RESUMO
We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage lambda pL promoter. Derepression of pL leads to a high frequency of mini-Mu transposition (5.6 X 10(-2) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.
Assuntos
Bacteriófago mu/genética , Elementos de DNA Transponíveis , Recombinação Genética , Clonagem Molecular , Conjugação Genética , DNA Viral/síntese química , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Genes , Plasmídeos , Proteínas Virais/genéticaRESUMO
Piperonyl butoxide has been found to act as potent inhibitor for potato spindle tuber viroid in Scopolia sinensis Hemsl plant.
Assuntos
Butóxido de Piperonila/farmacologia , Vírus de Plantas/efeitos dos fármacos , Nicotina/farmacologia , Doenças das Plantas , Vírus de RNA/efeitos dos fármacos , RNA Viral/biossínteseRESUMO
The existence of three infectious forms of potato spindle tuber viroid (PSTV) RNA from Scopolia sinensis was demonstrated by fractionation with high salt, by reverse phase and high pressure liquid chromatography, and by polyacrylamide gel electrophoresis. Purification of fraction II was achieved by the following steps: extraction of nucleic acid with phenol, precipitation of the RNA with cetyltrimethylammonium bromide, fractionation of the RNA with lithium chloride and isopropanol, and finally gel electrophoresis. A procedure using reverse phase chromatography was developed to obtain 70-90% recovery of RNA from polyacrylamide gels. Purified PSTV fraction II RNA was digested with ribonuclease A and T and labelled with [gamma-32P[ATP using polynucleotide kinase. The labelled digests were separated by the electrophoresis-homochromatography procedures of Sanger. About 20 and 30 spots were obtained with ribonuclease A and T, respectively.
Assuntos
Doenças das Plantas , Vírus de Plantas/análise , RNA Viral/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Desoxirribonucleases , Peso Molecular , Oligorribonucleotídeos/análise , RibonucleasesRESUMO
Without prior in vitro enzymatic ligation a DNA duplex was assembled successfully by directly transforming competent cells with a mixture containing six synthetic complimentary oligodeoxyribonucleotides and a linearized plasmid. One out of 100 transformants was positive in colony hybridization with one of the synthetic fragment probe. The sequence of the DNA duplex inserted into the plasmid was confirmed by dideoxy sequencing method.
Assuntos
DNA/síntese química , Sequência de Bases , Enzimas de Restrição do DNA/metabolismo , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , PlasmídeosRESUMO
A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesized and cloned by ligation of 26 deoxyribooligonucleotide fragments in two steps with a linearized plasmid followed by transformation. On selection by colony hybridization and DNA sequence analysis, clone pTLY.10 was identified to contain a complete T4 lysozyme synthetic DNA. On expression under lac-promoter, unfused T4 lysozyme was obtained in approximately 4-6% yield. The design and synthesis of two putative folding mutants, flexible (Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75, were based on hierarchical principles. Both mutants lost enzymatic activity of the wildtype. These results are readily understandable if the hierarchical organization of the structure is taken into account. A possible explanation is that the catalytic sites are blocked in both mutants.
Assuntos
Regulação da Expressão Gênica , Genes Sintéticos , Muramidase/genética , Mutação , Fagos T/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/síntese química , Plasmídeos , Conformação ProteicaRESUMO
A DNA of 495 bp coding for T4-lysozyme was chemically synthesized and cloned in Escherichia coli. On DNA sequence analysis, clones pTLY.10 and pTLY.9 were identified to contain identical and complete T4-lysozyme coding sequences except that pTLY.9 had an additional 23 bp inverted repeat DNA at the 3'-end of the coding sequence. On expression and purification under similar conditions, T4-lysozymes from these two clones showed different degrees of retention time on HPLC as well as in the rate of enzymatic reaction. We speculate that this difference could be due to the generation of a pause mutant of T4-lysozyme in pTLY.9 under the influence of 3'-inverted repeat DNA that alters the rate of protein synthesis.