DEVEMPMENTAL
BIOLOGY
Some Required
54, 201-213 (1976)
Events in Conidial
Germination
of Genetics,
University Accepted
crassa
Loo’
MELANIE Department
of Neurospora
of Washington, August
Seattle
Washington
98195
4,1976
A temperature-sensitive mutant of Neurospora cmssa, with reduced levels of protein synthesis at 37”C, was used to identify some essential events in conidial germination. Conidia of mutant strainpsi-1 were incubated for 2 hr at 37°C and then shifted to 20°C. Germination was inhibited at 37”C, but commenced after 1.5 hr at 20°C. Increases in aspartate transcarbamylase activity, cell wall synthesis, and nuclear number preceded germination. However, increases in glutamate dehydrogenase activity, amino acid uptake, and DNA synthesis were inhibited prior to germination. Although all of these events were correlated with germination in control cultures of the mutant at 20°C and of its parent strain at 20 and 37”C, some events were apparently not essential for germination. The requirement for aspartate transcarbamylase activity was demonstrated independently by the failure of strainpyr-3d (lacking the activity) to germinate in the absence of uridine. The dispensability of glutamate dehydrogenase activity and DNA synthesis for the germination of some conidia was verified by the germination of strain am-l (lacking glutamate dehydrogenase activity) in the absence of glutamate and by the germination of the parent strain in the presence of hydroxyurea (an inhibitor of DNA synthesis). These findings identify some landmarks in germination which may be useful in further studies of the regulation of a developmental program. They also provide preliminary evidence that the resting conidia may contain nuclei arrested at different stages of their division cycle. INTRODUCTION
The isolation of a temperature-sensitive mutant, psi-l (Loo, 19751, defective in protein synthesis at temperatures above 33°C provided a way of asking about the quantitative and qualitative requirements for protein synthesis during conidial germination in Neurosporu CF~SS~. The initial observation that mutant psi-l did not germinate at temperatures above 33°C was not surprising. Mirkes (1974) has shown that the formation of polysomes occurred within minutes after conidia were incubated for germination. A study of temperature-sensitive mutants by Inoue and Ishikawa (1970) has also shown that protein synthesis was required from the start of conidial incubation until the appearance of germ tubes-the visible definition of germination. This was a period of from 1.25 to 4 hr for different conidia in the same culture at 36°C. When cycloheximide was 1Current address: Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706.
added to inhibit protein synthesis at any time during this period, germination was blocked. Furthermore, several investigators have reported the appearance or increase of specific enzyme activities prior to germination. These activities included NADP-dependent glutamate dehydrogenase (NADP-GDH) (Tuveson, et al. 1967), isoleucine-valine synthetic enzymes and aspartate transcarbamylase (ATC) (Jobbagy and Wagner, 1973), tRNA methylases (Wang, et al., 19711, and amino acid transport proteins (Tisdale and Debusk, 1970). The addition of cycloheximide was shown to prevent the appearance of all of these activities. It might be concluded that the above proteins, synthesized during pregermination, were required for germination. However, the previous studies only demonstrated a coincidence between enzyme synthesis and germination. If one wished to approach germination as the outcome of a developmental program, it would be necessary to show that certain events are indis201
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Q 1976 by Academic Press, of reproduction in any form
Inc. reserved.
202
DEVELOPMENTAL BIOLOGY
pensable and not just correlates or secondary effects. Earlier experiments characterizing the temperature-sensitive defect in strain psi1 (Loo, 1975)showed that protein synthesis was inhibited at temperatures above 33°C. Conidia shifted from 20 to 37°C during the pregermination period exhibited a 20% decrease in synthesis 10 min after the shift and a 60% decrease 60 min after the shift. Despite this gradual and incomplete inhibition of protein synthesis, virtually no conidia germinated at 37°C. Even conidia which would have germinated shortly if kept at 20°C failed to do so when shifted to 37°C. It seemed that some event just prior to germination required uninhibited protein synthesis; conidia could not coast into germination on residual synthesis at 37°C. It was also observed that mutant conidia incubated directly at 37°C doubled in dry weight after about 11 hr, but remained ungerminated. These observations suggested that germination was not simply the result of conidia increasing in mass and outgrowing their spore walls. Some of the proteins synthesized prior to germination seemed to have a more important developmental role than others. It was therefore hoped that mutant psi-l could add to information on events required for germination by providing a specific and reversible restriction of protein synthesis. Moreover, once required events were identified, it was hoped that their programming might be investigated. The basic experiment, designed to screen out events that were not required for germination, was the shift-down experiment. Specific events were monitored in cultures of strain psi-l at 37”C, when protein synthesis was restricted, and after a shift down to 20°C while protein synthesis recovered. At the same time, germination was monitored. If germination occurred before a given event, it could not be deemed a requirement. But its correlation with germination would be strengthened. The activities of NADP-GDH, ATC, and
VOLUME 54. 1976
amino acid transport proteins were measured in shift-down experiments. In addition, DNA synthesis, nuclear division, and the incorporation of [14C]glucose into cell walls were monitored. MATERIALS
AND
METHODS
Strains. Mutant strain psi-l was isolated by the inositolless-death enrichment technique (Lester and Gross, 1959) after ultraviolet mutagenesis of strain inos a (FGSC No. 89601). For details, see Loo (1975). Media and culture conditions. Standard Neurosporu media and culture conditions were used and are described in Loo (1975). The only alteration was that the restrictive temperature used in these experiments was 37°C. Enzyme assays. NADP-GDH (L-glutamate:NADP oxidoreductase, EC 1.4.1.4) was assayed in crude extracts as described by Barratt (1963). Volumes were reduced by 40% to allow assays to be done in 1.5-ml quartz cuvettes. ATC (r,-aspartate carbamoyl transferase, EC 2.1.3.2) was assayed in crude extracts as described by Williams and Davis (1970). Crude extracts were prepared by filtering samples of cultures and scraping pads of cells into chilled mortars. The pads were ground with twice the volume of washed sea sand and l-2 ml of appropriate buffer. Homogenates were centrifuged at 12,000g for 10 min and supernatants were removed to be used in enzyme assays. All procedures were carried out at 4°C. Protein in crude extracts was assayed by the method of Lowry et al., (1951). Amino acid uptake was measured in whole cells. One-milliliter samples of cultures were withdrawn into tubes containing 0.1 ml of L-[4,5-3Hlleucine (1 $X/ml, 19 Ci/mmole, Amersham/Searle). After 5 min, the contents of the tubes were filtered onto glass fiber filters (Schleicher and Schuell) and washed with ice-cold distilled water, containing an excess of unlabeled
MELANIE
Loo
Conidial
Germination
leucine. Filters were dried under a heat lamp and counted in vials containing toluene-based scintillation fluid (Omnifluor). Other techniques. Germination was monitored by microscopic observation. Any conidium with a protuberance away from its knobbed ends was considered germinated. Cell wall synthesis and DNA synthesis were monitored through the use of radioisotopes n-[14Clglucose (8 mCi/mmole, Schwarz BioResearch) and [2-*4Cluracil (52 mCi/mmole, Schwarz BioResearch) as described in Loo (1975). The fluorometric assay of DNA was a modification of the method of Kissaine and Robins (1958), as described by Hopper et al. (1974). Triplicate samples were used for each determination. Nuclear staining was performed by the basic Giemsa technique, as modified for fungal cells. Cells were fixed in 3.7% formaldehyde in 0.9% NaCl for at least 30 min. Cells were pelleted and washed once with distilled water. A heavy suspension of the washed cells was spread over microscope slides coated with a thin layer of egg white. After air-drying, these were rinsed in 70% ethanol and redried. Slides were then immersed in 1% NaCl at 60°C for 30 min and 1 M HCl at 60°C for 15 min. The
RELATIVE
ENZYME
ACTIVITIES
IN STRAIN
Neurospora
203
crassa
slides were rinsed in buffer (0.067 M) potassium phosphate, pH 7.2) and immersed in stain (10 parts buffer: 1 part stock Giemsa stain, Fischer Scientific Co.) for l2 hr. Slides were rinsed in distilled water and then buffer until no leaching of the stain was visible. Coverslips were mounted over a drop of buffer and sealed with clear nail polish. Such preparations were not permanent, but could be examined for 2-3 days. For determinations of nuclear distributions, over 200 cells were examined. RESULTS
Enzyme Activities
Increases in enzyme activities during germination of the progenitor strain inos a agreed with those reported in the literature. At both 20 and 37°C the specific activities of NADP-GDH, ATC, and amino acid uptake enzymes increased severalfold (Table 1). The increases were more rapid at 37 than at 20°C. When 50% of the conidia had germinated, the specific activity of NADP-GDH has increased about lofold at 20°C and l&fold at 37°C and that of ATC had increased 2.5-fold at 20°C and 4fold at 37°C. The increases in amino acid uptake activity were not normalized to total cell protein. But since dry weight in-
TABLE Activitya
of
1
inos a DURING
“C
PREGERMINATION Time
1
2 4 13.2
AND GERMINATION
of incubation 3
4
5
5.2 19.7
8.8 23.2
10.8 -
NADP-GDH
20 37
1.3 4.2
ATC
20 37
0.96 1.58
1.99 3.93
2.15 4.32
Amino acid uptake*
20
2
6
8
37
13.8
27.6
235
1.75 4.21 12 235
3.11 6.17 12 235
(1 Activities are normalized to the specific activity at t = 0. Specific activity of NADP-GDH at t = 0 was about 15 units/mg of protein, where 1 unit equals a change in absorbance at 340 nm of 0.02lmin. Specific activity of ATC at t = 0 was about 13 punits/mg of protein, where 1 unit equals a change in absorbance at 650 nm of 70/min. * Amino acid uptake was assayed in whole cells and is expressed as relative activity per cell.
