in each vector with the same cut site, creating asymmetric and complementary cohesive ends (5'-CCCC and 5'-GGGG). Multimers were made by: (i) cloning a target DNA into the class-IIS restriction enzyme cut site of each vector; (ii) excision of the monomeric insert by digestion with the class-IIS restriction enzyme; (iii) isolation of the fragments; (iv) self-ligation of the fragments; (v) cloning into the original vector digested with the class-IIS restriction enzyme; and (vi) repeating steps (i) through (v) to generate higher-order multimers. Various-sized multimers of a 93-bp DNA fragment encoding magainin, an antimicrobial peptide, were obtained with the gene amplification vector, pBBS1. Larger multimers, up to about 108 copies, were constructed from the monomer by the sequential amplification procedure. Of six different Escherichia coli hosts examined for the stability of multimers, the multimers were the most stable in E. coli D1210. The gene amplification vector system described here is very efficient and can be applied in the construction of tandem multimers of any kind of DNA, as long as the cloned DNA does not contain the cut site of the class-IIS restriction enzyme to be utilized.
Assuntos
DNA Recombinante/genética , Amplificação de Genes , Vetores Genéticos/genética , Antibacterianos , Anti-Infecciosos , Clonagem Molecular , Desoxirribonucleases de Sítio Específico do Tipo II , Escherichia coli/genética , Peptídeos/genética , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
Three proteins, yeast transcription regulatory protein GCN4, M.FokI DNA methyltransferase and R.FokI restriction endonuclease (ENase) were used to attain specific cleavage of DNA at the 18-20-bp GCN4 recognition site. This is a novel version of the 'Achilles' heel cleavage' (AC) technique [Koob et al., Science 241 (1988) 1084-1086]. Since the method employs a class-IIS ENase (R.FokI), which cleaves the DNA outside of its recognition sequence, it leaves the overlapping GCN4-binding intact. Thus, the same GCN4 site can be used in consecutive cleavage reactions. This novel GCN4-IIS-AC technique was applied to study the protein-DNA interaction. Quantitative analysis of the effect of temperature, reaction time, and GCN4 and M.FokI concentrations allowed determination of the GCN4-DNA complex half-life, which was found to be 7 h at 30 degrees C, 18 h at 22 degrees C and over 24 h at 10 degrees C. In addition, conditions for controlled, partial GCN4-IIS-AC digestion of DNA were determined, and applied to the physical mapping of large genomes.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Bases , Sítios de Ligação , DNA Recombinante/metabolismo , Eletroforese em Gel de Campo Pulsado , Meia-Vida , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
The restriction endonuclease AvcI, an isoschizomer of Sau96I [Sussenbach et al., Nucleic Acids Res. 5 (1978) 1153-1163], was purified from Actinomyces cristalomycini. AvcI recognizes a 5-bp palindromic sequence, 5'-G decreases GNCC and cleaves it after the first G residue producing a 3-nucleotide 5'-overhang.
Assuntos
Actinomyces/enzimologia , Desoxirribonucleases de Sítio Específico do Tipo II/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Cromatografia de Afinidade , Cromatografia por Troca Iônica , DNA/química , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/isolamento & purificação , Oligodesoxirribonucleotídeos/metabolismo , Especificidade por SubstratoRESUMO
SacNI, an isoschizomer of the restriction endonuclease, BanII [Sugisaki et al., Nucleic Acids Res. 10 (1982) 5747-5752], has been isolated from Streptomyces achromogenes N-J-H. SacNI recognizes the palindromic sequence, 5'-GRGCY/C, and cleaves within the recognition sequence, generating a 3' protruding RGCY end (where R = A or G, and Y = C or G).