Translation. an early stage of morphogenesis in the slime. mold Dictyostelium discoideum, but it does. not affect the incorporation of "4C-amino acids

Transcription

1 JOURNAL OF BACTERIOLOGY, May 1972, p Copyright American Society for Microbiology Vol. 110, No. 2 Printed in U.S.A. Regulation of Nitrate Reductase in Neurospora crassa: Regulation of Transcription and Translation K. N. SUBRAMANIAN' AND G. J. SORGER Department of Biology, McMaster University, Hamilton, Ontario, Canada Received for publication 3 November 1971 A technique employing cycloheximide and actinomycin D has been used for the separation of transcription and translation during the induction of nitrate reductase in Neurospora crassa. Nitrate reductase is found to be synthesized in low efficiency when nitrate is not provided during both transcription and translation. Nitrate reductase synthesis is enhanced by nitrate. Nitrate is found to induce nitrate reductase by enhancing the increase of the capacity to synthesize nitrate reductase, and ammonia is found to repress nitrate reductase, by inhibiting the induced increase of the capacity to make the enzyme, or by making it unstable in vivo, or both. The effect of ammonia is partially reversed by nitrate. The addition of ammonium or the removal of nitrate during translation of the induced capacity to synthesize nitrate reductase is found to result in the inactivation of nitrate reductase in vivo. A low level of nitrate in the medium is found to be sufficient for enhancing the induced increase of the capacity to synthesize nitrate reductase, but a higher level of nitrate is required to stabilize the enzyme after its formation. The induced capacity to synthesize nitrate reductase is relatively stable in the presence or absence of nitrate, but not in the presence of ammonia. Neurospora crassa mycelia grown with ammonia as the sole nitrogen source do not contain nitrate reductase activity. Transfer of the mycelia to a medium containing nitrate results in the induction of nitrate reductase. Purine-, pyrimidine-, and amino acid-requiring mutants of Neurospora all require their respective supplements for the induction of nitrate reductase (11), which shows that the synthesis of ribonucleic acid (RNA) and protein are required during the induction of this enzyme. Nitrate and ammonia could exert their effects on transcription, translation, or stability of nitrate reductase during its induction. The antibiotic cycloheximide is known to inhibit protein synthesis completely but to allow some synthesis of RNA in eukaryotic cells. The type of RNA that accumulates during treatment with cycloheximide is predominantly deoxyribonucleic acid (DNA)-like, is labile in vivo, and is believed to be messenger RNA (mrna) (3, 6). In germinating cotton seeds, the capacity for the synthesis of a proteolytic enzyme appears during treatment ' Present address: Department of Botany, University of Michigan, Ann Arbor, Mich with cycloheximide, and actinomycin D blocks this capacity (7). Turner et al. (14) describe the accumulation of mrna specific for kynureninase when N. crassa is treated with cycloheximide and an a.ppropriate inducer of the enzyme. The accumulation of mrna, but not its expression, is sensitive to actinomycin D. Actinomycin D is found to prevent the development of amoebae and the incorporation of 3Huridine into RNA of whole cells when added at an early stage of morphogenesis in the slime mold Dictyostelium discoideum, but it does not affect the incorporation of "4C-amino acids into protein in this organism (9). In the experiments described below, we have attempted to separate transcription and translation during the induction of nitrate reductase in N. crassa by using the antibiotics, cycloheximide and actinomycin D, and have looked at the role of nitrate and ammonia in each individual stage during this induction process. MATERIALS AND METHODS Strains. N. crassa wild-type strain 74A was used in this study. Chemicals. Actinomycin D and cycloheximide 547

