Enzyme patterns and protein synthesis during synchronous conidiation in Neurospora crassa

DEVELOPMENTAL

BIOLOGY

Enzyme

26, 17-27

Patterns

(1971)

and Protein

Conidiation

Synthesis

during

in Neurospora

crassa

Synchronous

JOHN C. UREY Department

of Biology,

Wheaton College, Norton,

Massachusetts

02766

Accepted April 4, 1971 Both the growth of aerial hyphae and the differentiation of conidia from existing aerials were inhibited by cycloheximide or ethionine. These results suggest a requirement for protein synthesis throughout conidiogenesis. Several results are presented which suggest NADase may be required for the processes culminating in the differentiation of macroconidia. In particular, using a new technique to isolate aerial hyphae and conidia, I discovered that the increase in NADase activity during conidiogenesis was confined to the newly formed aerial hyphae and conidia. No NADase was detected in the old mycelium. In sharp contrast, aryl-8.glucosidase and cellobiase activities increased in both the mycelium and the aerials and conidia. The increases in all three enzyme activities during conidiogenesis were inhibited by cycloheximide and ethionine, a result suggesting the de nouo synthesis of these enzymes. Other results show that aryl-@glucosidase, cellobiase, and a fourth enzyme, trehalase, were not required to form normal numbers of conidia.

1970). Thus it is reasonable to suppose that both aerial growth and conidiation are dependent upon continuing protein synthesis, as I show here they are. The second objective of this paper is to report the time courses of enzymatic changes and associate these with morphological changes occurring in particular parts of the developing organism. Finally, I present evidence suggesting that one of the enzymes, NADase, is required for the events which culminate in conidiation (cf. Combepine and Turian, 1970).

INTRODUCTION

Neurospora crassa grows by the elongation of vegetative hyphae which regularly branch, fuse, and branch again to produce the characteristic mycelial pad. Under appropriate conditions, hyphae within the mycelial mass will grow upward becoming aerial hyphae, and from the tips of these, asexual spores, called macroconidia, are formed. The development of aerial hyphae and conidia is an attractive example of differentiation in a eukaryote because the process appears to be relatively simple. This view is supported by the facts that radical morphological changes do not occur (Weiss and Turian, 1966; Lowry et al., 1967; Richmond et al., 1967; Manocha, 1968) and that only a handful of genes are presently known to interrupt or disturb the process (Barratt and Garnjobst 1949; DelVecchio and Turian, 1968; Grigg, 1958, 1960). Central to the objectives of the present study is the fact that comparisons of the enzymes of vegetative hyphae and conidia have shown that these structures are biochemically distinct (Turian, 1966; Eberhart, 1961; Zalokar and Cochrane, 1956; Stine 1967, 1968; Oulevey-Matikian and Turian, 1968; Comb&pine and Turian,

MATERIALS

AND

METHODS

Strains. Wild-type cultures used throughout were vegetative isolates of a single ascospore obtained from the cross of standard wild types St74A and 74-OR8 la; this strain is designated 74-6A. Davis and Mora (1968) isolated the aconidial mutant UM723 which permits the double mutant arg-5, arg-12” to use exogenous omithine as its arginine source. The mutant CM62, called glut-2, was isolated by Myers and Eberhart (1966) and makes less than 1% of the normal amount of aryl@glucosidase. Growth media. Stocks were maintained 17

