ABSTRACT
A sequence-specific M1GS ribozyme (M1-T3) was constructed by covalently linking an oligonucleotide (guide sequence,GS) to the 3' terminus of M1 RNA ,the catalytic subunit of RNase P from Escherichia coli. The engineered ribozyme is targeted to the mRNA sequence encoding a protein kinase (UL97) of HCMV and could effectively cleave the mRNA segment in vitro. Further studies about the significance of some structural elements in the M1 GS (e.g. the 3' CCA tail sequence and a bridge sequence between the 3' terminus of M1 RNA and the 5' terminus of the GS) were carried out. The results showed that the bridge sequence of 88 nucleotides in a mutated M1 GS (i.e. M1-T3*) dramatically increased the cleavage activity to the substrate in vitro. Moreover, the 3'CCA tail sequence was confirmed to be a necessary element for the cleavage activity of M1 GS ribozyme. These data we got in the study will help in understanding the interaction between the M1 GS RNA and its substrate,and will markedly facilitate the research of a general gene targeting agent for anti-HCMV applications.
Subject(s)
5' Untranslated Regions/metabolism , Escherichia coli Proteins/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , RNA, Catalytic/metabolism , Ribonuclease P/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemical synthesis , Phosphotransferases (Alcohol Group Acceptor)/metabolism , RNA, Viral/metabolism , Ribonuclease P/chemical synthesis , RNA, Small UntranslatedABSTRACT
In this paper, the determination of Ba using oxygen enriched air-acetylene flame in AAS has been studied and the effect of acetylene flow, oxygen flow and the ratio of O2/C2H2 on the absorbance have been compared. When the amount of oxygen is 52% in mixed gas and the ratio of O2/C2H2 is about 0.72, higher sensitivity for Ba can be obtained. In this flame acids and most of elements did not interfere. By adding 1,000 mg.L-1 K the interference can be minimized and the sensitivity for the determination of Ba can be increased. The sensitivity of the method is 0.16 mg.L-1/1%, the detection limit is 0.034 mg.L-1, and the relative standard deviation is 2.5%. The analytical results of the standard reference material (GBW08301 and GSD-6) by this method are consistent with the certified values, and it was proved that this method are reliable and is suitable for the determination of Ba in mineral water.