Ribozyme-mediated suppression of gene expression in S. cerevisiae = 리보핵산요소의 유전자 발현억제

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To identify the function of hammerhead ribozymes in vivo, we have evaluated their catalytic activity and substrate specificity in S. cerevisiae. An assay system based on the use of X/lacZ fusion gene as a reporter was established. Subsequently either active or inactive ribozyme was fused in-frame to the amino terminus of the X/lacZ. The catalytic activity of ribozyme was analyzed using a quantitative β-galactosidase assay indirectly and was verified by the direct detection of the cleavage product using primer extension analysis. There were significant differences between an active Rz and an inactive Rz. By primer extension analysis, target X mRNA was cleaved with the catalytically active ribozyme whereas no detectable specific cleavage was observed with an inactive mutant. However, both an active and an inactive ribozyme resulted in a remarkable reduction in β-galactosidase activity. For elucidating the gene inactivation by an inactive ribozyme, the detailed experiment was performed with several Rz4 variants. Rz4 variants with alterations in the catalytic domain, stem I, or stem II-tetra loop moiety have been examined for their catalytic activity and gene suppression in vivo. An extent of the gene suppression was CUG (96.1%), G5A (92.1%), U7G (99.8%), Rz4f (75.7%), Rz4Δtetra (93%), Rz4As (90%), and Rz4Δsl (0%). A considerable decrease by 90% could be explained as an antisense effect. Nearly complete inhibition of gene expression among various constructs except Rz4f and Rz4Δsl might be due to an antisense effects mainly rather than a translational inefficiancy caused by a stable sten II-tetra loop structure. Moreover, it might be distinct that the slightly enhanced activity caused by Rz4f and the complete increase caused by Rz4Δsl implied the involvement of an active conformation for the gene suppression concomitant with the cleavage. It does matter whether the catalytic motif can be made or not, for the cleavage function. The folding into an active conformation...
Kang, Chang-Wonresearcher강창원researcher
한국과학기술원 : 생물과학과,
Issue Date
144191/325007 / 000945355

학위논문(박사) - 한국과학기술원 : 생물과학과, 1998.8, [ ix, 107 p. ]

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