204
DEVELOPMENTAL
BIOLOGY
creases only about twofold during germination, the specific uptake activity increased considerably. Uptake activity per cell had increased about 12-fold at 20°C and greater than 30-fold at 37”C, when 50% of the conidia had germinated. The absolute level of specific ATC activity was slightly lower than that reported by Jobbagy and Wagner (1973), but this may have been due to differences in genetic background, culture conditions, and assay techniques. As previously described, shift-down experiments with strain psi-l were designed to test the requirements for particular enzyme activities by showing whether significant levels of activity appeared before germination, under conditions of limited protein synthesis. Significant levels were defined as those attained by control cultures when 50% of the population had germinated. Accordingly, cultures were incubated for 2 hr at 37°C and then shifted to 20°C. Germination and enzyme activities were monitored hourly in these cultures and in control cultures of the mutant conidia at 20°C. In all cases, germination was completely inhibited at 37°C and began about 1.5 hr after the shift to 20°C (Fig. 1). Amino acid uptake and NADP-GDH activities were also inhibited at 37°C and increased more slowly in down-shifted cultures than in control cultures (Table 2). When 50% of the conidia had germinated, amino acid TABLE RELATIVE
INCREASES
IN ENZYME
ACTIVITIES
VOLUME
54, 1976
uptake per cell had increased about 20-fold in control cultures and about sixfold in shifted cultures; NADP-GDH specific activity had increased about lo-fold in control cultures and only about threefold in shifted cultures. The specific activity of ATC had increased about twofold in both the control and shifted cultures at 50% germination. However, this activity had increased markedly in the first hour of incubation at 37°C fallen just prior to the shift, and risen again (Table 2). These data were interpreted to rule out increases in amino acid uptake and NADP-GDH activities as requirements for germination. But an increase in ATC activity could not be ruled out. Admittedly, the lack of germination synchrony
#m- -+ I
2
4
3 TIME
-.-
-p
-m 6
5
(hrs)
FIG. 1. Germination of strainpsi-1 in cultures at 20”~ (o- - -o), 37°C (W - -w), and in a culture shifted from 37 to 20°C after 2 hr c&m). 2
IN STRAIN psi-l
DURING
AND GERMINATION
“C 1
2
3
4
5
NADP-GDH
20 37 + 20
1.8 1
3.5 1.5
7.8 3.2
10.9 2.9
9.8 4.4
ATC
20 37 + 20
1.72 5.6
2.57 1.32
2.78 0.9
Amino acid uptakeb
20 37 -+ 20
3 3
7.5 4.2
(1 Activities * Relative
are normalized to t = 0. amino acid uptake activity
Time
PREGERMINATION
Activity”
per whole
cell.
of incubation
15 1.8
3.5 2.21 24 5.1
3.94 2.3 27 16.2
MELANIE
Loo
Conidial
Germination
weakened this interpretation, as did the arbitrary assignment of significant levels of activity. It was entirely possible that the observed levels in control cultures were in excess of those required for germination. To verify the interpretation of shiftdown experiments, germination was monitored under conditions which eliminated the functions in question. Since conidia were routinely germinated in media without exogenous amino acids, and mutants lacking uptake activities have been shown to germinate (Tisdale and Debusk, 1970), the function of uptake proteins could not be required. Mutant strains am-l, lacking NADP-GDH activity (Fincham and Codding-ton, 1963), and pyrSd, lacking ATC activity (Davis, 1965), were obtained from the Fungal Genetics Stock Center. They were tested for their ability to germinate in the presence and absence of appropriate supplements. Strain am-l germinated both in the presence and in the absence of glutamic acid (Fig. 2). On the other hand, strain pyr9d germinated only when supplemented with pyrimidines (Fig. 2). Thus, the conclusions of the shift-down experiments were supported independently. NADP-GDH and its metabolic product glutamic acid were not essential for germination, whereas the metabolic products of ATC, pyrimidines, were essential. Cell Wall Synthesis, Nuclear Division
DNA
Synthesis,
and
Besides specific enzyme activities, other processes related to cell growth were monitored in shift-down experiments. Cell wall synthesis seemed an obvious requirement, at least for germ-tube extension. Synthesis was monitored by the incorporation of [14Clglucose into trichloroacetic acid (TCA)-precipitable material made in 4min labeling periods. Control experiments showed that in short periods, labeled glucose was incorporated almost completely into material resistant to dissolution by NaOH and ethanol at 100°C. From the work of Gooday (1971), this was assumed
of Neurospora
crassa
205
FIG. 2. Germination at 37°C of strain am-l in the presence (O- - -0) and absence (W-W) of glutamic acid and of strain pyrG?d in the presence (O- - -0) and absence (M----W of uridine.
FIG. 3. Relative [W]glucose incorporation into TCA-precipitable material in a I-min pulse during germination of strain inos a at 20°C (O- - -0) and 37°C (O-Cl) and of strainpsi-l at 20°C (O- - -0) and in a culture shifted from 37 to 20°C after 2 hr (m---m). Incorporation is normalized to the amount in the first pulse period at 20°C.
to be cell wall material. The incorporation of glucose by conidia of strain inos a reached an early pek about 45 min after the start of incubation. Then it fell briefly and rose again as conidia began to germinate (Fig. 3). The same pattern was observed at 20 and 37”C, though the incorporation was higher at 37°C. Conidia of strainpsi-1 showed the same early pattern of incorporation at both 20 and 37°C (Fig. 3). While the second peak was observed at 2O”C, mutant conidia showed a lag in synthesis after the shift-down. As with ATC activity, the appearance of the early peak at 37°C makes it impossible to rule out cell wall synthesis asa requirement for germination.
206
DEVELOPMENTAL
BIOLOGY
The relationships of DNA synthesis and nuclear division to germination were of interest because they are requirements for cell division. Since Neurospora grows as a syncytium, it was possible that cytoplasmic growth did not require immediate nuclear proliferation. Weijer and Koopmans (1964) had reported that DNA synthesis preceded germ-tube formation. However, germination was very slow in their cultures, and they relied on measurements of extracted DNA. In the present studies, DNA synthesis was first monitored by the incorporation of [14Cluracil into NaOH-resistant, TCA-precipitable material. (There is no specific label for DNA in Neurospora, since thymine is converted into precursors for both RNA and DNA.) Conidia of strain inos a began synthesizing DNA just prior to germ-tube formation at 20 and 37°C. It was not clear whether enough DNA was synthesized to account for synthesis by all of the germinated conidia. Conidia of strain psi-l also began DNA synthesis near the time of germ-tube formation at 20°C (Fig. 41, but they synthesized no DNA during the 2 hr of incubation at 37°C. After the shift from 37 to 2o”C, there was a 2-hr lag before DNA synthesis began (Fig. 4). It appeared that some germination was occurring in the absence of DNA synthesis. The weakness of this interpretation was that labeling of new DNA required the
FIG. 4. DNA synthesis and germination in strain psi-l at 20°C (0) and in a culture shifted from 37 to 20°C after 2 hr (M). DNA synthesis c----j was measured by the increase in TCA-precipitable, NaOHresistant, incorporated [Y!luracil. Germination (- -1 was monitored microscopically.
VOLUME
54. 1976
1
2
4
3 TIME
5
6
(hrs)
FIG. 5. DNA synthesis and germination in strain psi-l at 20°C (0) and in a culture shifted from 37 and 20°C after 2 hr (m). DNA synthesis (---) was measured by the increase in DABA-reactive material Germination (- -1 was monitored microscopically.
proper uptake of labeled precursors, in addition to DNA synthesis. And it was possible that uptake was secondarily affected by the inhibition of protein synthesis. Hence, shift-down experiments were repeated with fluorometric assays of DNA. Triplicate samples of whole conidia were treated with diaminobenzoic acid (DABA), which reacts with released purine deoxyribosides to produce a fluorescent compound. The same patterns of DNA synthesis observed in labeling studies were observed using the fluorometric assays (Fig. 5). DNA synthesis was also monitored fluorometrically in cultures of strain inos a in the presence of hydroxyurea (HU). HU has been shown to inhibit DNA synthesis in yeast at a concentration of 0.075 M, presumably by inhibiting ribonucleotide reductase @later, 1973). In the presence of 0.075 M HU, DNA synthesis was also inhibited in conidia of strain inos a for at least 4 hr. There was no increase in labeled material or DABA-reactive material. After 4 hr, some synthesis was observed (Fig. 6). Protein and RNA synthesis were also partially inhibited by HU. However, germination of at least 40% of the conidia proceeded in the absence of DNA synthesis. Hence, both the shift down and HU experiments demonstrated that DNA synthesis was not required for germination in all conidia. Nuclear division was monitored by fixing conidia after different times of incuba-
MELANIE
1
Conidial
Loo
2
3
Germination
4
FIG. 6. Germination and macromolecular synthesis in conidia of strain inos a at 37°C in the presence (closed symbols) and absence (open symbols of 0.075 M hydroxyurea. Germination was monitored microscopically. DNA synthesis was monitored both by the increase in TCA-precipitable, NaOH-resistant, incorporated [‘YIJluracil (A) and by the increase in DABA-reactive material (a). RNA (0) and protein (W) synthesis were monitored by the incorporation of [%]uracil and t3Hlleucine. Hydroxyurea and labeled precursors were added at the start of conidial incubation.
