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We wished to identify metabolic signals governing changes in ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity in Neurospora crassa. By manipulations of the ornithine supply and by the use of inhibitors of the polyamine pathway, we found that spermidine negatively governs formation of active ornithine decarboxylase and that putrescine promotes inactivation of the enzyme. Direct addition of putrescine or spermidine to cycloheximide-treated cells confirmed the role of putrescine in enzyme inactivation and showed that spermidine had no effect on this process. Increases in ornithine decarboxylase activity caused by blocking spermidine synthesis occurred prior to a significant decrease in the spermidine pool. This is consistent with our prevous finding that only 10-20% of the spermidine pool is freely diffusible within N. crassa cells. We presume that only this small fraction of the pool is active in regulation.Ornithine decarboxylase (OrnDCase; L-ornithine carboxylyase, EC 4.1.1.17) is a key enzyme of the synthesis of polyamines (putrescine, spermidine, and spermine) in most fungi and animals (1-3). While the roles of polyamines in vivo are still uncertain, these compounds are indispensable in eukaryotes (2). An increase of OrnDCase activity and an increased rate of polyamine synthesis are correlated with the onset of rapid growth, of transformation to the neoplastic state, and of periods of cell differentiation in most organisms (see refs. in ref. 2). Moreover, OrnDCase is rapidly inactivated upon the cessation of growth or upon addition of polyamines to cells. The regulation of the amount of active enzyme is the major mechanism of control; feedback and product inhibition are weak or absent.A test of polyamine pathway intermediates ( Fig. 1) for their roles in regulating the OrnDCase of Neurospora crassa is reported here. We identify spermidine and putrescine as regulatory signals governing, respectively, the formation and inactivation of OrnDCase catalytic activity. The work is discussed in connection with our previous observations (4-6) that polyamines are highly compartmentalized in cells of N.crassa. MATERIALS AND METHODSStrains, Growth, and Sampling. The strain of N. crassa used was IC3, a prototrophic strain carrying the arginase-less (aga) allele, UM-906. Vogel's medium N (7) was used for growth. Cultures were grown exponentially in 1-liter aerated cultures (8) for determinations of growth rate, enzyme activity, and polyamine pools. Samples (20 ml) for dry weight were collected on Whatman no. 1 filter circles and dried with acetone. Mycelial samples (10 ml) for polyamine determinations were collected and washed on membrane filters (pore size, 5 ,um). The pad was extracted with 1 ml of 0.4 M perchloric acid with 1 mM EDTA. As an internal standard, 100 nmol of 1,7-diaminoheptane was added before centrifugation; the supernatant was saved and frozen until use. Mycelial samples (5 ml) for enzyme assay were permeabilized with a toluene/ethanol solution, followed by freezing (9).OrnD...

To define the structural gene for ornithine decarboxylase (ODC) (25). The polyamine pathway appears to become active in cells undergoing rapid growth, differentiation, and conversion to the neoplastic state (2,4,15,28). Despite the relationship of the pathway to the growth process, the roles of polyamines and the elaborate regulation of ODC in most organisms are only recently becoming understood at the molecular level (4,14,20). Mutants affecting ODC have been recovered in a number of microbial and higher eucaryotic systems (5, 6, 10, 18, 20-23, 23a, 24, 27) dine. 3HCI. Techniques and media for crosses and complementation tests have been described previously (7). Growth of strains for the determination of growth rate, enzyme activity, and polyamine pools was carried out in 1-liter cultures as described previously (7). Stationary growth in liquid media for dry-weight determinations with different supplements was done in 10 ml of media in 50-ml Erlenmeyer flasks at 25 and 35°C.Mutant selection. Conidia of strain IC-40 (aga inl), grown previously in 1 mM arginine, inositol (50 jig/ml), and 0.05 rnM spermidine, were used. In this strain, arginine creates a polyamine requirement through ornithine starvation (8), and the small amount of spermidine was enough to support growth while limiting endogenous pools of polyamines. Conidia were irradiated with UV irradiation to 50% survival. They were then suspended in 100 ml of minimal medium (in a 500-ml Erlenmeyer flask) with 2% sucrose and myo-inositol (0.025 ,ug/ml) and put in a shaking water bath. The small amount of inositol was designed to support limited growth until the small endogenous polyamine pools (and the inositol in the medium) were exhausted. As shaking continued, auxotrophs unable to grow further survived, whereas prototrophs suffered inositol-less death. Periodic filtrations through cheesecloth were necessary in the earlier stages to remove clumps of mycelia.

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To define the structural gene for ornithine decarboxylase (ODC) in Neurospora crassa, we sought mutants with kinetically altered enzyme. Four mutants, PE4, PE7, PE69, and PE85, were isolated. They were able to grow slowly at 25 degrees C on minimal medium but required putrescine or spermidine supplementation for growth at 35 degrees C. The mutants did not complement with one another or with ODC-less spe-1 mutants isolated in earlier studies. In all of the mutants isolated to date, the mutations map at the spe-1 locus on linkage group V. Strains carrying mutations PE4, PE7, and PE85 displayed a small amount of residual ODC activity in extracts. None of them had a temperature-sensitive enzyme. The enzyme of the PE85 mutant had a 25-fold higher Km for ornithine (5mM) than did the enzyme of wild-type or the PE4 mutant (ca. 0.2 mM). The enzyme of this mutant was more stable to heat than was the wild-type enzyme. These characteristics were normal in the mutant carrying allele PE4. The mutant carrying PE85 was able to grow well at 25 degrees C and weakly at 35 degrees C with ornithine supplementation. This mutant and three ODC-less mutants isolated previously displayed a polypeptide corresponding to ODC in Western immunoblots with antibody raised to purified wild-type ODC. We conclude that spe-1 is the structural gene for the ODC.