18

DEVELOPMENTAL BIOLOGY

on agar slants of medium N (Vogel 1956) containing sucrose (1% w/v) and glycerol (1% w/v). Cultures used for the induction of aerial hyphae and conidia were grown on liquid medium N containing sucrose (1.5% w/v), sodium acetate (0.23% w/v), and Tween 80 (0.01% w/v). Chemicals. Reagent grade chemicals and glass redistilled water were used in all media and solutions. Tween 80 was obtained from Atlas Chemical Co. Cycloheximide was purchased from Nutritional Biochemical Corp. Amino acids were bought from Calbiochem. Sigma Chemical Comp-nitrophenyl-8-nglucoPanY supplied pyranoside. Induction of conidiogenesis. A brief recapitulation of the method set forth by Siegel et al. (1968) is given here. Sixtyeight-hour-old mycelial pads (8 cm diameter), formed in stationary liquid culture, were washed with 0.1 M sodium phosphate buffer pH 6.1 and then the center of each was cut out, leaving a ring about 0.5 cm wide. Rings were transferred on filter paper to 8-inch fingerbowls which were sealed with glass plates. When anhydrous calcium chloride was added to the fingerbowl before it was sealed, the mycelial ring formed aerial hyphae and conidia under conditions of very low humidity. Aerial hyphae and conidia were formed at a high relative humidity when the mycelial ring was transferred to a buffer-soaked filter paper and sealed in a fingerbowl with a reservoir of phosphate buffer. The period during which the mycelial mass was sealed in the fingerbowls is referred to as the induction period. Figures 1 and 2 graph the growth of aerial hyphae and the differentiation of conidia during the induction period. In several experiments it was important to isolate the aerial hyphae from the ring of vegetative hyphae from which they had grown. The method used depended on the fact that aerials readily grew through a filter paper placed over the ring just before it was transferred to the fingerbowl. Eight

VOLUME 26, 1971

hours later, when the aerials had grown up through both layers of filter paper and had just begun to form conidia, the upper paper, carrying the aerials, was carefully removed. When such detached aerial hypahe were placed in a second fingerbowl at high humidity, it was found that they produced normal numbers of conidia. Cycloheximide, a known inhibitor of protein synthesis in Neurospora (Pall, 1966) and ethionine, an analog of methionine, were used to inhibit protein synthesis during the development of aerial hyphae and conidia. A ring of mycelia or of isolated aerial hyphae was cut into several equal sections, some of which were treated with various concentrations of the inhibitor or analog dissolved in 1 ml of 0.1 M phosphate buffer pH 6.1, while others, treated with buffer alone or with methionine, served as controls. The material was transferred to fresh fingerbowls and the subsequent amount of development was measured. Since the treatment of isolated aerial hyphae was started after the beginning of conidiation, one section from each culture was used to determine the number of conidia present at the start of the treatment. Measurement of aerial growth and conid&ion. Macroconidia, isolated by the method of Siegel et al. (1968), were counted in a hemacytometer. The most convenient and reliable method devised for measuring the growth of aerial hyphae during the period of induction was based upon the fact that aerials will grow up-and adhere to-coverslips held perpendicular to the surface of the mycelial ring. In detail, coverslips placed on radii across the rings at the start of the induction period were removed at predetermined intervals and exposed to osmium tetroxide vapors to fix the Each coverslip was adhering aerials. mounted on a slide, examined with a microscope fitted with a micrometer grid, and the number of squares containing aerial material was recorded. Normally no aerial material is lost during these procedures. Extraction and assay of enzymes. Cul-

