Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom st...Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom strand; indicates nicking on the top strand）. However, wild type Dralll shows significant star activity. In this study, it was found that the prominent star site is CATSGTT;GTG, consisting of a star 5＇ half （CAT） and a canonical 3＇ half （GTG）. Dralll nicks the 3＇ canonical half site at a faster rate than the 5＇ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the Dralll protein was solved. It indicated, as supported by mutagenesis, that Dralll possesses a ~13a- metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site- directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.展开更多
The influence of factors on the subst rate-specificity of Pst Ⅰ restric tion endonuclease has been studied with the method of electrophoresis. The resu lts show that, the specificity of Pst Ⅰ almost can not be inf...The influence of factors on the subst rate-specificity of Pst Ⅰ restric tion endonuclease has been studied with the method of electrophoresis. The resu lts show that, the specificity of Pst Ⅰ almost can not be influenced by th e sin gle alteration of the concentration of Tris·HCl, Mg 2+ or Na+ in the rea ction s ystem, but it can be altered by the reduction of any two of them. The specificit y can not be altered by the single alteration of pH or the replacement of Mg 2+ w ith Mn 2+. The addition of glycerol or dimethylsulphoxide (DMSO) to the rea ction system results in the relaxation of the substrate-specificity of Pst Ⅰ , b ut dim ethylmethylformide, glycol and ethyl alcohol can not bring about the alteration of Pst Ⅰ specificity. Through the method of cloning and sequencing, the nuc leot ides of No.1 and 6 in the recognition sequence of Pst Ⅰ have changed (1C→A or 6 G→T). Used with the enzyme analysis of an artificially synthetic DNA segment co ntaining a special sequence, the nucleotides of No.1 and 6 have both changed (1C →A and 6G→T). The recognition sequence of Pst Ⅰ is speculated to be chan ged from CTGCA↓G to TGCA↓.展开更多
研究了温度、酶浓度、反应时间、几种有机溶剂等因素对PstⅠ星号活力的影响,并在有利于星号活力的反应条件下测定了星号活力及原活力的Km 分别为1.46×10- 7 m ol/L和1.02×10- 8 m ol/L,Vm ax 分别为2.86×10- 16 m ol/s和...研究了温度、酶浓度、反应时间、几种有机溶剂等因素对PstⅠ星号活力的影响,并在有利于星号活力的反应条件下测定了星号活力及原活力的Km 分别为1.46×10- 7 m ol/L和1.02×10- 8 m ol/L,Vm ax 分别为2.86×10- 16 m ol/s和3.06×10- 15 m ol/s.展开更多
Objective:To isolate and characterize the saponin from Persian Gulf brittle star(Ophiocoma erinaceus)and to evaluate its hemolytic and cytotoxic potential. Methods:In an attempt to prepare saponin from brittle star,co...Objective:To isolate and characterize the saponin from Persian Gulf brittle star(Ophiocoma erinaceus)and to evaluate its hemolytic and cytotoxic potential. Methods:In an attempt to prepare saponin from brittle star,collected samples were minced and extracted with ethanol,dichloromethane,n-buthanol.Then,concentrated n-butanol extract were loaded on HP-20 resin and washed with dionized water,80%ethanol and 100%ethanol respectively.Subsequently,detection of saponin was performed by foaming property,fourier transform infrared spectroscopy and hemolytic analysis on thin layer chromatography.The cytotoxic activity on HeLa cells was evaluated through 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide(MTT)assay and under invert microscopy.Results:The existence of saponin in Ophiocoma erinaceus were approved by phytochemical method.The presence of C-H bond,C-O-C and OH in fourier transform infrared spectrum of fraction 80%ethanol is characteristic feature in the many of saponin compounds.Hemolytic assay revealed HD_(50) value was 500μg/mL.MTT assay exhibited that saponin extracted in IC_(50) value of 25μg/mL inducsd potent cytotoxic activity against HeLa cells in 24 h and 12.5μg/mL in 48 h,meanwhile in lower concentration did not have considerable effect against HeLa cells.Conclusions:These findings showed that only 80%ethanol fraction Persian Gulf brittle star contained saponin like compounds with hemolytic activity which can be detected simply by phytochemical that can be appreciable for future anticancer research.展开更多
文摘Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom strand; indicates nicking on the top strand）. However, wild type Dralll shows significant star activity. In this study, it was found that the prominent star site is CATSGTT;GTG, consisting of a star 5＇ half （CAT） and a canonical 3＇ half （GTG）. Dralll nicks the 3＇ canonical half site at a faster rate than the 5＇ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the Dralll protein was solved. It indicated, as supported by mutagenesis, that Dralll possesses a ~13a- metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site- directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.
