THE RANA-CATESBEIANA RCR GENE ENCODING A CYTOTOXIC RIBONUCLEASE - TISSUE DISTRIBUTION, CLONING, PURIFICATION, CYTOTOXICITY, AND ACTIVE RESIDUES FOR RNASE ACTIVITY
Hc. Huang et al., THE RANA-CATESBEIANA RCR GENE ENCODING A CYTOTOXIC RIBONUCLEASE - TISSUE DISTRIBUTION, CLONING, PURIFICATION, CYTOTOXICITY, AND ACTIVE RESIDUES FOR RNASE ACTIVITY, The Journal of biological chemistry, 273(11), 1998, pp. 6395-6401
Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine seque
nce-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes.
It possesses both ribonuclease activity and cytoxicity against tumor c
ells, We report here for the first time the cloning of RC-RNase cDNA f
rom liver rather than from oocytes where RC-RNase is stored, An intern
al fragment of cDNA was obtained by reverse transcription-PCR using de
duced oligonucleotides as primers. Full-length cDNA was obtained by 5'
- and S'-RACE technique. The cDNA clone, named rcr gene, contained a 5
'-untranslated region, a putative signal peptide (22 amino acids), a m
ature protein (111 amino acids), a 3'-untranslated region, and a polya
denylation site. The cDNA which encoded the mature protein was fused u
pstream with a modified pelB signal peptide DNA and inserted into pET1
1d for expression in Escherichia coli strain BL21(DE3). The secretory
RC-RNase in the culture medium was enzymatically active and was purifi
ed to homogeneity. The recombinant RC-RNase had the same amino acid se
quence, specific activity, substrate specificity, antigenicity, and cy
totoxicity as that of native RC-RNase from frog oocytes. Amino acid re
sidues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic
activity. Ribonucleolytic activity was involved in and may be essentia
l for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNas
e had approximately 45% identity to that of RNase superfamily genes. T
his indicates that RC-RNase is a distinct ribonuclease gene in the RNa
se superfamily.