UREY

Enzyme Patterns during Conidiation

19

Incorporation of leucine- 3H into protein. tures were frozen upon harvesting on dry ice, lyophilized, and weighed. They were Protein synthesis in mycelia and isolated ground with acid-washed sand in a cold aerial hyphae was monitored by the incormortar with pestle and extracted with cold poration of leucine-3H into cold TCA-in0.02 M 2-(N-morpholino)ethanesulfonic soluble material (Roberts, 1964). The acid-potassium hydroxide (MESK) buffer Neurospora was submerged in 2.0 ml 0.1 pH 6.0. Preparations were centrifuged for M phosphate buffer pH 6.1 containing lo- ’ 5 mm at 10,000g and the supernatants were M labeled leucine (0.5 &i/ml) for 1 hr; in stored at - 90°C until assayed. certain experiments, the buffer also conTo ensure adequate amounts of aerial tamed cycloheximide or ethionine. Badiohyphae and conidia for enzyme extraction, activity incorporated was measured with a these structures were harvested from intact Beckman LS-233 liquid scintillation counmycelial pads rather than from rings. The ter and is reported with zero-time controls aerial mass was allowed to grow through subtracted. filter paper (see above) and then was RESULTS shaved from the surface of the paper with a razor blade and placed in 20 ml 0.1 M Aerial Growth and Conidiation during the phosphate buffer pH 6.1. The conidia were Period of Induction freed from the aerial hyphae by vigorous The growth of aerial hyphae during the agitation on a vortex mixer and the preparation was transferred to a cheesecloth period of induction is shown in Fig. 1. The filter. The conidia, after passing through rate was constant and linear for at least 30 hr for cultures maintained at high humidthe filter, were collected by centrifugation and the aerial hyphae were removed from ity, whereas at low humidity the aerials the surface of the cheesecloth. The conidia, grew rapidly for 6 hr and then at a aerial hyphae, and the buffer used in se- markedly reduced rate. Aerial hyphae parating them, were frozen at -90°C until were never observed before the first hour of the induction period. Figure 2 shows the assayed. NADase (NADglycohydrase EC 3.2.2.5) rates at which conidia were produced unwas assayed by the method of Kaplan et al. der conditions of low and high humidity. (1951) and the activity is reported as micromoles NAD destroyed per minute. Me- Protein Synthesis and Conidiogenesis thods for assaying thermolabile cellobiase Table 1 shows that mycelial cultures ( p-n-glucoside glucohydrolase EC 3.2.- treated from the start of the induction 1.21) and thermostable aryl-&glucosidase period with either cycloheximide or ethi(@glucoside glucohydrolase EC 3.2.- onine and incubated in low humidity 1.21) are given by Eberhart (1961). The formed drastically reduced numbers of activity of each enzyme is reported as nano- conidia. Indeed, higher concentrations of moles of nitrophenol liberated per minute. cycloheximide prohibited formation of Trehalase (trehalose-1-glucohydrase EC aerial hyphae. When treatment was started 3.2.1.28) was assayed by the method of soon after the aerial hyphae began to difHill and Sussman (1963) in a 0.05 M ferentiate conidia, there was a significant, MESK buffer pH 5.5. The glucose formed but less dramatic, inhibition of conidiation. was measured by the Somoygi-Nelson The table shows inhibition in cultures which method (Somoygi, 1952). The activity is re- had formed either small numbers or large ported as the absorbance change at 520 nm numbers of conidia prior to treatment. per minute. Protein was measured by the Cycloheximide and ethionine inhibited inmethod of Lowry et al. (1951) using bovine corporation of leucine into protein and serum albumin fraction V as the standard. brought about reduced levels of the specific

20

DEVELOPMENTAL

BIOLOGY

VOLUME

26, 1971

7000 6000 5000

0 : 4000Le rr .-L $000 %; ; 20004 1000 2

4

6

8

IO

12 HOU US

IS

24

30

FIG. 1. Aerial hyphal growth during the induction period. The amount of hyphal material adhering to coverslips placed at the start of induction perpendicular to the surface of the mycelial ring was measured. X--X induction at low humidity (data of R. E. Nelson); O--O induction at high humidity.

activities of NADase, aryl-p-glucosidase, and cellobiase (see Table 2). Changes in Enzyme Activities during Development of Aerial Hyphae and Conidia Figures 3 and 4 show the changes in the specific activities of aryl- j+glucosidase, cellobiase, NADase, and trehalase during the period of induction. During the first hour, there was no detectable growth of aerial hyphae (Fig. 1) nor was there a significant change in enzyme activity. The next 5 hrs was a period of active aerial growth at low humidity and the levels of all four enzymes rose sharply. From the sixth to ninth hour, the period of conidiation, the activities of aryl- &glucosidase, cellobiase, and trehalase continued to increase. In contrast, NADase activity did not increase after the sixth hour. The time course of changes in NADase and cellobiase were the same for cultures induced under conditions of high humidity as at low humidity. In contrast, at high humidity, trehalase activity

did not increase at all and aryl-P-glucosidase increased sharply only early during the period of hyphal growth. These large increases in specific activity should be interpreted as increases in the total enzyme activity present since the amount of extractable protein decreased less than 10% during induction. In the case of NADase, the activity reported for myCelia1 extracts is less than the total activity made because the enzyme is easily washed out of hyphae and conidia (Zalokar and Cochrane, 1956). Making the reasonable assumption that the proportion of the total enzyme leaving the mycelium per unit time is constant throughout the induction period, it can be concluded that NADase activity appears during early aerial hyphal growth. When preparations with low and high enzymatic activities were mixed, the activity of the mixture was the sum of the activities of each component. Therefore, it is not likely that the changes in enzyme activity accompanying aerial growth and