tion and staining their nuclei. The numbers of conidia with one, two, three, and more nuclei and the percentage of germinated conidia were determined for each time point. A distribution of nuclear numbers and average nuclear number per conidium was calculated for ungerminated conidia, germinated conidia, and the population as a whole. In control cultures of strain inos a there was a small but steady increase in the average nuclear number per ungerminated conidium at both 20 and 37°C (Tables 3 and 4). Germinated conidia had an average nuclear number at least one greater than ungerminated conidia. The
of
Neurospora
crassa
207
greater average nuclear number in germinated conidia could have been due to the preferential germination of conidia with more nuclei, the occurrence of nuclear division before germination, or a combination of the two. The increase in average nuclear number in cultures with no germinated conidia suggested that nuclear division preceded germination. In cultures with germinated and ungerminated conidia, the overall increase in average nuclear number was sufficiently large that all germinated conidia could have had at least one nucleus which had divided. Because the starting population comprised conidia with variable numbers of nuclei, it was impossible to tell whether germinated conidia with two and three nuclei originally contained those nuclei or whether they had gained them by nuclear division. However, the percentage of germinated conidia containing four or more nuclei was greater than the original percentage of those conidia. Hence, some of the germinated conidia with four or more nuclei must have completed nuclear division before germinating. Moreover, almost no germinated uninucleate conidia were observed, though the percentage of ungerminated uninucleate conidia decreased. These two observations supported the idea that nuclear division did precede germination. Nuclear division and germination were also monitored in conidia of strain psi-l (Table 3). In 20°C control cultures of strain psi-l, germination and nuclear division were more rapid than in cultures of strain inos a. The average nuclear number per germinated conidium of strain psi-l was also larger than for an ungerminated conidium. In fact, the average nuclear number per ungerminated conidium decreased slightly. This indicated that there was some preferential germination of conidia with more nuclei. However, the decrease in uninucleate and binucleate conidia in the whole population suggested that many of the germinated conidia had undergone
TABLE DISTRIBUTIONS
OF NUCLEAR
NUMBERS
20°C AND
AND
STRAIN
Culture
AVERAGE
psi-l
Percentage
inos 1 (20°C) t=o t=1 t=2 t=3 t = 4 (90%) UG t = 4 (lO%)G Total
psi-l (20°C) t=o t = 2 (71%) UG t = 2 (29%) G Total
t = 3 (53%) UG t = 3 (47%) G Total
t = 4 (31%) UG t = 4 (69%) G Total
psi-l (37 --* 20) t=3 t = 4 (38%) UG t = 4 (62%) G Total
3 NUCLEAR
OF NUCLEAR
t=2 t = 3 (71%) UG t = 3 (29%) G Total
t = 4 (24%) UG t = 4 (76%) G Total
inos
3
24
45.4 41.2 34.6 35.3 29.1 1.9 26.3
48.7 51.6 53.2 50.5 57.2 27.4 54.3
4.9 6.3 10.4 11 11.8 39.5 14.5
0.9 0.9 1.8 3.2 1.9 32.2 4.9
1.61 1.67 1.79 1.84 1.87 3.15 1.99
27.2 23.7 0 16.5 27.7 0.4 14.7 29.8 0.4 8.9
46.1 49.7 9 37.5 56.8 11.2 35.3 55 14.7 26.9
13.9 18 21.7 19.1 13.6 25.4 19.1 13.2 25.1 29.6
12.9 8.6 68.3 26.9 1.9 63 30.9 2 59.8 42.6
2.2 2.14 5.14 3.01 1.9 4.54 3.14 1.88 4.4 3.62
16.2 23.6 2.3 10.4
45.8 60.7 26.8 39.7
25.5 14.1 32.2 25.3
12.5 1.6 38.7 24.6
2.44 1.94 3.46 2.88
NUMBERS 37°C IN THE
AND AVERAGE PRESENCE AND
Percentage
4 NUCLEAR ABSENCE
of conidia
with
NUMBERS IN CONIDIA OF HYDROXYUREA
nuclear
number:
OF STRAIN
inos
t=1
3
24
37.3 37.3 31.1 29.9 0 21.2 23.5 1 6.4
47.2 43.2 50.3 50 20 41.3 54.4 11.4 21.7
12.6 14.4 14.2 14.7 26.2 18 16.9 24.7 22.8
2.7 5.1 4.4 5.4 53.8 19.5 5.2 62.9 49.1
1.81 1.87 1.95 1.98 4.25 2.66 2.04 4.57 3.96
36.9 35.9
50 44.8 46.5 34.2 42.8 53.6 43.3 48.3
11.6 15.8 13 34.2 19.4 10.3 26.4 18.4
1.4 3.2 3 25.8 9.8 2.9 22.1 12.5
1.8 1.86 1.82 3.02 2.18 1.85 2.83 2.35
Total
t = 4 (48%) UG t = 4 (52%) G Total
37.5 5.7 28 33.2 8.2 20.5
208
a AT
Average nuclear number
inos a (37°C) + HU t=2 t = 3 (70%) UG t = 3 (30%) G
a AT
Average nuclear number
2
1
t=1
OF STRAIN
1
Culture
inos a (37°C) t=o
IN CONIDIA
AND AFTER A SHIFT-DOWN of conidia with nuclear number:
TABLE DISTRIBUTIONS
NUMBERS
AT 20°C
MELANIE
Loo
Conidial
Germination
nuclear division before germinating. Again, there was a scarcity of germinated uninucleate conidia and an increase in the total percentage of conidia with four or more nuclei. In shift-down experiments with strain psi-l, as much as 60% of the population germinated within 2 hr after the shift. The germinated conidia again had a higher average nuclear number than ungerminated conidia. Although the overall increase in nuclear number was less than that in control cultures, it was sufficient that every germinated conidium could have undergone nuclear division. So, even when nuclear division was restricted, there was a striking correlation between germination and higher nuclear numbers. Part of the correlation may have been due to the preferential germination of conidia with more nuclei, but part of it must have been due to the occurrence of nuclear division before germination. Hence, nuclear division could not be ruled out as a requirement for germination It was also apparent in shift-down experiments that conidia of strain psi-l had increased their nuclear numbers before DNA synthesis had fully recovered (see Figs. 4 and 5). It seemed that some nuclei could divide without undergoing DNA replication. To verify this possibility, nuclear numbers were monitored in conidia of strain inos a in the presence of HU. Table 4 shows that even when DNA synthesis was inhibited by HU (see Fig. 6), there was an increase in nuclear number. As with down-shifted conidia of strain psi-l, there was a striking coincidence of germination and higher nuclear numbers in the same conidia. The increased incidence of germinated uninucleate conidia in HUtreated cultures of strain inos a and in down-shifted cultures of strain psi-l indicated that nuclear division was, at best, a loose requirement for germination. But both kinds of cultures confirmed that about 30% of the nuclei in conidia were arrested at a stage where they may divide
of Neurospora
crassa
209
without undergoing DNA synthesis. The methods used to quantitate DNA synthesis were sufficiently insensitive that some nuclei might have synthesized a small amount of DNA before dividing. But the simplest assumption is that some nuclei in resting conidia contained two genomes’ worth of DNA. That is, nuclei in conidia may be arrested in Gl or S (prior to the completion of DNA replication) or in G2 or early M (after replication, but before mitosis). Cell Wall Synthesis and Nuclear Division in Strain pyr-3d The identification of three possible requirements for germination made it possible to ask if there were some regulatory interactions among them. More specifically, one could see whether the absence of one activity, ATC, led to the absence of cell wall synthesis and nuclear division. Events were monitored, as before, in cultures of strain pyr-3d in minimal medium and medium supplemented with uridine. The early peak in cell wall synthesis was observed in both supplemented and unsupplemented cultures (Fig. 7), but the level of synthesis in the absence of uridine quickly fell below that in the presence of uridine. Nuclear division was also greatly reduced in conidia of strain pyr3d, but there was a significant increase in the average nuclear number after 4 hr of incubation in the absence of uridine (Table 5). In supplemented cultures, nuclear numbers increased, as they had in control cultures of strain inos a. In all cases, there was no germination in the absence of uridine (Table 5). In other systems, there is evidence that single protein may have both enzymatic and regulatory functions (Nathans and Kozak, 1972). The above experiments indicated that at least the activity of ATC is not required for positive regulation of cell wall synthesis and nuclear division. That is, the absence of ATC activity did not inhibit the initiation of those two events in
210
DEVELOPMENTAL
BIOLOGY
the absence of uridine. It is likely that reduced ATC activity affected cell wall synthesis and nuclear division simply because it affected levels of uridine, which is used both for nucleic acid synthesis and as a glucose carrier for cell wall synthesis. DISCUSSION
Shift-down experiments, in which mutant psi-l was used to restrict protein synthesis and delay germination, have shown that not all of the protein synthesis associated with germination is required for germination. Increases in amino acid uptake, NADP-dependent glutamate dehydrogen-
ii H -
6 I
TIME
(hrs)
FIG. 7. Relative glucose incorporation by conidia of strain pyr-3d during the first 2 hr of germination at 37°C in the presence (O- - -0) and absence (w-m) of uridine. Incorporation in I-min pulses was normalized to that in the first pulse O-4 min) in the presence of uridine. TABLE DISTRIBUTIONS
OF NUCLEAR
VOLUME
54, 1976
ase activity, DNA synthesis, aspartate transcarbamylase activity, cell wall synthesis, and nuclear division were observed prior to germination in control cultures. However, only the last three preceded germination in cultures of strain psi-l shifted from 37 to 20°C. That the synthesis of amino acid transport proteins, NADP-GDH, and DNA were not required for germination was confirmed by other observations. The germination of conidia in medium without exogenous amino acids showed that the function of uptake proteins was dispensable. Similarly, the germination of strain am-l in the absence of glutamic acid, and the germination of strain inos a when DNA synthesis was inhibited by hydroxyurea, confirmed that the enzymatic function of NADP-GDH and DNA synthesis were not required for germination. It was only possible to demonstrate that the function of ATC was required for germination. Strain pyr9d, which lacks that activity, would not germinate without exogenous uridine. Because of the apparent dispensability of DNA synthesis, the requirement for pyrimidines may reflect an increased need for sugar nucleotides in RNA and cell wall synthesis. Cell wall synthesis and nuclear division were not temporally uncoupled from germination in shift-down experiments, but they were not proved to be requirements. Such proof entails showing that germination is inhibited when each event is specifically in5
NUMBERS AND AVERAGE NUMBERS IN CONIDIA OF STRAIN pyr-3d THE PRESENCE AND ABSENCE OF UURIDINE AT 37°C
Percentage of conidia with nuclear number:
GROWN
Average nuclear number
1
2
3
24
37.5
45.9
12.5
4
1.83
9
Total
0 2.8
37 13 20.4
27 30 29.1
27 57 47.7
2.89 3.74 3.47
t = 4 - UdR
18.2
54.1
21.1
6.6
2.17
t=o t = 4 + UdR 31% UG 69% G
IN
MELANIE
Loo
Conidial
Germination
hibited. Nevertheless, cell wall synthesis seems a likely requirement for germination, since there is an increase in cell size and a change in cell wall composition (Mahadevan and Rao, 1970) during germination. Although it was assumed that the incorporation of labeled glucose into TCAprecipitable material reflected cell wall synthesis, it must also be allowed that other internal changes in glucose utilization might affect the uptake and incorporation of labeled glucose. Cyclic changes in glucose utilization have been observed during the yeast cell cycle (Golombek and Wintersberger, 19741, where they are unrelated to a program for germination. The correlation between nuclear division and germination was more striking: Even when the overall increase in nuclear number was limited, the average nuclear number per germinated conidium was at least one greater than that for an ungerminated conidium. A small percentage of germinated uninucleate conidia disallows the conclusion that nuclear division is a strict germination requirement. It may be possible to test the requirement for nuclear division by monitoring germination in cultures treated with griseofulvin, which is reported to inhibit nuclear division in other fungi (Gull and Trinci, 1974). The identification of these possible grmination requirements marked them as likely targets of regulation in a developmental program. That is, if a mutant were found which was specifically blocked in an early stage of germination, it might coordinately affect ATC synthesis, cell wall synthesis, and nuclear division. No evidence of immediate regulatory interaction among these three activities was found. Cell wall synthesis and nuclear division were limited, but not abolished, in strain pyr-3d in the absence of uridine. Their execution in the presence of added uridine indicated that a mutation affecting the activity of ATC did not necessarily affect a regulatory site. Since in a few cases single proteins have been shown to have both
of Neurospora
cmssa
211
catalytic and regulatory functions, it would be interesting to see whether cell wall synthesis and nuclear division occur in a pyr-3d-deletion strain. The execution of those activities would indicate that the transcriptional and translational products of the pyr-3d locus have no regulatory function in germination. Besides identifying the possibility of a developmental program, this study pointed out some of the preparations made by conidia for the resumption of metabolic activity. For example, the early synthesis of ATC and lack of GDH synthesis under restrictive conditions (in strain psi-l at 37°C) suggested that proteins required for germination may be preferentially synthesized. This could be accomplished by transcriptional or translational control or by the storage of preformed messenger RNA in conidia. Such storage may account for the rapid formation of polysomes in conidia (Mirkes, 1974) and is consistent with reports of conserved RNA species in freshly harvested and germinating conidia (Bhagwat and Mahadevan, 1970). The difference in requirements for ATC and GDH also indicated that conidia may have large reserves of glutamic acid and its amino acid derivatives, while having smaller reserves of nucleosides. Indeed, Schmit and Brody (1975) have reported that the pool of glutamic acid is fourfold higher in conidia than in mycelia, while Schiltz and Terry (1970) have reported that nucleoside uptake activity increases during germination. Hence, conidia seem to be primed for immediate protein synthesis, though any RNA species packaged into conidia is not sufficient for germination. This is, in part, supported by Holloman’s (1970) findings that considerable RNA synthesis was associated with germination and that even a partial inhibition of RNA synthesis inhibited germination. The synthesis of NADP-GDH during germination in control cultures suggested a further difference in the regulatory organization of conidia as opposed to mycelia.