基金Supported by the Research Fund for the Doctoral Program of Higher Education.
文摘The influence of factors on the subst rate-specificity of Pst Ⅰ restric tion endonuclease has been studied with the method of electrophoresis. The resu lts show that, the specificity of Pst Ⅰ almost can not be influenced by th e sin gle alteration of the concentration of Tris·HCl, Mg 2+ or Na+ in the rea ction s ystem, but it can be altered by the reduction of any two of them. The specificit y can not be altered by the single alteration of pH or the replacement of Mg 2+ w ith Mn 2+. The addition of glycerol or dimethylsulphoxide (DMSO) to the rea ction system results in the relaxation of the substrate-specificity of Pst Ⅰ , b ut dim ethylmethylformide, glycol and ethyl alcohol can not bring about the alteration of Pst Ⅰ specificity. Through the method of cloning and sequencing, the nuc leot ides of No.1 and 6 in the recognition sequence of Pst Ⅰ have changed (1C→A or 6 G→T). Used with the enzyme analysis of an artificially synthetic DNA segment co ntaining a special sequence, the nucleotides of No.1 and 6 have both changed (1C →A and 6G→T). The recognition sequence of Pst Ⅰ is speculated to be chan ged from CTGCA↓G to TGCA↓.
文摘研究了温度、酶浓度、反应时间、几种有机溶剂等因素对PstⅠ星号活力的影响,并在有利于星号活力的反应条件下测定了星号活力及原活力的Km 分别为1.46×10- 7 m ol/L和1.02×10- 8 m ol/L,Vm ax 分别为2.86×10- 16 m ol/s和3.06×10- 15 m ol/s.
文摘Objective:To isolate and characterize the saponin from Persian Gulf brittle star(Ophiocoma erinaceus)and to evaluate its hemolytic and cytotoxic potential. Methods:In an attempt to prepare saponin from brittle star,collected samples were minced and extracted with ethanol,dichloromethane,n-buthanol.Then,concentrated n-butanol extract were loaded on HP-20 resin and washed with dionized water,80%ethanol and 100%ethanol respectively.Subsequently,detection of saponin was performed by foaming property,fourier transform infrared spectroscopy and hemolytic analysis on thin layer chromatography.The cytotoxic activity on HeLa cells was evaluated through 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide(MTT)assay and under invert microscopy.Results:The existence of saponin in Ophiocoma erinaceus were approved by phytochemical method.The presence of C-H bond,C-O-C and OH in fourier transform infrared spectrum of fraction 80%ethanol is characteristic feature in the many of saponin compounds.Hemolytic assay revealed HD_(50) value was 500μg/mL.MTT assay exhibited that saponin extracted in IC_(50) value of 25μg/mL inducsd potent cytotoxic activity against HeLa cells in 24 h and 12.5μg/mL in 48 h,meanwhile in lower concentration did not have considerable effect against HeLa cells.Conclusions:These findings showed that only 80%ethanol fraction Persian Gulf brittle star contained saponin like compounds with hemolytic activity which can be detected simply by phytochemical that can be appreciable for future anticancer research.