2 548 SUBRAMANIAN AND SORGER J. BACTERIOL. were purchased from Sigma Chemical Co., St. Louis, Mo.; a-amanitine was obtained as a gift from T. Wieland, Max Planck Institut for Zellchimie, Heidelberg, Germany. Permeabilization of mycelia. Mycelia were made permeable to actinomycin D by pretreatment with 0.1 M phosphate buffer (ph 6.2) containing 0.5% disodium et,hylenediaminetetraacetate (EDTA), essentially as described by Urey and Horowitz (15). Mycelial pads were washed with water and subsequently incubated for 7 min in 20 ml of buffer containing 0.5% EDTA, with frequent swirling. The buffer was decanted and the mycelial pads were incubated for an additional 6 min with swirling in fresh EDTAcontaining buffer. The mycelia were finally washed five times with double-distilled water. Method for the intended separation of transcription and translation. The procedure used in this study is outlined in Table 1. Mycelia were grown in ammonium medium in stationary cultures for 40 hr, washed with water, transferred to medium containing cycloheximide (1 gg/ml), and shaken in a rotary shaker at 27 C for up to 60 min (Step I). They were then washed with double-distilled water and subsequently treated with buffer containing EDTA as described above. The pads were then washed thoroughly with double-distilled water, transferred to medium containing both actinomycin D (50 gg/ml) and cycloheximide (1 jg/ml), and shaken at 27 C for up to 150 min (Step II). They were then washed with water, transferred to medium containing only actinomycin D (50 gg/ml), and shaken as before for 105 min (Step III). At the end of Step III, mycelia were washed thoroughly with icecold water and pressed between folds of filter paper, and each mycelial pad was extracted by grinding with twice its weight of powdered silica with 2 ml of ice-cold 0.1 M potassium phosphate buffer (ph 7.0). The homogenate was centrifuged at 500 x g for 10 min, and the supernatant fluid was used for the enzyme assays. Enzyme assays. Nitrate reductase was assayed as described earlier (13). Protein was estimated by using the biuret method (2). One unit of nitrate reductase activity is defined as the amount of enzyme catalyzing the formation of 1 nmole of nitrite per min. Specific activity is expressed as units per milligram of protein. RESULTS Cycloheximide, an inhibitor of protein synthesis in eukaryotic cells, is known to be very effective in inhibiting the synthesis of nitrate reductase, at very low concentrations (12, 13). The action of cycloheximide is instantaneous, and the mycelia do not need to be preincubated with this antibiotic for the synthesis of nitrate reductase to be stopped. In this study many inhibitors of RNA synthesis, including the antibiotics actinomycin D, a-amanitine, and rifampin, and the purine analogues, 6- methyl purine and 8-azaguanine, have been examined for their effect on the induction of nitrate reductase. Of these, a-amanitine, which is known to inhibit nuclear RNA polymerase of eukaryotes (5), rifampin, which is known to inhibit RNA polymerase of bacteria (1), and 8-azaguanine, have been found to have absolutely no effect on the synthesis of nitrate reductase in N. crassa (results not shown). The lack of inhibition by a-amanitine is particularly surprising since Neurospora is a eukaryote. 6-Methyl purine inhibits to some extent but is not quite satisfactory, and after the mycelia are preincubated with this inhibitor they become refractory to it (Table 2). Actinomycin D, which is known to inhibit DNA-dependent RNA synthesis in both prokaryotes and eukaryotes (10), is found to be an effective inhibitor of the synthesis of nitrate reductase (Table 2). Actinomycin D, at a concentration of 50 gg/ml, stops nitrate reductase synthesis almost completely after the mycelia have been preincubated with this antibiotic. Therefore, in the experiments to be described below, actinomycin D is used as the inhibitor of choice. The mycelia have to be pretreated with buffer containing EDTA for actinomycin D to be effective in blocking the induction of nitrate reductase. Turner et al. (14) found a much lower amount of actinomycin D (2 gg/ml) to be effective in preventing the induction of kynu- TABLE 1. Technique used to manipulate different stages in the induction of.trate reductase Durationa Antibiotic(s) present in Step (min) medium Process(es) inhibited Process studied I 60 Cycloheximide Translation Transcription in the absence of translation II 150 Cycloheximide and Transcription and mrna stability actinomycin D translation III 105 Actinomycin D Transcription Translation in the absence of transcription aunless otherwise stated.

