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Nucleic Acids Research, 1992, Vol. 20, No. 15 3919-3924
© 1992

MOLECULAR BIOLOGY

Random mutations to evaluate the role of bases at two important single-stranded regions of genomic HDV ribozyme

P.K.R. Kumar1, Young-Ah Suh1, Hirotsugu Miyashiro2, Fumiko Nishikawa1, Junji Kawakami1,3, Kazunari Taira1 and Satoshi Nishikawa1,*

1Department of Cellular and Molecular Biology, Fermentation Research Institute, Agency of Industrial Science {wedge} Technology MITI, 1-1-3 Higashi, Tsukuba Science City, Ibaraki 305 2Research Institute for WAKAN-YAKU, Toyama Medical and Pharmaceutical University Toyama 930-01 3Faculty of Pharmaceutical Sciences, Hokkaido Universtiy Sapporo 060, Japan

*To whom correpondence should be addressed

Received July 7, 1992. Revised July 7, 1992. In elucidating function of two Important single-stranded regions [SSrA (726–731 nt) and SSrB (762–766 nt)] derived mainly from three secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several random mutants at those two regions on the HDV88 molecule (683–770 nt) by oligonucleotidedirected mutagenesis. When self-cleavage activities were compared among mutants, at the region SSrA, G726 was found to play an important role during cleavage reaction since substitutions of the base to A (mutant A20) or C (mutant A16) or U (mutant A23), reduced the ribozyme activity to very low levels suggesting the importance of G726 position. C763 at SSrB region was found to play a more significant role during catalysis than G726 (at region SSrA) since any substitutions at C763 completely inactivated the ribozyme. Other bases located In these two regions could be substituted to other bases at the expense of some self-cleavage activity. The results presented here together with our previous deletion analysis indicate that these two regions may play an important role during cleavage process.


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