Vol. 156, No. 1JOURNAL OF BACTERIOLOGY, OCt. 1983, p. 155-1600021-9193/83/100155-06$02.00/0Copyright © 1983, American Society for Microbiology

Kinetics of the Nuclear Division Cycle of Aspergillus nidulansLAWRENCE G. BERGEN* AND N. RONALD MORRIS

Department ofPharmacology, UMDNJ-Rutgers Medical School, Piscataway, New Jersey 08854

Received 3 March 1983/Accepted 18 July 1983

We have analyzed the cell cycle kinetics of Aspergillus nidulans by using theDNA synthesis inhibitor hydroxyurea (HU) and a temperature-sensitive cell cyclemutant nimT that blocks in G2. HU rapidly inhibits DNA synthesis (S), and as aconsequence progression beyond S to mitosis (M) is blocked. Upon removal ofHU the inhibition is rapidly reversible. Conidia (asexual spores) of nimT weregerminated at restrictive temperature to synchronize germlings in G2 and thendownshifted to permissive temperature in the presence of HU. This proceduresynchronizes the germlings at the beginning of S in the second cell cycle afterspore germination. We have measured the total duration of S, G2, and M as thetime required for these cells to recover from the HU block and undergo the nextnuclear division. The duration of S was defined by the time course of sensitivity toreintroduction of HU during recovery from the initial HU block. The cell cycletime was measured as the nuclear doubling time, and the duration of mitosis wasdetermined from the mitotic index. The duration of G, was calculated bysubtracting the combined durations of S, G2, and M from the nuclear doublingtime, and the length of G2 was calculated by subtracting S and M from theaggregate length of S, G2, and M. We have also determined the duration of thephases of the cell cycle during the first cycle after spore germination. In theseexperiments spores were germinated directly in HU without first being blocked inG2. Because the durations of G1, S, G2, and M for the first cell cycle after sporegermination were identical with those previously determined for spores presynch-ronized at the beginning of S in the second cell cycle, we conclude that dormantconidia of A. nidulans are arrested at, or before, the start of S.

Aspergillus nidulans is well suited for analy-ses of the cell cycle and mitosis since it has ashort cell cycle (90 to 120 min), grows on definedmedia, and has well-developed genetics includ-ing a large number of mutations that affect thecell cycle. These mutations fall into two catego-ries. The first category consists of temperature-sensitive cell cycle mutants (6, 12; Mortimoreand Upshall, manuscript in preparation) ofwhich there are two types, those blocked ininterphase (Morris' nim mutants [6]) and thoseblocked in mitosis (Morris' bim mutants [6]).The second category consists of anti-microtu-bule drug-resistant and temperature-sensitivemutations in the structural gene loci for a- and P-tubulin (8, 16). Some of these mutations affectmitosis and nuclear migration (9, 10). Mitosis inA. nidulans has been characterized morphologi-cally (11, 13) and is restricted to 3 to 4% of thecell cycle. This may be contrasted with mitosisin Saccharomyces cerevisiae in which the mitot-ic spindle is present during most of the cell cycle(1, 2). A. nidulans is easily grown from uninucle-ate asexual spores to form coenocytic germlingsin which mitosis of all nuclei is synchronous

(14). As a consequence of this mitotic synchronywe can deduce the cell cycle history of eachgermling from the number of nuclei in the cell.For example, the appearance of binucleate cellsdocuments the completion of the first cell cycleafter germination, tetranucleate cells indicatecompletion of the second cell cycle, etc.

Hereford and Hartwell have analyzed theblock points of cell cycle mutants of S. cerevisi-ae in relation to the S phase by using the DNAsynthesis inhibitor hydroxyurea (HU) (4). Tocarry out a similar analysis of the block points ofthe A. nidulans nim mutants we need to deter-mine whether HU inhibits DNA synthesis in A.nidulans and to define the consequent effects ofHU upon the cell cycle. HU has been shown inmany organisms to inhibit DNA synthesis bysuppressing the conversion of ribose triphos-phates to deoxyribose triphosphates (17, 18),and it is probably the most specific and revers-ible inhibitor of fungal DNA synthesis (15). Inthis report we demonstrate that HU also inhibitsDNA synthesis in A. nidulans and can be used toalign cells at the beginning of the S phase. Afterremoval of HU, cells enter S phase synchro-

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156 BERGEN AND MORRIS

nously. With this synchronization method andlight microscopic examination of the number ofnuclei per germling we have determined that A.nidulans contains discrete G1, S, G2, and Mphases, and we have estimated the duration ofeach phase. In addition we have shown that thedormant asexual spores enter the cell cycle at,or before, the start of DNA synthesis.