UREY

Enzyme Patterns during Conidiation

conidiation were due to changes in the levels of enzyme activators or inhibitors. The Distribution of the Enzymes within the Developing Organism

The next observations were designed to associate enzymatic activities with particular parts of the developing organism. Table 3 reports the distribution of the activities of three enzymes among conidia, aerial hyphae, and nonaerial hyphae. NADase activity was present only in the aerials and conidia. No increase occurred in the ring of vegetative mycelia during the induction period. On the other hand, aryl-gglucosidase and cellobiase activity appeared in all three parts. Part of the activity of each enzyme was washed out of the aerials and

21

conidia by the buffer used in separating them (see Materials and Methods) and is reported separately in Table 3. The relative contribution of the enzyme pools in the aerials and conidia to the activity in the buffer is unknown. The increases in aryl-/3-glucosidase and cellobiase in the mycelial pad suggested that these enzymes might be formed under conditions which prohibit the formation of aerial hyphae and conidia. Furthermore, since NADase was confined to the aerials and conidia, its activity might not increase in the absence of aerial development. In the experiment reported in Table 4, an attempt was made to reproduce two impoftant aspects of the induction conditions, namely a metabolic stepdown from growth

FIG. 2. The number of conidia formed during the induction period. x--X, induction at low humidity (taken from Siegel et al., 1968)., O--O, induction at high humidity, - - from same culturesused to obtain data in Fig. 1.

22

DEVELOPMENTAL BIOLOGY

VOLUME 26, 1971

TABLE 1 INHIBITION OF CONIDIOGENESISBY CYCLOHEXIMIDE AND BY ETHIONINE” Material Mycelial

and stage treated

Additions

rings at the start of induction

Isolated aerial hyphae during early conidiation

to buffer

Number of conidia formed ~.-

None Cycloheximide 0.10 pg/ml L-Methionine 100 pg/ml nL-Ethionine, 100 pg/ml

5.4 5 2.6 4

x x x x

10’ 10’ lo8 10’

None Cycloheximide, 0.10 pglml None L-Methionine, 50 pg/ml nL-Ethionine, 50 pg/ml

3.0 7 2.0 2.1 5.1

x x x x x

108 10’ 10’ 107 108

a Mycelial rings were induced at low humidity after being washed with 0.1 M phosphate buffer pH 6.1 containing the substances listed in the table. The rings were harvested after 12 hr induction. Aerial hyphae were isolated as described in the Materials and Methods. They were placed in a fingerbowl at high humidity and allowed to complete differentiation in the presence of phosphate buffer containing the substances shown in the table. The aerials treated with cycloheximide had made 6 X lo3 conidia per ring and those with ethionine 9 X lo5 conidia prior to their isolation. TABLE 2 INHIBITION OF PROTEIN SYNTHESIS AND ENZYME INCREASE BY CYCLOHEXIMIDE AND ETHIONINE~

Additions buffer

to

T

^

._.

Specific activity (units/mg protein)

TCA insoluble Leu- $H (cpm) 1NADast

1$&!zCelloidase

biase

___ None Cycloheximide, 1 pglml L-Methionine, 50 g/ml m-Ethionine, 50 rglml “Mycelial rings phosphate buffer shown at the start and the rings were tion of leucine into sured.

7700 2290

1.38 0.25

6.4 0.88

11.0 5.0

8990

2.90

5.10

14.5

6620

0.22

2.30

10.0

of 74-6A were washed with 0.1 M pH 6.1 containing the inhibitors of induction under low humidity, harvested after 8 hr. The incorporasections of a ring for 1 hr was mea-

medium to buffer and an increase in available oxygen. The washed mycelial rings were submerged in phosphate buffer through which pure oxygen was bubbled. Under these conditions, which prohibited aerial development and conidiation, the activities of aryl-@-glucosidase and cellobiase increased while that of NADase did not.