212
DEVELOPMENTAL
BIOLOGY
In mycelia, NADP-GDH synthesis is repressed by glutamate and maximally derepressed by ammonium at 0.065 M (Barratt, 1963). The basal medium used in these experiments contained 0.008 M nitrate and ammonium as the nitrogen source, and the conidia apparently contained enough glutamate to carry out germination. Hence, it is puzzling that NADP-GDH synthesis was derepressed. It is possible that active regulatory forms of nitrogen were sequestered during germination or that a different regulatory program superseded the usual metabolic program. While no definite relationship between nuclear division and germination could be drawn from these studies, some interesting observations on nuclear behavior were made. First, while the increase in nuclear number was variable under different conditions, there was always a minimal increase associated with germination. This increase was just large enough that every germinated conidium could have undergone nuclear division. Second, nuclear division was apparently not synchronous within individual conidia. If it had been, a single round of division would have abolished the class of trinucleate conidia. In fact, this class increased slightly. This means that the presence of a nondividing nucleus did not inhibit the division of a second nucleus. A similar observation was made by Rosenberger and Kesel (1967) that nuclear division in Aspergillus was asynchronous when growth was slow. On the other hand, nuclei in the plasmodium of Physarum divide synchronously. Even when plasmodia in different stages of nuclear division were fused, nuclear division was synchronous (Rusch et al., 1966). In this case, and in the case of artificial mammalian cell heterokaryons (Rae and Johnson, 19701, synchrony resulted from the acceleration of DNA synthesis by one population of nuclei and from the retardation of entry into mitosis by the other population of nuclei. Since the nuclear mem-
VOLUME
54, 1976
brane of Physarum remains intact throughout division, the maintenance of independent division cycles by nuclei of Neurospora and Aspergillus is not just due to the persistence of their nuclear membranes. The maintenance of independent nuclear division cycles in filamentous syncytial organisms might be an adaptation facilitating linear growth and might be accomplished by reducing the competition between nuclei for division substrates. Finally, during germination, some nuclei did not require DNA synthesis to enter mitosis (as evidenced by the increase in nuclear number when DNA synthesis was inhibited), while other nuclei did require DNA synthesis before mitosis (as evidenced by the reduced increase in nuclear number when synthesis was inhibited). Apparently, nuclei in conidia were in different stages of nuclear division: G2 or M (gap phase or mitosis following DNA synthesis) and Gl or S (gap phase or synthetic phase preceding completion of DNA synthesis). It is not clear whether nuclei were packaged at different stages during conidiation or whether they were packaged at a uniform stage and some fraction proceeded to a later stage. Whatever the case, this again demonstrated the independent behavior of two nuclei in the same cytoplasm. Moreover, it suggested that nuclei in resting cells need not be in Gl. In most mammalian cell systems, dormant cells have been shown to have nuclei in Gl (Epifanova and Terskikh, 1969). Regenerating cells were only rarely observed to divide prior to DNA synthesis, as in the case of mouse ear epithelium (Gelfant, 1966). The presence of G2 nuclei in conidia does not appear to result from the multinculeate condition, for uninucleate conidia could also complete nuclear division in the absence of DNA synthesis. This suggests that the usual appearance of Gl nuclei in resting cells is not due to any cell-arresting substance made in Gl, but is more likely due to the susceptibility of Gl nuclei to dwindling metabolic activities.
MELANIE
The author wishes to thank information on ATC assays and advice and encouragement. This by an NIH training grant to the ington and an NSF grant to D.
Loo
Conidial
Gel pmination
L. G. Williams for D. R. Stadler for his work as supported University of WashR. Stadler.
REFERENCES BARRATI, R. W. (1963). Effect of environmental conditions on NADP-specific glutamic acid dehydrogenase in Neurospora crassa. J. Gen. Microbial. 33, 33-42. BHAGWAT, A. S., and MAHADEVAN, P. R. (1970). Conserved mRNA from the conidia of Neurospora crussa. Mol Gen. Genet. 109, 142-151. DAVIS, R. H. (1965). Carbamyl phosphate synthesis in Neurosporu. II. Genetics, metabolic position, and regulation of arginine-specific carbamyl phosphokinase. Biochim. Biophys. Acta 107, 54-68. EPIFANOVA, 0. I., and TERSKIKH, V. V. (1969). On the resting periods in the cell life cycle. Cell Tiss. Kinet. 2, 75-93. FINCHAM, J. R. S., and CODDINGTON, A. (1963). Complementation at the am locus of Neurospora crussa: A reaction between different mutant forms of glutamate dehydrogenase. J. Mol. Biol. 6,361373. GELFANT, S. (1966). Patterns of cell division: The demonstration of discrete cell populations. In “Methods in Cell Physiology” (D. M. Prescott, ed.), Vol. 2, p. 359. Academic Press, New York. GOLDMBEK, J., and WINTERSBERGER, E. (1974). Glucose uptake in the cell cycle of Saccharaomyces cerevisiae. Exp. Cell Res. 86, 199-202. GOODAY, G. W. (1971). Autoradiographic study of hyphal growth of some fungi. J. Gen. Microbial. 67, 125-133. GULL, K., and TRINCI, A. P. J. (1974). Effects of griseofulvin on the mitotic cycle of the fungus Basidiobolus ranarum. Arch. Microbial. 95,57-65. HOLLOMON, D. W. (1970). Ribonucleic acid synthesis during fungal spore germination. J. Gen. Microbiol. 62, 75-87. HOPPER, A. K., MAGEE, P. T., WELCH, S. K., FRIEDMAN, M., and HALL, B. D. (1974). Macromolecule synthesis and breakdown in relation to sporulation and meiosis in yeast. J. Bacterial. 119, 619628. INOUE, H., and ISHIKAWA, T. (1970). Macromolecule synthesis and germination of conidia in temperature-sensitive mutants of Neurospora crassa. Japun J. Genet. 45, 357-369. JOBBAGY, A. J., and WAGNER, R. P. (19731. Changes in enzyme activities of germinating conidia of Neurospora crassa. Develop. Biol. 31, 264-274. KISSANE, J. M., and ROBBINS, E. (1958). The fluorometric measurement of deoxyribonucleic acid in
of Neurospora
crassa
213
animal tissues with special reference to the central nervous system. J. Biol. Chem. 233, 184-188. LESTER, H. E., and GROSS, S. R. (1959). Efficient method for selection of auxotrophic mutants of Neurospora. Science 129, 572. LOO, M. (1975). A temperature-sensitive mutant of Neurospora apparently defective in the initiation of protein synthesis. J. Bacterial. 121, 286-295. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,255265. MAHADEVAN, P. R., and RAO, S. R. (1970). Enzyme degradation of conidial walls during germination. Zndian J. Exp. Biol. 8, 293-297. MIRKES, P. M. (1974). Polysomes, ribonucleic acid, and protein synthesis during germination of Neurospora crassa conidia. J. Bacterial. 177, 196-202. NATHANS, D., and KOZAK, M. (1972). Translation of the genome of a ribonucleic acid bacteriophage. Bacterial. Rev. 36, 109-134. RAO, P. N., and JOHNSON, R. T. (1970). Mammalian cell fusion: Studies on the regulation of DNA synthesis and mitosis. Nature (London) 255, 159-164. ROSENBERGER, R. F., and KESEL, M. (1967). Synchrony of nuclear replication in individual hyphae of Aspergillis nidulans. J. Bacterial. 94, 14641469. RUSCH, H. P., SACHSENMEIER, W., BEHRENS, K., and GRUTER, V. (1966). Synchronization of mitosis by the fusion of the plasmodia of Physarum polycephalum. J. Cell Biol. 31, 204-209. SCHILTZ, J. R., and TERRY K. D. (1970). Nucleoside uptake during the germination of Neurospora crassa conidia. Biochim. Biophys. Actu 209, 278288. SCHMIT, J. C., and BRODY, S. (1975). Neurospora crassa conidial germination: Role of endogenous amino acid pools. J. Bacterial. 124, 232-242. SLATER, M. L. (1973). Effect of reversible inhibition of deoxyribonucleic acid synthesis on the yeast cell cycle. J. Bacterial. 113, 263-270. TISDALE, J. H., and DEBUSK, A. G. (1970). Developmental regulation of amino acid transport in Neurospora crussa. J. Bacterial. 104, 689-697. TUVESON, R. W., WEST, D. J., and BARRATP, R. W. (1967). Glutamic acid dehydrogenases in quiescent and germinating conidia of Neurospora crassa. J. Gen. Microbial. 48, 235-248. WEIJER, J., and KOOPIKANS, A. (1964). Karyokinesis of somatic nuclei of Neurospora crassa. II. DNA replication in synchronously dividing conidial nuclei. Canad. J. Genet. Cytol. 6, 426-430. WONG, R. S. L., SCARBROUGH, G. A., and BOREK, E. (1971). Transfer ribonucleic acid methylases during the germination of Neurospora crassa. J. Bacteriol. 108, 446-450.