MATERIALS AND METHODSStrains, media, and incubation conditions. Most ex-

periments were performed with FGSC187 (pabaAlyA2; Fungal Genetics Stock Center, Arcata, Calif.).To analyze the kinetics of the second cell cycle weused the ts cell cycle mutant nimT23(ts911), the parentstrain of which is FGSC154, (adE20 biAl WA2 cnxE16sC12 methGl nicA2 lacA3 choAl chaAl) (6). Thismutation is known to block in G2 when incubated atrestrictive temperature (Bergen et al., submitted forpublication).

Asexual spores (conidia) were isolated and stored aspreviously described (7; Bergen et al., submitted forpublication) and used within 2 months of isolation.Spores were germinated in YG (0.5% yeast extract,2% glucose) plus 0.2% Tween 80 (Fisher ScientificCo.). Tween 80 is a mild, nonionic detergent thatsuppresses clumping of germlings and synchronizesgermination. For a typical experiment a 25-ml flaskwith 10 ml of YG plus 2.5 ml of 1% Tween 80 wasinoculated at 4 x 106 spores per ml and incubated in atemperature-controlled, shaking water bath (modelG76; New Brunswick Scientific Co.) with vigorousagitation.

[3H]adenine incorporation into RNA and DNA. RNAand DNA synthesis was determined by a modificationof the method of Orr and Rosenberger (12) in whichcells were incubated with [8-3H]adenine and nucleicacid synthesis was determined as radioactivity intrichloroacetic acid (TCA) precipitate. [3H]adeninewas chosen because A. nidulans lacks thymidine ki-nase and consequently exogenous radioactive thymi-dine does not label DNA (3). Since adenine may beconverted to histidine in A. nidulans, incorporation oflabel into protein was inhibited by adding 25 ,ug ofhistidine per ml to the medium. To measure RNAsynthesis, cells were grown in YG containing 15 to 20,uCi of [3H]adenine (New England Nuclear Corp; 6 ,ugof adenine per ,Ci), samples were washed withwater and centrifuged (Beckman microfuge B, 15 s)repetitively until the wash water contained no countsabove background, and then the cells were incubatedwith 1 M NaOH for 1 to 2 days at room temperature tohydrolyze RNA. Carrier DNA (salmon sperm, 30,g/ml) was added, and macromolecules were precip-itated by mixing samples with equal volumes of ice-cold 25% TCA. Counts solubilized by this step werefrom the hydrolyzed RNA. To measure DNA synthe-sis, the TCA precipitate was washed three times with10 ml of ice-cold 10% TCA to remove residual RNAcounts and then boiled for 15 min in 1 ml of 10% TCAto hydrolyze the DNA. The sample was then cooled,the remaining precipitate was removed, and the super-natant was counted. When determinations of RNAsynthesis were not required, DNA synthesis was de-termined directly by mixing each sample with an equal

volume of 2 M NaOH and hydrolyzing as above.Tritium was detected by liquid scintillation with "Han-difluor" (Mallinckrodt) in a Packard model 3375.

Fluorescent staining of nuclei. To determine thenumber of nuclei in germlings, samples (0.5 ml) werefixed with equal volumes of 2% glutaraldehyde for 5min, and the cells were removed from the glutaralde-hyde by centrifugation and suspended in 0.5 ml ofwater. This fixed material could be stored for severaldays at 4°C. Nuclei were stained with either 10 p.g ofmithramycin per ml or 1 ,ug of 2,4-diamidino-2-phenyl-indole (Sigma Chemical Co.) per ml in 2% Nonidet P-40 as described previously (7). To study mithramycinfluorescence we used the optical filter system ofOakley and Morris (9). To analyze for 2,4-diamidino-2-phenylindole fluorescence we used a 50-W mercuryfluorescent lamp and Zeiss filters (UG5 as an excitor,BG38 for red suppression, LP470 as an interferencebarrier filter, and LP460 as a dichroic reflector).Neofluar optics (Zeiss) were required for epifluores-cence with 2,4-diamidino-2-phenylidole since planapochromatic lenses absorb the excitation wave-lengths. Micrographs were taken with either Tri-X orPlus-X film (Eastman Kodak Co.) and developed to1,600 or 400 ASA, respectively, with Diafine develop-er (Acufine).

RESULTSSince the effects of HU on A. nidulans were

not known, we have examined the influence ofthis drug on nucleic acid synthesis, nucleardivision, and growth. We found 15 mM HU toeffectively inhibit DNA synthesis (Fig. 1). Incontrast, RNA synthesis was unaffected by HU.After 8 h of germination in the presence of 3H-adenine, 23,000 cpm/106 nuclei was incorporatedinto RNA in the presence of HU and 24,300cpm/106 nuclei was incorporated in the absenceof HU. RNA synthesis was calculated per nucle-us to compensate for the inhibitory effect ofHUon replication of the DNA template. The onsetof HU inhibition on DNA synthesis was rapid,within 5 min, and recovery from HU was alsorapid, within 10 min after washing the cells freefrom drug (Fig. 2).