3 VOL. 110, 1972 TRANSCRIPTION OF NITRATE REDUCTASE549 TABLE 2. Effect of inhibitors of RNA synthesis on the induction of nitrate reductase Final concn of Fnlcnno pcfcatvt Inhibitor EDTA treat- inhibitor in pre- Preincubation ment incubation time (hr) ifnhibltconicn inf Speciftcactireity ion in-i of nitate mediuma duction medium ductase None No None No None Yes Methyl purine No x 10-4 M Methyl purine No 5 x 10-4 M x 10-4 M 64.5 Actinomycin D Yes 10 tig/ml ug/ml 30.1 Actinomycin D Yes 10 gg/ml ,g/ml 13.0 Actinomycin D Yes 20 Ag/ml ug/ml 20.9 Actinomycin D Yes 20,g/ml tsg/ml 6.5 Actinomycin D Yes 50 Ag/ml ,gg/ml 12.8 Actinomycin D Yes 50 jig/ml ,ug/ml 0.3 a Ammonia-grown mycelial pads were harvested, treated with EDTA-containing buffer (or not), and preincubated in media containing no nitrogen source in the presence or absence of inhibitors (preincubation medium). They were then transferred to nitrate media with or without the addition of inhibitors (induction medium). After incubation for 2.5 hr, the nitrate reductase activities of the mycelia were determined. reninase in N. crassa. They could employ the lower amount because they exposed the mycelia to EDTA throughout the induction procedure and made them more permeable to actinomycin D. In our studies we find that nitrate reductase is not induced when EDTA is present throughout the induction procedure. Consequently, we have had to give the mycelia a brief treatment with EDTA and then expose them to a high level of actinomycin D to ensure that at least a small fraction of it gets into the cells. The level of actinomycin D that we have used is comparable to that used by Lewis and Fincham (8) in their studies on nitrate reductase in Ustilago maydis. EDTA treatment alone, as described in this study, does not inhibit the capacity of the mycelia to synthesize nitrate reductase (Table 2). Separation of different stages of induction. The procedure used to attempt the separation of transcription from translation during the induction of nitrate reductase is outlined in Table 1. The mrna specific for nitrate reductase should accumulate in Step I in the presence of inducer, but translation of this mrna should not be allowed in this step because of the presence of cycloheximide. In Step II, the mycelia are preincubated with actinomycin D, and, because of the continued presence of cycloheximide, translation to form protein should not take place. In Step III, only acintomycin D is present, and, even though it should stop the formation of new mrna, the translation of existing mrna should occur. The effect of the nitrogen source on the individual stages of induction of nitrate reductase is shown in Table 3, panels A and B. When nitrate is present throughout Steps I, II, and III, nitrate reductase is made. When mycelia are not given the Step I treatment and are preincubated with actinomycin D in medium containing cycloheximide but no nitrogen source, and are then treated with nitrate and actinomycin D in Step III, they possess negligible nitrate reductase activity. This result emphasizes the effectiveness of actinomycin D after preincubation of the mycelia with it, since, in spite of the presence of nitrate in Step III, the synthesis of nitrate reductase does not occur. This result also indicates that the induced increase in the capacity to make nitrate reductase occurs mainly in Step I. Mycelia that are treated with nitrate and cycloheximide in Step I and are preincubated with actinomycin D in Step II in medium containing nitrate and cycloheximide, and are then harvested without being given the treatment in Step III, are found to have negligible nitrate reductase activity. This observation shows that, as long as cycloheximide is present, no protein synthesis can take place, and that translation should occur only in Step Ill, in which cycloheximide is not present. Role of nitrate and ammonia in the individual stages during the induction of nitrate reductase. The nitrate reductase activity of the mycelia is found to be maximal when nitrate is present as the nitrogen source