Mutants with Low Levels of Aryl-&lucosidase and of NADase Myers and Eberhart (1966) reported that the mutant CM 62, called glut-2, makes less than 1% the wild-type __ amount of aryl- P-glucosidase and has conidia. The data in Table 5 confirm his observations, for they show that induced cultures of this strain made numbers of conidia comparable to the wild-type controls, yet aryl-/3-glucosidase activity was not detected. The mutant UM 723 was reported to be an aconidial strain by Davis and Mora (1968). I found that vegetative cultures of this strain form very few aerial hyphae and no conidia when transferred to our standard conditions for induction. Assays for NADase revealed no increase in activity during the induction period. DISCUSSION

Events culminating in the production of macroconidia can be conveniently divided into three phases. While these events and stages are readily observed when vegetative mycelia are induced according to the methods used here, characteristically different results may be found under other conditions. There is an initial lag phase

UREY

Enzyme Patterns during Conidiation

lasting about an hour during which neither aerial hyphal growth nor enzymatic changes were detected. This lag may be analogous to the lag in bacterial growth following a “stepdown” from a rich medium to a poor one. The second phase is marked by extensive aerial growth and dramatic changes in the rates of enzyme synthesis. It is a 3-4 hr period during which

0

2

4

23

important changes evidently occur in aerial hyphae and vegetative mycelia for in both structures the levels of aryl-gglucosidase and cellobiase. show marked increases. On the other hand, the fact that NADase activity increased in the aerials but not in the vegetative hyphae demonstrates the existence of spatial differentiation within the developing system. These data con-

8

10

FIG. 3. Changes in enzyme activities during the induction period at low humidity. Mycelial rings, which had been induced at low humidity, were harvested at the times shown, and the soluble enzymes extracted and assayed. NADase; O--O, aryl-@-glucosidase; x--x, O--O, trehalase; A--A, cellobiase.

24

DEVELOPMENTAL

5

BIOLOGY

4

B

VOLUME

B

26, 1971

lb

1’2

HOURS

FIG. 4. Changes in enzyme activities during the induction period at high humidity., Mycelial rings, which had been induced at high humidity, were harvested at the times shown and the soluble enzymes extracted and assayed. X--X, NADase; O--O, aryl-gglucosidase; O--O, trehalase; A---A, cellobiase.

firm and extend Zalokar’s (1959) earlier report of morphological and biochemical differentiation within a growing mycelial mass. The third and final phase is characterized by the rapid formation of free macroconidia. As before, protein synthesis is required during this phase, strongly suggesting that conidiation involves de nouo enzyme synthesis. In addition, new structural proteins, such as those which may serve to complete the septa between adjacent conidia are probably synthesized during this phase. With the onset of conidi-

ation, new aerial growth is virtually abolished in cultures induced at low humidity. But at high humidity, conidiation is accompanied by the undiminished elaboration of aerial hyphae. Since the growth of aerial hyphae and the differentiation of conidia are inhibited by cycloheximide and by ethionine, both processes probably require continuing protein synthesis. This conclusion is strengthened by the fact that cycloheximide (Ennis and Lubin, 1964; Siegel and Sisler, 1964) and ethionine (Kappy and Metzenberg, 1965)

UREY

25

Enzyme Patterns during Conidiution

tell me which, if any, of these enzymatic activities is essential for development. Trehalase activity has been associated with conidiation here and by Hanks and Length AeriCon- Buf- Sussman (1969). My finding that mycelia MYEnzyme 0 f induc idia Celia als ferb induced at high humidity produced normal t ion (hr) / numbers of conidia with no increase in tre8 NADase, u/mE : 0.02 1.1 halase activity confirms and strengthens 24 Protein 0.03 0.45 , 2.1 .94 their conclusion that trehalase is not essen8 Aryl-@-gluco50 22 tial to conidiogenesis. This is proved by the sidase, isolation of Sargent and Braymer (1969) u/mg pro24 32 42 14 14 tein of trehalase-less mutants which form Cellobiase, 8 29 18 conidia. Cellobiase (Mahadevan and Eberu/mg pro24 37 33 17 5.5 hart, 1964) occurs in the conidia produced tein __ under our conditions of induction. How“Whole mycelial pads of 74-6A were induced ever, Eberhart’s report (Eberhart et al., under conditions of high humidity for 8 or 24 hr. 1964), confirmed in this laboratory, that the TABLE 3 ENZYME DISTRIBUTION AMONG THE MYCELIA, AERIAL. HYPHAE, AND CONIDIA DURING INDUCEJJ CONIDIATION AT HIGH HUM~ITP

