The coding region for the cytotoxin a-sarcin from Aspergillus giganteus has been chemically synthesized by the ligation of 19 overlapping oligodeoxyribonucleotides. An Escherichia coli clone producing the cytotoxin was constructed by inserting the synthesized gene directly downstream to the region coding for the signal peptide of the OmpA protein ( a major outer membrane protein of E. coli), using the secretion cloning vector PIN-111-OmpA2. The enzyme encoded by the chemically synthesized gene expressed in E. coli displayed properties identical to those of native a-sarcin isolated from A . gipunteus with respect to its chemistry, antigenicity and ribonucleolytic activity in qualitative assays.a-Sarcin is a basic cytotoxic protein produced by the mold Aspergillus giganteus [ 11; it suppresses protein synthesis in yeast and wheat germ extracts due to its ribonucleolytic activity which introduces a specific cut in the 28s rRNA of isolated ribosomes [2]. Most remarkably, it also exhibits anti-tumor activity against different types of tumors at doses that are nontoxic [3]. The toxin is composed of a single polypeptide chain which is neither glycosylated nor modified in any respect. The amino acid sequence of a-sarcin [4] is very similar (86%) to the Aspergillus restrictus toxins mitogillin and restrictocin [5], and similar in limited regions to the purine-specific RNases isolated from other fungi. It may also harbor structural elements which are closely related to the structure of the RNase T1 from Aspergillus oryzae [6, 71.The ribosome-inactivating effects of a-sarcin, restrictocin and mitogillin have been studied in great detail [S]. The position of the cleavage site on 28s rRNA from rat liver ribosomes was shown to be 393 nucleotides from the 3' end of the 28s rRNA, yielding 3'-phosphate and 5'-hydroxyl termini [9]. a-Sarcin injected into Xenopus laevis oocytes cleaves a single phosphodiester bond at the putative a-sarcin recognition sequence [lo] and causes a rapid decline in oocyte protein synthesis of soluble cytoplasmic proteins similar to the effect of injecting cyclohexamide or puromycin [ll]. MATERIALS AND METHODS MaterialsChemicals used for thc automated synthesis of the deoxyoligonucleotides were from Applied Biosystems (Weiterstadt, TnlO (tetr) [F:traD36, proAB, l a d q , lacZ A4151 and E. coli CJ 236 dut, ung, thi, relA; pCJ105 (Cmr) were supplied as part of the mutagenesis kit from Bio-Rad. Pseudomonas putida 2440 is a derivative of the strain mt-2, defective in host-specific restriction enzymes [12]. The bacterial strains were grown at 37°C in LB medium [13] containing ampicillin (100 pg/ml) if necessary. Cells were grown to the mid-(log phase) (AGo0 = 0.2), expression was induced by the addition of IPTG (0.5 mM) and the cells were harvested in the late stationary phase the next morning. Synthesis and purification of oligodeoxyribonucleotidesThe oligodeoxyribonucleotides were synthesized on an Applied Biosystems DNA synthesizer model 380 A following the phosphotriester method [I41 and purified b...