BIOLOGY
Some Required
54, 201-213 (1976)
Events in Conidial
Germination
of Genetics,
University Accepted
crassa
Loo’
MELANIE Department
of Neurospora
of Washington, August
Seattle
Washington
98195
4,1976
A temperature-sensitive mutant of Neurospora cmssa, with reduced levels of protein synthesis at 37”C, was used to identify some essential events in conidial germination. Conidia of mutant strainpsi-1 were incubated for 2 hr at 37°C and then shifted to 20°C. Germination was inhibited at 37”C, but commenced after 1.5 hr at 20°C. Increases in aspartate transcarbamylase activity, cell wall synthesis, and nuclear number preceded germination. However, increases in glutamate dehydrogenase activity, amino acid uptake, and DNA synthesis were inhibited prior to germination. Although all of these events were correlated with germination in control cultures of the mutant at 20°C and of its parent strain at 20 and 37”C, some events were apparently not essential for germination. The requirement for aspartate transcarbamylase activity was demonstrated independently by the failure of strainpyr-3d (lacking the activity) to germinate in the absence of uridine. The dispensability of glutamate dehydrogenase activity and DNA synthesis for the germination of some conidia was verified by the germination of strain am-l (lacking glutamate dehydrogenase activity) in the absence of glutamate and by the germination of the parent strain in the presence of hydroxyurea (an inhibitor of DNA synthesis). These findings identify some landmarks in germination which may be useful in further studies of the regulation of a developmental program. They also provide preliminary evidence that the resting conidia may contain nuclei arrested at different stages of their division cycle. INTRODUCTION
The isolation of a temperature-sensitive mutant, psi-l (Loo, 19751, defective in protein synthesis at temperatures above 33°C provided a way of asking about the quantitative and qualitative requirements for protein synthesis during conidial germination in Neurosporu CF~SS~. The initial observation that mutant psi-l did not germinate at temperatures above 33°C was not surprising. Mirkes (1974) has shown that the formation of polysomes occurred within minutes after conidia were incubated for germination. A study of temperature-sensitive mutants by Inoue and Ishikawa (1970) has also shown that protein synthesis was required from the start of conidial incubation until the appearance of germ tubes-the visible definition of germination. This was a period of from 1.25 to 4 hr for different conidia in the same culture at 36°C. When cycloheximide was 1Current address: Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706.
added to inhibit protein synthesis at any time during this period, germination was blocked. Furthermore, several investigators have reported the appearance or increase of specific enzyme activities prior to germination. These activities included NADP-dependent glutamate dehydrogenase (NADP-GDH) (Tuveson, et al. 1967), isoleucine-valine synthetic enzymes and aspartate transcarbamylase (ATC) (Jobbagy and Wagner, 1973), tRNA methylases (Wang, et al., 19711, and amino acid transport proteins (Tisdale and Debusk, 1970). The addition of cycloheximide was shown to prevent the appearance of all of these activities. It might be concluded that the above proteins, synthesized during pregermination, were required for germination. However, the previous studies only demonstrated a coincidence between enzyme synthesis and germination. If one wished to approach germination as the outcome of a developmental program, it would be necessary to show that certain events are indis201
Copyright All rights
Q 1976 by Academic Press, of reproduction in any form
Inc. reserved.
202
DEVELOPMENTAL BIOLOGY
pensable and not just correlates or secondary effects. Earlier experiments characterizing the temperature-sensitive defect in strain psi1 (Loo, 1975)showed that protein synthesis was inhibited at temperatures above 33°C. Conidia shifted from 20 to 37°C during the pregermination period exhibited a 20% decrease in synthesis 10 min after the shift and a 60% decrease 60 min after the shift. Despite this gradual and incomplete inhibition of protein synthesis, virtually no conidia germinated at 37°C. Even conidia which would have germinated shortly if kept at 20°C failed to do so when shifted to 37°C. It seemed that some event just prior to germination required uninhibited protein synthesis; conidia could not coast into germination on residual synthesis at 37°C. It was also observed that mutant conidia incubated directly at 37°C doubled in dry weight after about 11 hr, but remained ungerminated. These observations suggested that germination was not simply the result of conidia increasing in mass and outgrowing their spore walls. Some of the proteins synthesized prior to germination seemed to have a more important developmental role than others. It was therefore hoped that mutant psi-l could add to information on events required for germination by providing a specific and reversible restriction of protein synthesis. Moreover, once required events were identified, it was hoped that their programming might be investigated. The basic experiment, designed to screen out events that were not required for germination, was the shift-down experiment. Specific events were monitored in cultures of strain psi-l at 37”C, when protein synthesis was restricted, and after a shift down to 20°C while protein synthesis recovered. At the same time, germination was monitored. If germination occurred before a given event, it could not be deemed a requirement. But its correlation with germination would be strengthened. The activities of NADP-GDH, ATC, and
VOLUME 54. 1976
amino acid transport proteins were measured in shift-down experiments. In addition, DNA synthesis, nuclear division, and the incorporation of [14C]glucose into cell walls were monitored. MATERIALS
AND
METHODS
Strains. Mutant strain psi-l was isolated by the inositolless-death enrichment technique (Lester and Gross, 1959) after ultraviolet mutagenesis of strain inos a (FGSC No. 89601). For details, see Loo (1975). Media and culture conditions. Standard Neurosporu media and culture conditions were used and are described in Loo (1975). The only alteration was that the restrictive temperature used in these experiments was 37°C. Enzyme assays. NADP-GDH (L-glutamate:NADP oxidoreductase, EC 1.4.1.4) was assayed in crude extracts as described by Barratt (1963). Volumes were reduced by 40% to allow assays to be done in 1.5-ml quartz cuvettes. ATC (r,-aspartate carbamoyl transferase, EC 2.1.3.2) was assayed in crude extracts as described by Williams and Davis (1970). Crude extracts were prepared by filtering samples of cultures and scraping pads of cells into chilled mortars. The pads were ground with twice the volume of washed sea sand and l-2 ml of appropriate buffer. Homogenates were centrifuged at 12,000g for 10 min and supernatants were removed to be used in enzyme assays. All procedures were carried out at 4°C. Protein in crude extracts was assayed by the method of Lowry et al., (1951). Amino acid uptake was measured in whole cells. One-milliliter samples of cultures were withdrawn into tubes containing 0.1 ml of L-[4,5-3Hlleucine (1 $X/ml, 19 Ci/mmole, Amersham/Searle). After 5 min, the contents of the tubes were filtered onto glass fiber filters (Schleicher and Schuell) and washed with ice-cold distilled water, containing an excess of unlabeled
MELANIE
Loo
Conidial
Germination
leucine. Filters were dried under a heat lamp and counted in vials containing toluene-based scintillation fluid (Omnifluor). Other techniques. Germination was monitored by microscopic observation. Any conidium with a protuberance away from its knobbed ends was considered germinated. Cell wall synthesis and DNA synthesis were monitored through the use of radioisotopes n-[14Clglucose (8 mCi/mmole, Schwarz BioResearch) and [2-*4Cluracil (52 mCi/mmole, Schwarz BioResearch) as described in Loo (1975). The fluorometric assay of DNA was a modification of the method of Kissaine and Robins (1958), as described by Hopper et al. (1974). Triplicate samples were used for each determination. Nuclear staining was performed by the basic Giemsa technique, as modified for fungal cells. Cells were fixed in 3.7% formaldehyde in 0.9% NaCl for at least 30 min. Cells were pelleted and washed once with distilled water. A heavy suspension of the washed cells was spread over microscope slides coated with a thin layer of egg white. After air-drying, these were rinsed in 70% ethanol and redried. Slides were then immersed in 1% NaCl at 60°C for 30 min and 1 M HCl at 60°C for 15 min. The
RELATIVE
ENZYME
ACTIVITIES
IN STRAIN
Neurospora
203
crassa
slides were rinsed in buffer (0.067 M) potassium phosphate, pH 7.2) and immersed in stain (10 parts buffer: 1 part stock Giemsa stain, Fischer Scientific Co.) for l2 hr. Slides were rinsed in distilled water and then buffer until no leaching of the stain was visible. Coverslips were mounted over a drop of buffer and sealed with clear nail polish. Such preparations were not permanent, but could be examined for 2-3 days. For determinations of nuclear distributions, over 200 cells were examined. RESULTS
Enzyme Activities
Increases in enzyme activities during germination of the progenitor strain inos a agreed with those reported in the literature. At both 20 and 37°C the specific activities of NADP-GDH, ATC, and amino acid uptake enzymes increased severalfold (Table 1). The increases were more rapid at 37 than at 20°C. When 50% of the conidia had germinated, the specific activity of NADP-GDH has increased about lofold at 20°C and l&fold at 37°C and that of ATC had increased 2.5-fold at 20°C and 4fold at 37°C. The increases in amino acid uptake activity were not normalized to total cell protein. But since dry weight in-
TABLE Activitya
of
1
inos a DURING
“C
PREGERMINATION Time
1
2 4 13.2
AND GERMINATION
of incubation 3
4
5
5.2 19.7
8.8 23.2
10.8 -
NADP-GDH
20 37
1.3 4.2
ATC
20 37
0.96 1.58
1.99 3.93
2.15 4.32
Amino acid uptake*
20
2
6
8
37
13.8
27.6
235
1.75 4.21 12 235
3.11 6.17 12 235
(1 Activities are normalized to the specific activity at t = 0. Specific activity of NADP-GDH at t = 0 was about 15 units/mg of protein, where 1 unit equals a change in absorbance at 340 nm of 0.02lmin. Specific activity of ATC at t = 0 was about 13 punits/mg of protein, where 1 unit equals a change in absorbance at 650 nm of 70/min. * Amino acid uptake was assayed in whole cells and is expressed as relative activity per cell.