4 550 SUBRAMANIAN AND SORGER J. BACTERIOL. TABLE 3. Effect of the nitrogen source on the individual stages of induction of nitrate reductase. ~~~~~~Duration Duato of Nitrogen sourcea employed in Specific Spcfi. ac-c trate re- Step I Step Step III Step I Step III ductase Panel treatment (min) tivity of ni- A No nitrogen source Nitrate Nitrate Nitrate B Nitrate Nitrate Nitrate No nitrogen source No nitrogen source No nitrogen source No nitrogen source No nitrogen source Nitrate No nitrogen source Nitrate Nitrate C Nitrate No nitrogen source Nitrate Nitrate No nitrogen source No nitrogen source Nitrate No nitrogen source No nitrogen source Nitrate No nitrogen source No nitrogen source D Nitrate + ammonium Nitrate + ammonium Nitrate Nitrate + ammonium Nitrate Nitrate Nitrate + ammonium No nitrogen source Nitrate Nitrate Nitrate + ammonium Nitrate Nitrate Nitrate Nitrate + ammonium Nitrate Nitrate Nitrate + ammonium E Nitrateb Nitrateb a Nitrate and ammonium, when provided as sources of nitrogen, were each present at 20 mm final concentration in the culture medium. b Ammonia-grown mycelia were EDTA-treated and were preincubated in medium containing no nitrogen source with actinomycin D for 150 min. They were then transferred to medium containing nitrate, cycloheximide, and actinomycin D and incubated for 60 min. They were subsequently transfeffed to medium containing nitrate and actinomycin D, incubated for 105 min, harvested, and extracted, and the extracts were assayed for nitrate reductase activity. in Steps I through III (Table 3, panel B). This maximum level of enzyme formed is about one-tenth of the maximum level induced when the mycelia are not treated with antibiotics (Table 2). The finding that nitrate reductase is synthesized even in the presence of actinomycin D in Step III shows that actinomycin D does not block the translation of the induced capacity to form nitrate reductase. When nitrate is omitted from Step I and the mycelia are incubated with cycloheximide in medium containing no nitrogen source, the amount of nitrate reductase synthesized is relatively low (Table 3, panel B) even when nitrate is present in Step II or Step Ill or in both. This shows that the induced increase in the capacity to make nitrate reductase occurs with high efficiency only if nitrate is present. The omission of nitrate from Step II, when nitrate is present in Steps I and III, is found to give rise to nearly as much enzyme synthesis as when nitrate is present in all the steps (Table 3, panel C). This result shows that the induced capacity to synthesize nitrate reductase is relatively stable in the absence of nitrate, even when no translation takes place. On the other hand, the omission of nitrate from Step III results in low nitrate reductase activity (Table 3, panel C). The longer the time of incubation in a medium containing no nitrogen source, the greater is the loss of activity. If nitrate is necessary only for the translation of preformed mrna, then the increasing fall in activity with increasing incubation period in Step III is difficult to explain. A simpler explanation is that nitrate has a stabilizing effect on the enzyme in vivo and that nitrate reductase undergoes inactivation in

