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J Biol Chem, Vol. 273, Issue 11, 6395-6401, March 13, 1998

The Rana catesbeiana rcr Gene Encoding a Cytotoxic Ribonuclease
TISSUE DISTRIBUTION, CLONING, PURIFICATION, CYTOTOXICITY, AND ACTIVE RESIDUES FOR RNase ACTIVITY

Huey-Chung Huang^§, Sui-Chi Wang, Ying-Jen Leu^¶, Shao-Chun Lu^§, and You-Di Liao^¶

From the  Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, the ^§ Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei 10016, and the ^¶ Institute of Radiation Biology, National Tsing-Hua University, Hsin-Chu 30043, Taiwan

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5'- and 3'-RACE technique. The cDNA clone, named rcr gene, contained a 5'-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3'-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.

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