204
DEVELOPMENTAL
BIOLOGY
creases only about twofold during germination, the specific uptake activity increased considerably. Uptake activity per cell had increased about 12-fold at 20°C and greater than 30-fold at 37”C, when 50% of the conidia had germinated. The absolute level of specific ATC activity was slightly lower than that reported by Jobbagy and Wagner (1973), but this may have been due to differences in genetic background, culture conditions, and assay techniques. As previously described, shift-down experiments with strain psi-l were designed to test the requirements for particular enzyme activities by showing whether significant levels of activity appeared before germination, under conditions of limited protein synthesis. Significant levels were defined as those attained by control cultures when 50% of the population had germinated. Accordingly, cultures were incubated for 2 hr at 37°C and then shifted to 20°C. Germination and enzyme activities were monitored hourly in these cultures and in control cultures of the mutant conidia at 20°C. In all cases, germination was completely inhibited at 37°C and began about 1.5 hr after the shift to 20°C (Fig. 1). Amino acid uptake and NADP-GDH activities were also inhibited at 37°C and increased more slowly in down-shifted cultures than in control cultures (Table 2). When 50% of the conidia had germinated, amino acid TABLE RELATIVE
INCREASES
IN ENZYME
ACTIVITIES
VOLUME
54, 1976
uptake per cell had increased about 20-fold in control cultures and about sixfold in shifted cultures; NADP-GDH specific activity had increased about lo-fold in control cultures and only about threefold in shifted cultures. The specific activity of ATC had increased about twofold in both the control and shifted cultures at 50% germination. However, this activity had increased markedly in the first hour of incubation at 37°C fallen just prior to the shift, and risen again (Table 2). These data were interpreted to rule out increases in amino acid uptake and NADP-GDH activities as requirements for germination. But an increase in ATC activity could not be ruled out. Admittedly, the lack of germination synchrony
#m- -+ I
2
4
3 TIME
-.-
-p
-m 6
5
(hrs)
FIG. 1. Germination of strainpsi-1 in cultures at 20”~ (o- - -o), 37°C (W - -w), and in a culture shifted from 37 to 20°C after 2 hr c&m). 2
IN STRAIN psi-l
DURING
AND GERMINATION
“C 1
2
3
4
5
NADP-GDH
20 37 + 20
1.8 1
3.5 1.5
7.8 3.2
10.9 2.9
9.8 4.4
ATC
20 37 + 20
1.72 5.6
2.57 1.32
2.78 0.9
Amino acid uptakeb
20 37 -+ 20
3 3
7.5 4.2
(1 Activities * Relative
are normalized to t = 0. amino acid uptake activity
Time
PREGERMINATION
Activity”
per whole
cell.
of incubation
15 1.8
3.5 2.21 24 5.1
3.94 2.3 27 16.2
MELANIE
Loo
Conidial
Germination
weakened this interpretation, as did the arbitrary assignment of significant levels of activity. It was entirely possible that the observed levels in control cultures were in excess of those required for germination. To verify the interpretation of shiftdown experiments, germination was monitored under conditions which eliminated the functions in question. Since conidia were routinely germinated in media without exogenous amino acids, and mutants lacking uptake activities have been shown to germinate (Tisdale and Debusk, 1970), the function of uptake proteins could not be required. Mutant strains am-l, lacking NADP-GDH activity (Fincham and Codding-ton, 1963), and pyrSd, lacking ATC activity (Davis, 1965), were obtained from the Fungal Genetics Stock Center. They were tested for their ability to germinate in the presence and absence of appropriate supplements. Strain am-l germinated both in the presence and in the absence of glutamic acid (Fig. 2). On the other hand, strain pyr9d germinated only when supplemented with pyrimidines (Fig. 2). Thus, the conclusions of the shift-down experiments were supported independently. NADP-GDH and its metabolic product glutamic acid were not essential for germination, whereas the metabolic products of ATC, pyrimidines, were essential. Cell Wall Synthesis, Nuclear Division
DNA
Synthesis,
and
Besides specific enzyme activities, other processes related to cell growth were monitored in shift-down experiments. Cell wall synthesis seemed an obvious requirement, at least for germ-tube extension. Synthesis was monitored by the incorporation of [14Clglucose into trichloroacetic acid (TCA)-precipitable material made in 4min labeling periods. Control experiments showed that in short periods, labeled glucose was incorporated almost completely into material resistant to dissolution by NaOH and ethanol at 100°C. From the work of Gooday (1971), this was assumed
of Neurospora
crassa
205
FIG. 2. Germination at 37°C of strain am-l in the presence (O- - -0) and absence (W-W) of glutamic acid and of strain pyrG?d in the presence (O- - -0) and absence (M----W of uridine.
FIG. 3. Relative [W]glucose incorporation into TCA-precipitable material in a I-min pulse during germination of strain inos a at 20°C (O- - -0) and 37°C (O-Cl) and of strainpsi-l at 20°C (O- - -0) and in a culture shifted from 37 to 20°C after 2 hr (m---m). Incorporation is normalized to the amount in the first pulse period at 20°C.
to be cell wall material. The incorporation of glucose by conidia of strain inos a reached an early pek about 45 min after the start of incubation. Then it fell briefly and rose again as conidia began to germinate (Fig. 3). The same pattern was observed at 20 and 37”C, though the incorporation was higher at 37°C. Conidia of strainpsi-1 showed the same early pattern of incorporation at both 20 and 37°C (Fig. 3). While the second peak was observed at 2O”C, mutant conidia showed a lag in synthesis after the shift-down. As with ATC activity, the appearance of the early peak at 37°C makes it impossible to rule out cell wall synthesis asa requirement for germination.
206
DEVELOPMENTAL
BIOLOGY
The relationships of DNA synthesis and nuclear division to germination were of interest because they are requirements for cell division. Since Neurospora grows as a syncytium, it was possible that cytoplasmic growth did not require immediate nuclear proliferation. Weijer and Koopmans (1964) had reported that DNA synthesis preceded germ-tube formation. However, germination was very slow in their cultures, and they relied on measurements of extracted DNA. In the present studies, DNA synthesis was first monitored by the incorporation of [14Cluracil into NaOH-resistant, TCA-precipitable material. (There is no specific label for DNA in Neurospora, since thymine is converted into precursors for both RNA and DNA.) Conidia of strain inos a began synthesizing DNA just prior to germ-tube formation at 20 and 37°C. It was not clear whether enough DNA was synthesized to account for synthesis by all of the germinated conidia. Conidia of strain psi-l also began DNA synthesis near the time of germ-tube formation at 20°C (Fig. 41, but they synthesized no DNA during the 2 hr of incubation at 37°C. After the shift from 37 to 2o”C, there was a 2-hr lag before DNA synthesis began (Fig. 4). It appeared that some germination was occurring in the absence of DNA synthesis. The weakness of this interpretation was that labeling of new DNA required the
FIG. 4. DNA synthesis and germination in strain psi-l at 20°C (0) and in a culture shifted from 37 to 20°C after 2 hr (M). DNA synthesis c----j was measured by the increase in TCA-precipitable, NaOHresistant, incorporated [Y!luracil. Germination (- -1 was monitored microscopically.
VOLUME
54. 1976
1
2
4
3 TIME
5
6
(hrs)
FIG. 5. DNA synthesis and germination in strain psi-l at 20°C (0) and in a culture shifted from 37 and 20°C after 2 hr (m). DNA synthesis (---) was measured by the increase in DABA-reactive material Germination (- -1 was monitored microscopically.
proper uptake of labeled precursors, in addition to DNA synthesis. And it was possible that uptake was secondarily affected by the inhibition of protein synthesis. Hence, shift-down experiments were repeated with fluorometric assays of DNA. Triplicate samples of whole conidia were treated with diaminobenzoic acid (DABA), which reacts with released purine deoxyribosides to produce a fluorescent compound. The same patterns of DNA synthesis observed in labeling studies were observed using the fluorometric assays (Fig. 5). DNA synthesis was also monitored fluorometrically in cultures of strain inos a in the presence of hydroxyurea (HU). HU has been shown to inhibit DNA synthesis in yeast at a concentration of 0.075 M, presumably by inhibiting ribonucleotide reductase @later, 1973). In the presence of 0.075 M HU, DNA synthesis was also inhibited in conidia of strain inos a for at least 4 hr. There was no increase in labeled material or DABA-reactive material. After 4 hr, some synthesis was observed (Fig. 6). Protein and RNA synthesis were also partially inhibited by HU. However, germination of at least 40% of the conidia proceeded in the absence of DNA synthesis. Hence, both the shift down and HU experiments demonstrated that DNA synthesis was not required for germination in all conidia. Nuclear division was monitored by fixing conidia after different times of incuba-
MELANIE
1
Conidial
Loo
2
3
Germination
4
FIG. 6. Germination and macromolecular synthesis in conidia of strain inos a at 37°C in the presence (closed symbols) and absence (open symbols of 0.075 M hydroxyurea. Germination was monitored microscopically. DNA synthesis was monitored both by the increase in TCA-precipitable, NaOH-resistant, incorporated [‘YIJluracil (A) and by the increase in DABA-reactive material (a). RNA (0) and protein (W) synthesis were monitored by the incorporation of [%]uracil and t3Hlleucine. Hydroxyurea and labeled precursors were added at the start of conidial incubation.