5 VOL. 110, 1972 TRANSCRIPTION OF NITRATE REDUCTASE 551 vivo when nitrate is not present in Step III during its formation. This finding supports the results reported in an earlier paper (12). The effect of the presence of ammonia in one or more of the individual steps on the synthesis of nitrate reductase is presented in Table 3, panel D. The presence of ammonia in Steps I or II is found to cause a large decrease in the synthesis of nitrate reductase. Thus, ammonia acts by inhibiting the increase in the induced capacity to synthesize nitrate reductase, or by making this capacity unstable in vivo, or both. It can be noted that the synthesis of nitrate reductase is less when ammonia is present in Step II than when it is present in Step I. Therefore, it seems probable that ammonia has a greater effect on the stability of the capacity to synthesize nitrate reductase than on the induced increase of this capacity. The presence of ammonia in Step III causes only a slight lowering of nitrate reductase activity. Again, with an increasing time of incubation with ammonia in Step III, there is an increasing loss of nitrate reductase activity. Thus, the data presented in the preceding paper (12) on the inactivation of nitrate reductase in vivo caused by ammonia are confirmed in this study. When ammonia-grown mycelia are preincubated with actinomycin D and then incubated with nitrate, cycloheximide, and actinomycin D, and are then finally incubated with only nitrate and actinomycin D, very little nitrate reductase is synthesized (Table 3, panel E). Thus, the capacity for the formation of nitrate reductase, synthesized in Step I, is effectively blocked by actinomycin D. Kinetics of induced increase in the capacity to synthesize nitrate reductase in the presence of cycloheximide. The kinetics of induced increase of the capacity to synthesize nitrate reductase in Step I is presented in Fig. 1. The extent of synthesis of the presumed specific mrna in Step I is measured by the activity of nitrate reductase synthesized in Step III due to the translation of the former. It can be seen that the capacity for the formation of nitrate reductase increases up to about 23 min of exposure to nitrate in presence of cycloheximide, and then remains constant. Kinetics of translation of induced capacity to synthesize nitrate reductase in the presence of actinomycin D. The kinetics of translation of the capacity to synthesize nitrate reductase is presented in Fig. 2. There is not much enzyme synthesis up to about 40 min after transfer of mycelia to Step III when the mycelia are recovering from cycloheximide toxicity. Then enzyme activity increases linearly up to about 163 min, after which it is constant. The maximal level of enzyme activity after 163 min of incubation in Step III (Fig. 2) is found to be almost one-fifth of the enzyme synthesized in a normal induction without the use of antibiotics (Table 2). Effect of various concentrations of nitrate and ammonia on the synthesis of nitrate reductase. The effect of various concentrations of nitrate in Steps I or III on the synthesis of nitrate reductase is shown in Table 4. The presence of nitrate in Step I at one-hundredth of its usual level does not affect significantly the maximal level of nitrate reductase synthesized. But decreasing the nitrate concentration in Step Ill is found to cause a significant reduction in the activity of nitrate reductase. It can be seen from the results presented in Table 3 that ammonia represses or antagonizes the induced increase in the capacity to synthesize nitrate reductase. The results of an experiment designed to investigate the effect of different nitrate concentrations on the ammonia u Ul U. Ul w IC w U) zi 4c c- U I~ J U I U U I Time in STEP I (minutes) FIG. 1. Kinetics of transcription of the mrna specific for nitrate reductase. Ammonia-grown mycelia were incubated in medium containing 20 mm nitrate and 1 gg of.cycloheximide per ml for various time intervals (Step 1). They were then treated with EDTA-containing buffer and incubated in nitrogenfree medium containing cycloheximide (1 usg/ml) and actinomycin D (50 jug/ml) for 150 min (Step II). They were finally transferred to medium containing nitrate (20 mm) and actinomycin D (50 ug/m() and incubated for 105 min (Step III). The nitrate reductase activity of the mycelia was then determined.

