From the complete nucleotide sequence of the genome of the avian sarcoma virus Y73, we have predicted amino acid sequence of p90gag-yes, the product of the transforming gene. Contrary to previous evidence from molecular hybridization d .90gag-yes f d h stu zes, p was 1 oun to ave much homology with the transforming gene product p60''c of Rous sarcoma virus, suggesting that the cellular counterparts of the two (c-yes and c-src) originated from a common prototype sequence.

An extracellular nuclease gene of Bacillus subtilis was cloned in the same organism by detecting the amplified enzyme activity, which was secreted from the transformant cells on an RNA-containing agar medium. An open reading frame encoding 289 amino acids was identified within the cloned fragment. The transcriptional initiation site was determined by nuclease S1 mapping and the promoter region showed similarity to the conserved recognition sequences for the EaA andjor Ea" RNA polymerases. The production of the nuclease by the B. subtilis transformants greatly depends on the liquid medium used. SDSIPAGE analysis of the purified enzyme showed two adjoining bands of molecular mass about 32 kDa. and the NH2-terminal amino acid sequence analysis suggested that the NH2-terminal portion of the nuclease was subjected to a limited proteolysis after or during secretion. The nuclease was uniquely characterized as a Mg'+-activated ribonuclease which hydrolyzes RNA apparently nonspecifically into oligonucleotides with 5'-terminal phosphate. The deduced amino acid sequence of this enzyme shows no obvious similarity with other nuclease sequences. To characterize the extracellular nuclease system of B. suhtilis both enzymatically and genetically, we attempted to clone genes coding for the nuclease. As we noted that the wild-type B. subtilis cell forms a very small but distinct clear zone on RNA-containing nuclease assay plates, we attempted to shot gun clone the nuclease gene(s) by means of the overproduction of the enzyme due to the gene dosage effect. The genusCorrespondence to A. Nakamura, Department of Biotechnology, Faculty of Agriculture, The University of Tokyo, Ydyoi 1-1-1, Bunkyo-ku, Tokyo 113, JapanAbbreviations. Bsn and bsn, Bacillus subtilis nuclease cloned in this study, and its gene, respectively; Cm, chloramphenicol.Enzvmes. Alkaline phosphatase (EC 3.1.3.1); deoxyribonuclease I (EC 3.1.21.1); nuclease S1 (EC 3.1.30.1); Escherichia coli DNA polymerase I (EC 2.7.7.7); nuclease PI (EC 3.1.30.1); ribonuclease Bsn from Barillus subtilis (EC 3.1.-.-); ribonuclease A from bovine pancreas (EC 3.1.27.5); ribonuclease Tz (EC 3.1.27.1); Thermus aquaticus DNA polymerase (EC 2.7.7.7); type-I1 restriction endonucleases BamHI, DraI, EcoRI, EcoRV, HindIII, NaeI, PstI, RsuI, Sun, SmaT and SspI (EC 3.1.21.4); T4 DNA kinase (EC 2.7.1.78); '14 DNA ligase (EC 6.5.1.1).Nofe. The nucleotide sequence data published here will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number D01097.In this paper, we describe the cloning and sequencing of the gene coding for an extracellular nuclease (Bsn) from B. subtilis, together with its unique enzymatic properties. MATERIALS AND METHODS Bacterial strains and plasmids

The mature portion of the TEM/J-lactamase (BLA) gene {bid) derived from pBR322 was fused with the promoter and signal region of Bacillus subtilis cellulase (BSC) gene {bsc), and the productivity was compared with that of the cloned native bsc gene, using a wild-type B. subtilis strain and a strain deficient in three proteases {i.e., extracellular serine protease, extracellular neutral protease, and the major intracellular serine protease) as hosts. The effects of the sen, sacQ, and prtR genes, carried on the same plasmids, were tested as for the productivities of BSCand BLA.The production of BSC was increased 9-fold by using the combination of the triple-protease deficient strain and the sacQ gene, compared with that by using the wild-type strain as a host, and no degradation of BSCwas observed in the triple-protease deficient strain. On the other hand, though the production of the BSC-BLAfusion protein increased 2.5-fold in the triple-protease deficient strain, the BLAactivity was decreased after the cell reached the stationary phase of growth, possibly due to some proteolysis. These observations showdifferent sensitivity of secretary proteins to cellular proteases, and suggest that BLAis decomposed by remaining minor proteases.

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