Abstract
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.
Highlights
Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana oocytes
Six clones with inserts of predicted size were obtained from the reverse transcription-PCR product of liver mRNA using oligonucleotides RC-I and RC-IIA as primers. The sequence of these six clones corresponded to amino acid residues 34 –107 of RC-RNase from frog oocytes
To obtain full-length cDNA, oligonucleotides RC-5Ј-RACE and RC-3Ј-RACE in combination with adaptor AP-1 were used as primers for PCR amplification of the 5Ј upstream and 3Ј downstream regions of the RC-RNase gene
Summary
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. 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. The ribonucleases from frog oocytes exert antitumor activity as well as ribonucleolytic activity, e.g. onconase from Rana pipiens (9 –11) and RC-RNase from Rana catesbeiana [12, 13]. We present for the first time the successful cloning of ribonuclease cDNA from frog liver rather than from oocytes where RC-RNase is stored.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have