6 552 SUBRAMANIAN AND SORGER J. BACTERIOL. mu ;6 I- a I-- 2c (1- I~ I~~~~~ I ISO Time in STEP m (minutes) FIG. 2. Kinetics of translation of preformed nitrate reductase mrna. Ammonia-grown mycelia were incubated in media containing nitrate (20 mm) and cycloheximide (1 jug/ml) for 60 min (Step 1). They were then treated with EDTA-containing buffer and given the Step II treatment for 150 min in nitrogen-free media. They were subsequently transferred to media containing nitrate (20 mm) and actinomycin D (50 jg/ml) (Step III), and at various time intervals, mycelia were harvested and their nitrate reductase activity was determined. effect are presented in Table 4. The repression by ammonia is maximal at the lowest nitrate concentration, and increasing the concentration of nitrate causes a partial reversal of the ammonia effect. DISCUSSION Turner et al. (14) have described the separation of transcription and translation during the induction of kynureninase in N. crassa. They treated logarithmically growing cells of N. crassa with the inducer kynurenine in medium containing cycloheximide and then washed the cells and transferred them to inducer-free medium. The mrna for kynureninase synthesized during exposure to the inducer in presence of cycloheximide is translated to give rise to kynureninase after transfer to inducer-free medium which does not contain cycloheximide. We have not been able to adopt a similar procedure for the separation of transcription and translation during the induction of nitrate reductase because nitrate reductase is rapidly inactivated in vivo in the absence of nitrate (12). Therefore it was essential to have nitrate in the system throughout Step III. In order to stop nitrate from starting another cycle of transcription and translation, it was necessary to have actinomycin D along with I TABLE 4. Effect of nitrate and ammonia levels on the synthesis of nitrate reductase Final nitrate concn Final concn of Specific activ- (mm) ammonium in ity of nitrate Step I Step IHa Step I (mm) reductase a Mycelia, after Step I treatment, were preincubated with actinomycin D in medium containing cycloheximide and no nitrogen source in Step II, and were then given the Step m treatment. nitrate in Step III (actinomycin D should not interfere with the translation of preformed mrna). Since actinomycin D is most effective only after the mycelia are preincubated with it, Step II was introduced between the steps controlling transcription and translation. Like the inducible enzyme systems in bacteria, nitrate appeared to induce the synthesis of nitrate reductase in N. crassa by increasing the capacity to synthesize nitrate reductase (presumeably by enhancing the synthesis of specific mrna). This capacity to synthesize the enzyme can then be translated in the presence or absence of inducer (Table 3, panel C). The removal of the inducer, nitrate, or the addition of the corepressor, ammonia, does not merely stop the further synthesis of nitrate reductase, but also results in the disappearance of existing enzyme activity. During the induction of nitrate reductase in N. crassa without the use of antibiotics, the enzyme activity increases from nothing in ammoniagrown mycelia to a high level in the nitrateinduced ones (12, 13). Such a high efficiency of induction appears to be similar to the induction of inducible enzymes in bacteria and is unlike the induction of enzymes in eukaryotic cells. The results of this paper reveal that the nitrate reductase of Neurospora is synthesized with low efficiency when mycelia are simply transferred from repressing to nonrepressing conditions (Table 3, panel B) and that nitrate augments this induction about sixfold. The above pattern of induction of nitrate reductase is like the pattern of induction of enzymes in eukaryotes and differs from that in bacteria. The concentration of nitrate required to give

