ELSEVIER Biochimica et Biophysica Acta 1218 (1994) 466-468 Biochi ~mic~a et BiophysicaA~ta Short Sequence-Paper Nucleotide sequence encoding human pancreatic ribonuclease Masaharu Seno "'*, Jun-ichiro Futami a, Megumi Kosaka a, Satimaru Seno b, Hidenori Yamada a a Department of Bioengineering Science, Faculty of Engineering, Okayama Unit~ersity, Tsushima-Naka, Okayama 700, Japan b Shigei Medical Research Institute, Yamada, Okayama 701-02, Japan Received 14 January 1994 Abstract A cDNA coding for human pancreatic ribonuclease was isolated from a pancreas cDNA library and sequenced. This cDNA (1620 bp) includes an entire open reading frame encoding mature protein (128 aa) following a signal peptide (28 aa) as well as 5'- and T-untranslated regions. Key words: Ribonuclease; pancreatic RNase; Pancreas; cDNA cloning; (Human) Bovine pancreatic ribonuclease (RNase) was an en- zyme central to biochemical research. RNase was the first enzyme studied by NMR [1]. Using RNase, Anfin- sen showed a model for protein folding [2]. RNases appear now to play important roles as factors control- ling growth and development in higher organisms since the roles of extracellular RNA molecules, the logical substrates for extracellular RNases, in cellular growth and developement have been confirmed (reviewed in Ref. 3). Pancreatic RNase from bovine was shown to suppress growth of tumor [4,5] and to inhibit DNA synthesis in cultured cells [6]. RNase from bovine semi- nal fluid has also long been known to be a potent inhibitor of tumor cell growth [7-9]. In contrast, stud- ies on RNases from human tissues are less active with the difficulties of obtaining the material. To make clear the biological significance of pancreatic RNase of hu- man, we cloned the cDNA by screening cDNA library prepared from human pancreas. First we designed three oligonucleotide primers, two forward and one reverse primer, from the primary structure of human pancreatic RNase [10]. One of the forward primers was a mix of 128 oligonucleotides of * Corresponding author. Fax: + 81 86 2535755. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases under the accession number D26129. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4781(94)00052-5 20-mer with the sequence of 5'-TTC/TCAA/GA/ CGA/C/G/TCAA/GCAC/TATGGA-3' (# 1) re- verse-translated from Phe-8-Asp-14 and the other a mix of 32 oligonucleotides of 20-mer with the sequence of 5'-TAC/TTGC/TAAC/TCAA/GATGATGA/ CG-3' (#2) from Tyr-25-Arg-31. The reverse primer was designed as a mix of 192 oligonucleotides of 20-mer with the sequence of 5'-CG/TA/GCAA/GTCA/C/ G/TGTA/G/TATA/GTGCAT-3' (#3), which was complementary to the one from Met-79-Arg-85. Poly- merase chain reaction on the phage solution of cDNA library using the pair of primers #1 and #3 showed an amplified DNA fragment of 230 bp, which could be a template for amplification of DNA fragment of 180 bp with the pair of primers #2 and #3. The sizes of both DNA fragments are consistent with the length of en- coded amino acid sequences. The library was screened using the DNA fragment of 230 bp as a probe. From about 300 positively hybridized plaques one of the phage clones with the longest inserted cDNA of around 1.5 kbp was selected, and the cDNA was subcloned into a phagemid vector pUCll9 [11] to construct the phagemid pBO23 and analyzed. The nucleotide se- quence analysis revealed 5'- and 3'-untranslated regions and the predicted signal sequence consisting of 28 amino acids followed by the primary structure identical to that of mature protein purified from pancreas [10] (Fig. 1). In the open reading frame which encodes the mature pancreatic RNase, only one ATG codon is