tion and staining their nuclei. The numbers of conidia with one, two, three, and more nuclei and the percentage of germinated conidia were determined for each time point. A distribution of nuclear numbers and average nuclear number per conidium was calculated for ungerminated conidia, germinated conidia, and the population as a whole. In control cultures of strain inos a there was a small but steady increase in the average nuclear number per ungerminated conidium at both 20 and 37°C (Tables 3 and 4). Germinated conidia had an average nuclear number at least one greater than ungerminated conidia. The
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greater average nuclear number in germinated conidia could have been due to the preferential germination of conidia with more nuclei, the occurrence of nuclear division before germination, or a combination of the two. The increase in average nuclear number in cultures with no germinated conidia suggested that nuclear division preceded germination. In cultures with germinated and ungerminated conidia, the overall increase in average nuclear number was sufficiently large that all germinated conidia could have had at least one nucleus which had divided. Because the starting population comprised conidia with variable numbers of nuclei, it was impossible to tell whether germinated conidia with two and three nuclei originally contained those nuclei or whether they had gained them by nuclear division. However, the percentage of germinated conidia containing four or more nuclei was greater than the original percentage of those conidia. Hence, some of the germinated conidia with four or more nuclei must have completed nuclear division before germinating. Moreover, almost no germinated uninucleate conidia were observed, though the percentage of ungerminated uninucleate conidia decreased. These two observations supported the idea that nuclear division did precede germination. Nuclear division and germination were also monitored in conidia of strain psi-l (Table 3). In 20°C control cultures of strain psi-l, germination and nuclear division were more rapid than in cultures of strain inos a. The average nuclear number per germinated conidium of strain psi-l was also larger than for an ungerminated conidium. In fact, the average nuclear number per ungerminated conidium decreased slightly. This indicated that there was some preferential germination of conidia with more nuclei. However, the decrease in uninucleate and binucleate conidia in the whole population suggested that many of the germinated conidia had undergone
TABLE DISTRIBUTIONS
OF NUCLEAR
NUMBERS
20°C AND
AND
STRAIN
Culture
AVERAGE
psi-l
Percentage
inos 1 (20°C) t=o t=1 t=2 t=3 t = 4 (90%) UG t = 4 (lO%)G Total
psi-l (20°C) t=o t = 2 (71%) UG t = 2 (29%) G Total
t = 3 (53%) UG t = 3 (47%) G Total
t = 4 (31%) UG t = 4 (69%) G Total
psi-l (37 --* 20) t=3 t = 4 (38%) UG t = 4 (62%) G Total
3 NUCLEAR
OF NUCLEAR
t=2 t = 3 (71%) UG t = 3 (29%) G Total
t = 4 (24%) UG t = 4 (76%) G Total
inos
3
24
45.4 41.2 34.6 35.3 29.1 1.9 26.3
48.7 51.6 53.2 50.5 57.2 27.4 54.3
4.9 6.3 10.4 11 11.8 39.5 14.5
0.9 0.9 1.8 3.2 1.9 32.2 4.9
1.61 1.67 1.79 1.84 1.87 3.15 1.99
27.2 23.7 0 16.5 27.7 0.4 14.7 29.8 0.4 8.9
46.1 49.7 9 37.5 56.8 11.2 35.3 55 14.7 26.9
13.9 18 21.7 19.1 13.6 25.4 19.1 13.2 25.1 29.6
12.9 8.6 68.3 26.9 1.9 63 30.9 2 59.8 42.6
2.2 2.14 5.14 3.01 1.9 4.54 3.14 1.88 4.4 3.62
16.2 23.6 2.3 10.4
45.8 60.7 26.8 39.7
25.5 14.1 32.2 25.3
12.5 1.6 38.7 24.6
2.44 1.94 3.46 2.88
NUMBERS 37°C IN THE
AND AVERAGE PRESENCE AND
Percentage
4 NUCLEAR ABSENCE
of conidia
with
NUMBERS IN CONIDIA OF HYDROXYUREA
nuclear
number:
OF STRAIN
inos
t=1
3
24
37.3 37.3 31.1 29.9 0 21.2 23.5 1 6.4
47.2 43.2 50.3 50 20 41.3 54.4 11.4 21.7
12.6 14.4 14.2 14.7 26.2 18 16.9 24.7 22.8
2.7 5.1 4.4 5.4 53.8 19.5 5.2 62.9 49.1
1.81 1.87 1.95 1.98 4.25 2.66 2.04 4.57 3.96
36.9 35.9
50 44.8 46.5 34.2 42.8 53.6 43.3 48.3
11.6 15.8 13 34.2 19.4 10.3 26.4 18.4
1.4 3.2 3 25.8 9.8 2.9 22.1 12.5
1.8 1.86 1.82 3.02 2.18 1.85 2.83 2.35
Total
t = 4 (48%) UG t = 4 (52%) G Total
37.5 5.7 28 33.2 8.2 20.5
208
a AT
Average nuclear number
inos a (37°C) + HU t=2 t = 3 (70%) UG t = 3 (30%) G
a AT
Average nuclear number
2
1
t=1
OF STRAIN
1
Culture
inos a (37°C) t=o
IN CONIDIA
AND AFTER A SHIFT-DOWN of conidia with nuclear number:
TABLE DISTRIBUTIONS
NUMBERS
AT 20°C
MELANIE
Loo
Conidial
Germination
nuclear division before germinating. Again, there was a scarcity of germinated uninucleate conidia and an increase in the total percentage of conidia with four or more nuclei. In shift-down experiments with strain psi-l, as much as 60% of the population germinated within 2 hr after the shift. The germinated conidia again had a higher average nuclear number than ungerminated conidia. Although the overall increase in nuclear number was less than that in control cultures, it was sufficient that every germinated conidium could have undergone nuclear division. So, even when nuclear division was restricted, there was a striking correlation between germination and higher nuclear numbers. Part of the correlation may have been due to the preferential germination of conidia with more nuclei, but part of it must have been due to the occurrence of nuclear division before germination. Hence, nuclear division could not be ruled out as a requirement for germination It was also apparent in shift-down experiments that conidia of strain psi-l had increased their nuclear numbers before DNA synthesis had fully recovered (see Figs. 4 and 5). It seemed that some nuclei could divide without undergoing DNA replication. To verify this possibility, nuclear numbers were monitored in conidia of strain inos a in the presence of HU. Table 4 shows that even when DNA synthesis was inhibited by HU (see Fig. 6), there was an increase in nuclear number. As with down-shifted conidia of strain psi-l, there was a striking coincidence of germination and higher nuclear numbers in the same conidia. The increased incidence of germinated uninucleate conidia in HUtreated cultures of strain inos a and in down-shifted cultures of strain psi-l indicated that nuclear division was, at best, a loose requirement for germination. But both kinds of cultures confirmed that about 30% of the nuclei in conidia were arrested at a stage where they may divide
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209
without undergoing DNA synthesis. The methods used to quantitate DNA synthesis were sufficiently insensitive that some nuclei might have synthesized a small amount of DNA before dividing. But the simplest assumption is that some nuclei in resting conidia contained two genomes’ worth of DNA. That is, nuclei in conidia may be arrested in Gl or S (prior to the completion of DNA replication) or in G2 or early M (after replication, but before mitosis). Cell Wall Synthesis and Nuclear Division in Strain pyr-3d The identification of three possible requirements for germination made it possible to ask if there were some regulatory interactions among them. More specifically, one could see whether the absence of one activity, ATC, led to the absence of cell wall synthesis and nuclear division. Events were monitored, as before, in cultures of strain pyr-3d in minimal medium and medium supplemented with uridine. The early peak in cell wall synthesis was observed in both supplemented and unsupplemented cultures (Fig. 7), but the level of synthesis in the absence of uridine quickly fell below that in the presence of uridine. Nuclear division was also greatly reduced in conidia of strain pyr3d, but there was a significant increase in the average nuclear number after 4 hr of incubation in the absence of uridine (Table 5). In supplemented cultures, nuclear numbers increased, as they had in control cultures of strain inos a. In all cases, there was no germination in the absence of uridine (Table 5). In other systems, there is evidence that single protein may have both enzymatic and regulatory functions (Nathans and Kozak, 1972). The above experiments indicated that at least the activity of ATC is not required for positive regulation of cell wall synthesis and nuclear division. That is, the absence of ATC activity did not inhibit the initiation of those two events in
210
DEVELOPMENTAL
BIOLOGY
the absence of uridine. It is likely that reduced ATC activity affected cell wall synthesis and nuclear division simply because it affected levels of uridine, which is used both for nucleic acid synthesis and as a glucose carrier for cell wall synthesis. DISCUSSION
Shift-down experiments, in which mutant psi-l was used to restrict protein synthesis and delay germination, have shown that not all of the protein synthesis associated with germination is required for germination. Increases in amino acid uptake, NADP-dependent glutamate dehydrogen-
ii H -
6 I
TIME
(hrs)
FIG. 7. Relative glucose incorporation by conidia of strain pyr-3d during the first 2 hr of germination at 37°C in the presence (O- - -0) and absence (w-m) of uridine. Incorporation in I-min pulses was normalized to that in the first pulse O-4 min) in the presence of uridine. TABLE DISTRIBUTIONS
OF NUCLEAR
VOLUME
54, 1976
ase activity, DNA synthesis, aspartate transcarbamylase activity, cell wall synthesis, and nuclear division were observed prior to germination in control cultures. However, only the last three preceded germination in cultures of strain psi-l shifted from 37 to 20°C. That the synthesis of amino acid transport proteins, NADP-GDH, and DNA were not required for germination was confirmed by other observations. The germination of conidia in medium without exogenous amino acids showed that the function of uptake proteins was dispensable. Similarly, the germination of strain am-l in the absence of glutamic acid, and the germination of strain inos a when DNA synthesis was inhibited by hydroxyurea, confirmed that the enzymatic function of NADP-GDH and DNA synthesis were not required for germination. It was only possible to demonstrate that the function of ATC was required for germination. Strain pyr9d, which lacks that activity, would not germinate without exogenous uridine. Because of the apparent dispensability of DNA synthesis, the requirement for pyrimidines may reflect an increased need for sugar nucleotides in RNA and cell wall synthesis. Cell wall synthesis and nuclear division were not temporally uncoupled from germination in shift-down experiments, but they were not proved to be requirements. Such proof entails showing that germination is inhibited when each event is specifically in5
NUMBERS AND AVERAGE NUMBERS IN CONIDIA OF STRAIN pyr-3d THE PRESENCE AND ABSENCE OF UURIDINE AT 37°C
Percentage of conidia with nuclear number:
GROWN
Average nuclear number
1
2
3
24
37.5
45.9
12.5
4
1.83
9
Total
0 2.8