7 VOL. 1 10, 1972 TRANSCRIPTION OF NITRATE REDUCTASE 553 a maximally induced increase in the capacity to synthesize nitrate reductase is found to be relatively low, whereas a relatively high concentration of nitrate is required to stabilize the enzyme during its formation by translation (Table 4). This would explain why the inducer, nitrate, appears to be required in relatively high amounts for the induction of nitrate reductase. Ammonia (i) represses or renders labile the capacity to synthesize nitrate reductase, or both, and (ii) causes the inactivation of the enzyme as it is formed (Table 3, panel D). Nitrate does not seem to overcome the effect of ammonia on enzyme degradation (12) but does seem to overcome partially the effect of ammonia on the induced increase or the stability of the capacity to synthesize nitrate reductase, or both (Table 4). The removal of a nitrogen source from the medium seems to have no effect on the stability of the capacity to synthesize nitrate reductase (Table 3, panel C), but it results in a very rapid inactivation of existing nitrate reductase (12). The data seem to suggest the following hypothesis. The capacity to synthesize nitrate reductase may be made labile by ammonia; the induced increase of this capacity is regulated antagonistically by nitrate and ammonia, the former acting as an enhancer and the latter as a depressant. The antagonistic action of ammonia and nitrate may operate through an allosteric aporepressor, but the effect on stability would have to be different. There seems to be no evidence that nitrate or ammonia, or both, affect the translation of the induced capacity to make nitrate reductase, but it is apparent that both these molecules control the stability of the enzyme in vivo, possibly by affecting the conformation of nitrate reductase and thus its recognition by a rapidly turningover protease (see reference 12). In the presence of cycloheximide, the accumulation of the induced capacity to synthesize nitrate reductase continues for about 23 min and stops (Fig. 1). Once formed, induced capacity to make the nitrate reductase appears to be relatively stable in the presence of actinomycin D for several hours. This stability may be due to the stabilizing effect of actinomycin D in Steps II and Ill, similar to the stabilizing effect of actinomycin D on mrna in rat liver (4). Our conclusions are therefore drawn with the qualification that acintomycin D could influence the effects of nitrate or ammonia, or both. ACKNOWLEDGMENTS We thank the National Research Council of Canada for grant no. A3649 which made this investigation possible. One of the authors (K. N. Subramanian) thanks McMaster University for a Teaching Postdoctoral Fellowship. LITERATURE CITED 1. Calvori, C., L. Frontali, L. Leano, and G. Tecce Effect of rifamycin on protein synthesis. Nature (London) 207: Dawson, R. M. C., D. C. Elliott, W. H. Elliott, and K. M. Jones Data for biochemical research, p Oxford University Press, New York. 3. de Kloet, S. R Accumulation of RNA with a DNA-like base composition in Saccharomyces carlsbergensis in the presence of cycloheximide. Biochem. Biophys. Res. Commun. 19: Endo, Y., H. Tominaga, and Y. Natori Effect of actinomycin D on turnover rate of messenger ribonucleic acid in rat liver. Biochim. Biophys. Acta 240: Fiume, L., and T. Wieland Amanitins. Chemistry and action. Fed. Eur. Biochem. Soc. Lett. 8: Fukuhara, H RNA synthesis of yeast in presence of cycloheximide. Biochem. Biophys. Res. Commun. 18: Ihle, J. N., and L. Dure m Synthesis of a protease in germinating cotton cotyledons catalyzed by mrna synthesized during embryogenesis. Biochem. Biophys. Res. Commun. 36: Lewis, C. M., and J. R. S. Fincham Regulation of nitrate reductase in the basidiomycete Ustilago maydis. J. Bacteriol. 103: Mizukami, Y., and M. Iwabuchi Effects of actinomycin D and cycloheximide on morphogenesis and syntheses of RNA and protein in the cellular slime mold, Dictyostelium discoideum. Exp. Cell Res. 63: Reich, E., and I. H. Goldberg Actinomycin and nucleic acid function, p In J. N. Davidson and W. E. Cohn (ed.), Progress in nucleic acid research and molecular biology, vol. 3. Academic Press Inc., New York. 11. Sorger, G. J Simultaneous induction and repression of nitrate reductase and TPNH cytochrome c reductase in Neurospora crassa. Biochim. Biophys. Acta 99: Subramanian, K. N., and G. J. Sorger Regulation of nitrate reductase in Neurospora crassa: stability in vivo. J. Bacteriol. 110: Subramanian, K. N., G. Padmanaban, and P. S. Sarma The regulation of nitrate reductase and catalase by amino acids in Neurospora crassa. Biochim. Biophys. Acta 151: Turner, J. R., K. Terry and W. H. Matchett Temporal separation of transcription and translation in Neurospora. J. Bacteriol. 103: Urey, J. C., and N. H. Horowitz Effects of EDTA on tyrosinase and L-amino acid oxidase induction in Neurospora crassa. Biochim. Biophys. Acta 132: