RESUMO
Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated 'reference plasmids'), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these 'reference plasmids' revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo I/análise , Plasmídeos/normas , Escherichia coli/enzimologia , Plasmídeos/química , Padrões de Referência , Especificidade por SubstratoRESUMO
OBJECTIVE: To map the regulatory domain of Escherichia coli T-protein. METHODS: Fragmentation cloning was employed in cloning of 11 fragments from T-protein. The regulatory activity of each fragment was determined respectively. RESULTS: The regulatory domain of T-protein was located in the C-terminal 270 amino acids, which was the same location as PDH domain. CONCLUSION: T-protein has no independent regulatory domain.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Clonagem Molecular , Desoxirribonucleases de Sítio Específico do Tipo I/análise , Proteínas de Escherichia coli/análise , Conformação de Ácido Nucleico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica/genética , Elementos Reguladores de Transcrição/genéticaRESUMO
Prenatal diagnosis of sickle cell anemia was carried out in four fetuses using DNA technology. Fetal chorionic villus specimen were obtained at the 10th week of pregnancy from women at risk of giving birth to children with sickle cell anemia. Whole cellular DNA was obtained and the part of the DNA presumed to have a mutation increased after PCR was performed. After the application of Dde I restriction enzyme, mini gel electrophoresis was performed. The study of the electrophoretic patterns of the DNA indicated that one of the four fetuses was unaffected, one was a carrier and the remaining two were affected.