37 13 20.4
27 30 29.1
27 57 47.7
2.89 3.74 3.47
t = 4 - UdR
18.2
54.1
21.1
6.6
2.17
t=o t = 4 + UdR 31% UG 69% G
IN
MELANIE
Loo
Conidial
Germination
hibited. Nevertheless, cell wall synthesis seems a likely requirement for germination, since there is an increase in cell size and a change in cell wall composition (Mahadevan and Rao, 1970) during germination. Although it was assumed that the incorporation of labeled glucose into TCAprecipitable material reflected cell wall synthesis, it must also be allowed that other internal changes in glucose utilization might affect the uptake and incorporation of labeled glucose. Cyclic changes in glucose utilization have been observed during the yeast cell cycle (Golombek and Wintersberger, 19741, where they are unrelated to a program for germination. The correlation between nuclear division and germination was more striking: Even when the overall increase in nuclear number was limited, the average nuclear number per germinated conidium was at least one greater than that for an ungerminated conidium. A small percentage of germinated uninucleate conidia disallows the conclusion that nuclear division is a strict germination requirement. It may be possible to test the requirement for nuclear division by monitoring germination in cultures treated with griseofulvin, which is reported to inhibit nuclear division in other fungi (Gull and Trinci, 1974). The identification of these possible grmination requirements marked them as likely targets of regulation in a developmental program. That is, if a mutant were found which was specifically blocked in an early stage of germination, it might coordinately affect ATC synthesis, cell wall synthesis, and nuclear division. No evidence of immediate regulatory interaction among these three activities was found. Cell wall synthesis and nuclear division were limited, but not abolished, in strain pyr-3d in the absence of uridine. Their execution in the presence of added uridine indicated that a mutation affecting the activity of ATC did not necessarily affect a regulatory site. Since in a few cases single proteins have been shown to have both
of Neurospora
cmssa
211
catalytic and regulatory functions, it would be interesting to see whether cell wall synthesis and nuclear division occur in a pyr-3d-deletion strain. The execution of those activities would indicate that the transcriptional and translational products of the pyr-3d locus have no regulatory function in germination. Besides identifying the possibility of a developmental program, this study pointed out some of the preparations made by conidia for the resumption of metabolic activity. For example, the early synthesis of ATC and lack of GDH synthesis under restrictive conditions (in strain psi-l at 37°C) suggested that proteins required for germination may be preferentially synthesized. This could be accomplished by transcriptional or translational control or by the storage of preformed messenger RNA in conidia. Such storage may account for the rapid formation of polysomes in conidia (Mirkes, 1974) and is consistent with reports of conserved RNA species in freshly harvested and germinating conidia (Bhagwat and Mahadevan, 1970). The difference in requirements for ATC and GDH also indicated that conidia may have large reserves of glutamic acid and its amino acid derivatives, while having smaller reserves of nucleosides. Indeed, Schmit and Brody (1975) have reported that the pool of glutamic acid is fourfold higher in conidia than in mycelia, while Schiltz and Terry (1970) have reported that nucleoside uptake activity increases during germination. Hence, conidia seem to be primed for immediate protein synthesis, though any RNA species packaged into conidia is not sufficient for germination. This is, in part, supported by Holloman’s (1970) findings that considerable RNA synthesis was associated with germination and that even a partial inhibition of RNA synthesis inhibited germination. The synthesis of NADP-GDH during germination in control cultures suggested a further difference in the regulatory organization of conidia as opposed to mycelia.
212
DEVELOPMENTAL
BIOLOGY
In mycelia, NADP-GDH synthesis is repressed by glutamate and maximally derepressed by ammonium at 0.065 M (Barratt, 1963). The basal medium used in these experiments contained 0.008 M nitrate and ammonium as the nitrogen source, and the conidia apparently contained enough glutamate to carry out germination. Hence, it is puzzling that NADP-GDH synthesis was derepressed. It is possible that active regulatory forms of nitrogen were sequestered during germination or that a different regulatory program superseded the usual metabolic program. While no definite relationship between nuclear division and germination could be drawn from these studies, some interesting observations on nuclear behavior were made. First, while the increase in nuclear number was variable under different conditions, there was always a minimal increase associated with germination. This increase was just large enough that every germinated conidium could have undergone nuclear division. Second, nuclear division was apparently not synchronous within individual conidia. If it had been, a single round of division would have abolished the class of trinucleate conidia. In fact, this class increased slightly. This means that the presence of a nondividing nucleus did not inhibit the division of a second nucleus. A similar observation was made by Rosenberger and Kesel (1967) that nuclear division in Aspergillus was asynchronous when growth was slow. On the other hand, nuclei in the plasmodium of Physarum divide synchronously. Even when plasmodia in different stages of nuclear division were fused, nuclear division was synchronous (Rusch et al., 1966). In this case, and in the case of artificial mammalian cell heterokaryons (Rae and Johnson, 19701, synchrony resulted from the acceleration of DNA synthesis by one population of nuclei and from the retardation of entry into mitosis by the other population of nuclei. Since the nuclear mem-
VOLUME
54, 1976
brane of Physarum remains intact throughout division, the maintenance of independent division cycles by nuclei of Neurospora and Aspergillus is not just due to the persistence of their nuclear membranes. The maintenance of independent nuclear division cycles in filamentous syncytial organisms might be an adaptation facilitating linear growth and might be accomplished by reducing the competition between nuclei for division substrates. Finally, during germination, some nuclei did not require DNA synthesis to enter mitosis (as evidenced by the increase in nuclear number when DNA synthesis was inhibited), while other nuclei did require DNA synthesis before mitosis (as evidenced by the reduced increase in nuclear number when synthesis was inhibited). Apparently, nuclei in conidia were in different stages of nuclear division: G2 or M (gap phase or mitosis following DNA synthesis) and Gl or S (gap phase or synthetic phase preceding completion of DNA synthesis). It is not clear whether nuclei were packaged at different stages during conidiation or whether they were packaged at a uniform stage and some fraction proceeded to a later stage. Whatever the case, this again demonstrated the independent behavior of two nuclei in the same cytoplasm. Moreover, it suggested that nuclei in resting cells need not be in Gl. In most mammalian cell systems, dormant cells have been shown to have nuclei in Gl (Epifanova and Terskikh, 1969). Regenerating cells were only rarely observed to divide prior to DNA synthesis, as in the case of mouse ear epithelium (Gelfant, 1966). The presence of G2 nuclei in conidia does not appear to result from the multinculeate condition, for uninucleate conidia could also complete nuclear division in the absence of DNA synthesis. This suggests that the usual appearance of Gl nuclei in resting cells is not due to any cell-arresting substance made in Gl, but is more likely due to the susceptibility of Gl nuclei to dwindling metabolic activities.
MELANIE
The author wishes to thank information on ATC assays and advice and encouragement. This by an NIH training grant to the ington and an NSF grant to D.
Loo
Conidial
Gel pmination
L. G. Williams for D. R. Stadler for his work as supported University of WashR. Stadler.
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animal tissues with special reference to the central nervous system. J. Biol. Chem. 233, 184-188. LESTER, H. E., and GROSS, S. R. (1959). Efficient method for selection of auxotrophic mutants of Neurospora. Science 129, 572. LOO, M. (1975). A temperature-sensitive mutant of Neurospora apparently defective in the initiation of protein synthesis. J. Bacterial. 121, 286-295. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,255265. MAHADEVAN, P. R., and RAO, S. R. (1970). Enzyme degradation of conidial walls during germination. Zndian J. Exp. Biol. 8, 293-297. MIRKES, P. M. (1974). Polysomes, ribonucleic acid, and protein synthesis during germination of Neurospora crassa conidia. J. Bacterial. 177, 196-202. NATHANS, D., and KOZAK, M. (1972). Translation of the genome of a ribonucleic acid bacteriophage. Bacterial. Rev. 36, 109-134. RAO, P. N., and JOHNSON, R. T. (1970). Mammalian cell fusion: Studies on the regulation of DNA synthesis and mitosis. Nature (London) 255, 159-164. ROSENBERGER, R. F., and KESEL, M. (1967). Synchrony of nuclear replication in individual hyphae of Aspergillis nidulans. J. Bacterial. 94, 14641469. RUSCH, H. P., SACHSENMEIER, W., BEHRENS, K., and GRUTER, V. (1966). Synchronization of mitosis by the fusion of the plasmodia of Physarum polycephalum. J. Cell Biol. 31, 204-209. SCHILTZ, J. R., and TERRY K. D. (1970). Nucleoside uptake during the germination of Neurospora crassa conidia. Biochim. Biophys. Actu 209, 278288. SCHMIT, J. C., and BRODY, S. (1975). Neurospora crassa conidial germination: Role of endogenous amino acid pools. J. Bacterial. 124, 232-242. SLATER, M. L. (1973). Effect of reversible inhibition of deoxyribonucleic acid synthesis on the yeast cell cycle. J. Bacterial. 113, 263-270. TISDALE, J. H., and DEBUSK, A. G. (1970). Developmental regulation of amino acid transport in Neurospora crussa. J. Bacterial. 104, 689-697. TUVESON, R. W., WEST, D. J., and BARRATP, R. W. (1967). Glutamic acid dehydrogenases in quiescent and germinating conidia of Neurospora crassa. J. Gen. Microbial. 48, 235-248. WEIJER, J., and KOOPIKANS, A. (1964). Karyokinesis of somatic nuclei of Neurospora crassa. II. DNA replication in synchronously dividing conidial nuclei. Canad. J. Genet. Cytol. 6, 426-430. WONG, R. S. L., SCARBROUGH, G. A., and BOREK, E. (1971). Transfer ribonucleic acid methylases during the germination of Neurospora crassa. J. Bacteriol. 108, 446-450.
