The Rana catesbeiana rcr gene encoding a cytotoxic ribonuclease. Tissue distribution, cloning, purification, cytotoxicity, and active residues for RNase activity.
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.
Huang
H C
HC
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
Wang
S C
SC
Leu
Y J
YJ
Lu
S C
SC
Liao
Y D
YD
D000595:Amino Acid Sequence; D029845:Amphibian Proteins; D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003603:Cytotoxins / Q000235:genetics* / Q000633:toxicity; D004527:Egg Proteins / Q000235:genetics* / Q000633:toxicity; D004722:Endoribonucleases / Q000235:genetics* / Q000633:toxicity; D005260:Female; D015870:Gene Expression; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology; D011892:Rana catesbeiana / Q000235:genetics*; D011994:Recombinant Proteins / Q000633:toxicity; D012260:Ribonucleases / Q000145:classification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D029845:Amphibian Proteins; D000970:Antineoplastic Agents; D003603:Cytotoxins; D004527:Egg Proteins; D011994:Recombinant Proteins; D004722:Endoribonucleases; D012260:Ribonucleases; C094067:pyrimidine-guanine sequence-specific ribonuclease, Rana catesbeiana