ELSEVIER Biochimica et Biophysica Acta 1261 (1995) 424-426 BB Biochi ~mic~a et Biophysica A~ta Short Sequence-Paper Molecular cloning and expression of human ribonuclease 4 cDNA " Masaharu Seno *, Jun-ichiro Futami, Yoshiaki Tsushima, Kazumi Akutagawa, Megumi Kosaka, Hiroko Tada, Hidenori Yamada Department of BioengineeringScience, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700, Japan Received 9 December 1994; revised 1 February 1995; accepted 2 February 1995 Abstract A cDNA coding for human ribonuclease 4 was isolated from a pancreas cDNA library and sequenced. This cDNA (996 bp) includes an entire open reading frame encoding mature protein (119 aa) following signal peptide (28 aa). Expression of mature protein in Escherichia coli showed an apparent molecular mass of about 16 kDa, which was slightly lower than the mature form of human RNase 1, in SDS-PAGE. Keywords: Pancreatic type ribonuclease family; RNase 4; cDNA cloning; Gene expression; (Pancreas); (Human) Pancreatic type ribonucleases (RNases) constitute a large superfamily crossing over species [1]. Bovine pancreatic RNase (RNase 1), largely known as RNase A, is classical and the first and best characterized. Members of this RNase family derived from amphibian, bovine and human have recently been found and characterized. Especially amphibian RNases, Onconase [2] and lectins [3], bovine seminal RNase [4] and human angiogenin (RNase 5) [5] appear to play important roles as factors controlling cellu- lar growth and development. In human, the counterparts of neither Onconase, amphibian lectins nor seminal RNase have been identified, while five homologues are found in various tissues. RNase 5 (angiogenin), which induces neo- vascularization on chicken embryo chorioallantoic mem- brane and rat cornea, is a unique homologue of RNase 1 with a ribonucleolytic activity markedly different from RNase 1 [6]. Another two members of the family, RNase 2 and 3, were originally isolated as neurotoxin from eosinophil and named eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), respectively [7]. Very recent study has revealed the existence of a novel member of RNase, which specifically cleaves on the 3' ~ The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases under the accession number D37931. * Corresponding author. Fax: + 81 86 2535755. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 016 7-4781 (95)00040- 2 side of uridine, called RNase 4 [8]. RNase 4 was originally isolated from the conditioned medium of human colon adenocarcinoma HT-29 [9], which was also the original source of RNase 5, and plasma [8], while the counterparts of RNase 4 in bovine and porcine were purified from liver [10,11] implying widespread distribution of RNase 4. To make clear the biological significance of heterogeneity of pancreatic type RNases, cDNA cloning for RNase 4 was carried out by screening cDNA library prepared from human pancreas [12] and a bacterial expression system was established. First we designed three oligonucleotide primers, two forward and one reverse primer, from the primary structure of human RNase 4 [8]. One of the forward primers was a mix of 128 oligonucleotides of 20-mer with the sequence of 5'-CAA/GGAC/TGGA/C/G/TATGTAC/TCAA/ GA/CG-3' (#1) reverse-translated from Q1-R7 and the other a mix of 48 oligonucleotides of 20-mer with the sequence of 5'-TAC/TTGC/TAAC/TC/TI'A/C/G/ TATGATGCA-Y (#2) from y24_Q30. The reverse primer was designed as a mix of 32 oligonucleotides of 20-mer with the sequence of 5'-CCC/TI'CA/GTGA/GCAA/ GTTCATC/TTT-3' (#3), which was complementary to the one from K68-Q 74. Polymerase chain reaction on the phage solution of cDNA library using the pair of primers #1 and #3 showed an amplified DNA fragment of 200 bp, which could be a template for amplification of DNA fragment of 130 bp with the pair of primers #2 and #3.