1

Aerial hyphae were removed and the conidia present at 24 hr were isolated as explained in the methods. bThe buffer used in separating the aerial hyphae and conidia.

act by entirely different mechanisms. It seems probable that the inhibitors act directly upon protein synthesis necessary for the process of conidiation per se, rather than indirectly through an effect on aerial growth; this is inferred from the observation that aerials detached from the mycelium normally form conidia but no more aerials (at high humidity), an event which is blocked by treatment with these inhibitors of protein synthesis. Thus these results confirm and extend, Strauss’s observation of the effect of ethionine on conidiation (Strauss, 1958). The characteristic increase in the specific activities of aryl-P-glucosidase, cellobiase, and NADase during the second phase can be inhibited by treatment with ethionine or cycloheximide. The interpretation that these enzymes, among others, are synthesized de nouo during induction is strengthened by the fact that extracts of the developing organism failed to yield evidence of either activators or inhibitors of these enzymes. But the observed correlations between morphogenetic changes and increases in units of enzyme does not

TABLE 4 ENZYME ACTIVITIES IN MYCELIAL FLINGSDURING “SUBMERGED INDUCTION” UNDER OXYGEN~

Conditions of induction

Specific activity (units/mg protein) NADase

Standard at high humidity Submerged with oxyge+

Aryl-B glucosidase

2.70

14

0.03

5

Cellobiase 10 6.6

n Both cultures were induced for 6 hr. b A mycelial ring of 74-6A was submerged in 50 ml of 0.1 M phosphate buffer pH 6.1 containing Tween 80 (0.01% w/v), and oxygen was bubbled vigorously through the buffer continuously. In addition to extracts of the mycelial rings, this buffer was assayed for each enzyme. TABLE 5 CONIDIA PRODUCTION AND ENZYME ACTIVITIES IN GLUC-2 DURING INDUCTIONS Specific activity (units/mg protein) Strain

Nxo;;;;aof

8.4 x 10’ 7.9 x 107

74-6A CM62 glut-2 “Each midity.

strain

was induced

Aryl- 8glucosidase

Cellobiase

28 <0.5

6.2 6.1

for 9 hr at high hu-

26

DEVELOPMENTAL

BIOLOGY

enzyme cannot be detected in conidia from slant cultures, together with the fact that the enzyme increased in submerged oxygenated hyphae which did not conidiate (see Table 4) all but excludes the possibility that it plays an important role in conidiation. Nor does it appear that aryl-fl-glucosidase is required for conidiation, since the mutant glut-2 could be induced to make normal numbers of conidia but no detectable enzyme. As in the case of cellobiase, its increase during the period of induction is probably a response to aerobic starvation. NADase appears to play an important, and perhaps an essential, role in the biology of the conidia. Following the discovery and characterization of the enzyme in Neurosporu (Kaplan et al., 1951), Zalokar and Cochrane (1956) found that activity insharply during conidiation; creased NADase concentration is highest in the conidia and is very low or absent in actively growing vegetative hyphae. The correlation between conidiation and rapid increase in NADase activity was confirmed by Stine (1968), who added the important facts that the activity begins to increase during the growth of aerial hyphae and that it decreases sharply as mature conidia germinate. Combepine and Turian (1970) have also concluded that NADase may be important in conidiogenesis. Because I was able to separate the aerial hyphae (conidiophores) from the mycleial pad, I have been able to demonstrate that all new NADase activity is confined to the developing aerial mass. The correlations between NADase and aerial hyphal growth are further strengthened by studies of the mutant UM723 which forms neither aerial hyphae nor NADase in our inducing system. On the other hand, there is no evidence to support the possibility that conidiation per se induces NADase synthesis since Stine (1968) has shown that the aconidial strain fluffy makes aerial hyphae, or sterile conidiophores, characterized by higher amounts of

