THE RANA-CATESBEIANA RCR GENE ENCODING A CYTOTOXIC RIBONUCLEASE - TISSUE DISTRIBUTION, CLONING, PURIFICATION, CYTOTOXICITY, AND ACTIVE RESIDUES FOR RNASE ACTIVITY

Citation
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
Citations number
40
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
273
Issue
11
Year of publication
1998
Pages
6395 - 6401
Database
ISI
SICI code
0021-9258(1998)273:11<6395:TRRGEA>2.0.ZU;2-P
Abstract
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.