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...

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Experiments on the newt, Notophthalmus viridescens, showed that 180" surgical rotation of the forelimb blastema could provoke supernumerary limb formation (Bryant and Iten, '76). The polar coordinate model predicts a high incidence of supernumerary limbs for angles of rotation at or near 180" and no supernumeraries for small angles of rotation. The results of recent experiments on axolotls (Maden and Turner, '78; Wallace, '78; Wallace and Watson, '79, Turner, pers. comm.), however, do not exhibit this pattern. In the majority of these experiments, supernumerary limbs could arise from any region of the limb circumference. The experiments described in this paper consist of a comprehensive series of ipsilateral rotations of newt forelimb blastemas performed at increments of 45" between 0" and 360". MATERIALS AND METHODSAdult male newts, Notophthalmus viridescens, obtained from Bill Lee's Newt Farm, Oak Ridge, Tennessee were used in these experiments. They were maintained at 25" 2 1" C, except as indicated below, and exposed to a 12-hour light cycle. Prior to any surgery, animals were anaesthetized by immersion in 30% chlorobutanol (Sigma). Both forelimbs of the newts were amputated at the level of the midhumerus and allowed to regenerate to the stage of three or four early digits (Iten and Bryant, '733. Each stump and blastema was then marked with a thin line a t the middorsal position (Fig. 1) using a drawn glass capillary and biological grade marking ink (Pelikan). These reference ink marks were used to position the blastemas and to subsequently monitor their orientation. Blastemas were amputated a t their interface with the stump, rotated by a specified angle, and replaced on the same stump as shown in Figure 1. To help hold each blastema in place during the initial stages of healing, small pieces of lens tissue were positioned alongside the grafts. When each operation was completed, the animal was positioned on a small cotton support in an individual finger bowl and placed in a refrigerator (-5" C). One day later, the cotton was removed, the newt was revived by immersion in cold water and left in the refrigerator for an additional three to four days to reduce activity and minimize graft loss. When removed from the refrigerator, animals were maintained individually at room temperature (15-20" C) and closely observed for signs of graft regression or derotation. After seven to ten days, animals were grouped in battery jars and placed in an incubator at 25" C. Each animal was examined once or twice per week until the graft and any supernumerary limbs were at the stage of late digits (usually about two months after grafting), at this time the limbs were fixed in Bouin's solution, stained with Victoria blue B, and cleared for skeletal analysis (Bryant and Iten, '74). No limbs with blastemas that regressed beyond