VOLUME

26; 1971

enzyme. A decisive answer to the question of the role of NADase in the life cycle of Neurosporu awaits the analysis of appropriate mutants. This work was carried out while the author was a Research Zoologist in the laboratory of Dr. R. W. Siegel at the University of California at Los Angeles. The work was supported by grants to Dr. Siegel from the National Science Foundation and the Cancer Research Funds of the University of California. Dr. Siegel was particularly helpful in the preparation of this manuscript, and his stimulating support is gratefully acknowledged. REFERENCES BARRA~, R. W., and GARNJOBST,L. (1949). Genetics of a colonial microconidiating strain of NeuFospOFa crassa. Genetics 34, 351-369. COMB~PINE, G., and TURIAN, G. (1970). Activites de quelques enzymes associes a la conidiogenese du Neurospom crassa. Arch. Mikrobiol. 72. 36-47. DAVIS, R. H., and MORA, J. (1968). Mutants of Neurospora crussa deficient in omithine-&transaminase. J. Bacterial. 96, 383-388. DELVECCHIO, V. G., and TURIAN, G. (1968). Intraconidial conidia in the spray mutant of Neurospora crassa. J. Gen. Microbial. 52, 461-465. EBERHART, B. (1961). Exogenous enzymes of Neurospora conidia and mycelia. J. Cell. Comp. Physiol. 53, 11-16. EBERHART, B., CROSS, D. F., and CHASE, L. R. (1964). @-glucosidase system of NeuFOspoFa crassa. I. & glucosidase and cellulase activities of mutant and wild-type strains. J. Eacteriol. 87, 761-770. ENNIS, H. L., and LUBIN, M. (1964). Cycloheximide: Aspects of inhibition of protein synthesis in mammalian cells. Science 146, 1474-1476. GREG, G. W. (1958). The genetic control of conidiation in a heterokaryon of Neurospora crassa. J. Gen. Microbial. 19, 15-22. GRIGG, G. W. (1960). Temperature-sensitive genes affecting conidiation in NeuFospora crassa. J. Gen. Microbial. 22, 667-670. HANKS, D. L., and SUSSMAN, A. S. (1969). The relation between growth, conidiation and trehalase activity in Neurospora crassa. Amer. J. Bat. 56, 1152-1159. HILL, E. P., and SUSSMAN, A. S. (1963). Purification and properties of trehalase(s) from Neurospora. Arch. Biochem. Biophys. 102, 389-396. KAPLAN, N. O., COLOWICK, S. P., and NASON, A. (1951). Neurospora diphosphopyridine nucleotidase. J. Biol. Chem. 191, 473-483. KAPPY, M. S., and METZENBERG, R. L. (1965). Studies on the basis of ethionine resistance in Neurospora. Biochim. Biophys. Acta 107, 425-433.

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Enzyme Patterns during Conidiation

LOWRY, 0. H., RIISEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. LOWRY, R. J., DURKEE, T. L., and SUSSMAN, A. S. (1967). Ultrastructural studies of microconidium formation in Neurospora crassa. J. Bacterial. 54, 17571763. MAHADEVAN, P. R., and EBERHART, B. (1964). The figlucosidase system of Neurospora. II. Purification and characterization of aryl-Bglucosidase. Arch. Biochem. Biophys. 108, 22-29. MANOCHA, M. S. (1968). Electron microscopy of the conidial protoplasts of Neurospora crassa. Can J. Bot. 46, 1561-1564. MYERS, M. G., and EBERHART, B. (1966). Regulation of cellulase and cellobiase in Neurospora crassa. Biothem. Biophys. Res. Commun. 24, 782-785. OULEVEY-MATIKIAN, N., and TURIAN, G. (1968). Controle metabolique et aspects ultrastructuraux de la conidiation (macroconidies) de Neurospora crassa. Arch. Mikrobiol. 60, 35-58. PALL, M. L. (1966). Cycloheximide as inhibitor of protein synthesis. Neurospora Newsletter 9, 16. RICHMOND, D. V., SOMERS, E., and MILLINGTON, P. F. (1967). Studies on the fungitoxicity of captan. V. Electron microscopy of captan-treated Neurospora crassa conidia. Ann. Appl. Biol. 59, 233-237. ROBERTS, R. B., ed. (1964). Studies of Macromolecular Biosynthesis, Carnegie Inst. Wash. Publ. 624. SARGENT,M. L., and BRAYMER, H. D. (1969). Selection of intramural enzyme mutants. Neurospora Newsletter 14, 11-12, and personal communication. SIEGEL, M. R., and SISLER, H. D. (1964). Site of ac-

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