nuclease activity of Neurospora crassa is accounted for by a number of T?: zymes distinguishable physically and catalytically. Of these, two nucleases specific for both DNA and RNA have been well characterized (LINN and LEHMAN 1965, 1966). More recently, an extracellular ribonuclease, RNase NI, and two intracellular ribonucleases, RNase N, and N" have been described (TAKAI, UCHIDA and EGAMI 1966, 1967a, b). Possible functions of nucleases in cells may be the degradation of nucleic acids

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... from the surrounding environment and the turnover of nucleic acids within the cells. There arises, however, much difficulty and confusion when one attempts to understand these functions of nucleases. Furthermore, the genetics of these enzymes as well as the intracellular regulation of nucleic acid metabolism remain relatively unexplored. One approach to the elucidation of the functions of nucleases and the genetic mechanism involved in nucleic acid metabolism is to find mutants which lack one or more kinds of nuclease activity. Such mutants have been previously obtained in Escherichia coli ( GESTELAND 1966; DURWALD and HOFFMANN-BERLING 1968) . The present paper describes a simple screening technique for the isolation of mutants of Neurospora with the ability to digest neither DNA nor RNA and demonstrates that two genes are responsible for the activity of a particular kind of nuclease. This nuclease appears to be a complex consisting of a nuclease and an inhibitor. Preliminary reports of this work have been published (ISHIKAWA, HASUNUMA and TOH-E 1967; ISHIKAWA, TOH-E, HASUNUMA and UNO 1968). MATERIALS A N D METHODS Materials: The wild-type strain 74A (ST. LAWRENCE) of neurospora crmsa was used to induce nuclease mutants and as a source of normal nucleases. Mutants 74A-Y234-M394 (ad-I) and 74A-T28-M4 (unknown locus), which require adenine or hypoxanthine, were also used to induce nuclease mutants. The following purine or pyrimidine-requiring mutants were used to test growth characteristics in various media: adenine-requiring mutants, ad-4 (F2), ad-8 (E6), Qd-3A and ad-6; a guanine-requiring mutant, 74A-T51-M116 (ISHIKAWA, unpublished) ; pyrimidine-requiring mutants, pyr-I (H263), pyr-2 (38502), pyr-3 (KS36) and pyr-4 (36601). The pyrimidine-requiring mutants were kindly supplied from the Fungal Genetics Stock Center, Hanover, New Hampshire. Media: The phosphate-free medium was prepared by substituting KC1 (lg/l) in Fries minimal medium for KH,PO,. Appropriate amounts of RNA, DNA or mononucleotides were included in the sterile phosphate-$we medium as sole sources of phosphate. These media will be described in this paper as RNA medium, DNA medium or mononucleotide medium (e.g., 3'-AMP medium) depending on the supplement