In most systems mitosis is tightly coupled toDNA synthesis such that, if DNA synthesis isblocked, the cell in which it is blocked fails toundergo mitosis. This is also true in A. nidulans.In the absence ofHU, spores germinated at 32°Cfor 8 h underwent on the average 2.59 nucleardivisions, whereas spores germinated in thepresence of 15 mM HU completed only 0.03nuclear divisions (see Fig. 5 for the equationdefining nuclear divisions). In contrast, sporegermination and growth of the germ tubes wasnot inhibited by this concentration of drug,although the rate of germ tube extension wasslower.We have used HU inhibition of DNA synthe-

sis to define and to determine the durations ofthe various phases of the cell cycle in A. nidu-

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KINETICS OF A. NIDULANS NUCLEAR DIVISION CYCLE 157

o HU RECOVERY

* RE-ADDITION OF HU

TIME OF RECOVERY (hrs)

" 0 -4 6 8

TIME (hrs)FIG. 1. Effects of 15 mM HU on the incorporation

of [3H]adenine into DNA. Spores of FGSC187 (4 x 106spores per ml) were germinated in YG at 32°C in thepresence of 20 ,uCi of [3H]adenine with (0) or without(0) 15 mM HU. Samples (750 ,ul, 3 x 106 cells) wereincubated with an equal volume of 2 M NaOH at roomtemperature for 1 day to kill the cells and hydrolyzethe RNA. Carrier DNA (30 p.g/ml) was added, and theDNA was precipitated by the addition of an equalvolume of ice-cold 25% TCA. The DNA was washedthree times with ice-cold 10% TCA and then hydro-lyzed by boiling for 15 min in the TCA. The cell wallsand other nonhydrolyzed debris were removed bycentrifugation, and the samples were counted. Thedata shown are the averages of duplicate samples,which were in all cases within 20% of one another.

lans. The first step in this analysis was tosynchronize young germlings at the beginning ofthe S phase. HU would be expected to affectcells differently depending upon their position inthe cell cycle at the time the drug is applied.Cells in S when HU is introduced would becomeblocked and remain at the point in S at whichDNA synthesis was inhibited. In contrast, cellsin G1, G2, or M should continue through thecycle in the presence ofHU until they reach thebeginning of the subsequent S, at which timefurther progression would be blocked. Thus,except for those cells already in S, HU can beexpected to synchronize cells at the beginning ofS. We have used a temperature-sensitive mu-tant, nimT, that blocks in G2 (Bergen et al.,submitted for publication) to obtain S-phasesynchrony of A. nidulans. When spores of animT mutant were germinated at restrictive tem-perature (44°C) for 7 h, no nuclear divisionoccurred, since the cells became blocked in G2.The nimT temperature-sensitive block wasreadily reversible. When a blocked culture was

cooled to permissive temperature (32°C) in thepresence of 15 mM HU, 83% of the germlingswere able to complete one nuclear division

FIG. 2. Time course of DNA synthesis during re-

covery from (0) and readdition (0) of HU. Spores ofFGSC187 were germinated in the presence of [3H]a-denine and 15 mM HU as described in the legend toFig. 1. After 6 h of incubation at 32°C the culture wassampled (A, 500 ,ul, 2 x 106 cells), and the remainingcells were centrifuged and washed two times with YGto remove the HU. The cells were then returned tofresh YG with [3H]adenine (t = 0). Samples of therecovering culture were removed at 15-min intervals.After 1 h of recovery the culture was split in two, andHU (90 mM) was added to one flask. Samples fromthis reintroduction flask were taken every 15 min (0).All samples were analyzed for 3H incorporation inDNA.

(forming binucleate cells), but not undergo asecond nuclear division, since HU blocked thesecells at the beginning of the next S phase. Weused this synchronization method to measurethe durations of the various cell phases.

Cells were synchronized at the start of thesecond S-phase (binucleate) by first germinatingat the restrictive temperature and then shiftingto the permissive temperature in the presence ofHU. The length of time between the removal ofHU and the appearance of tetranucleate cellsrepresents the time required to reinitiate DNAsynthesis plus the aggregate duration of S, G2,and M. At 32°C, 50% of the cells completed thecell cycle and underwent division in 95 min (Fig.3). Considering that the cells require approxi-mately 10 min to reinitiate DNA synthesis afterremoval of HU (Fig. 2) we conclude that theaggregate time for S, G2, and M at 32°C isapproximately 85 min.The number of nuclei per cell reflects the

nuclear division history of that cell; thereforegermlings with two or four nuclei have complet-ed one or two cell cycles, respectively (Fig. 4).The length of the entire nuclear division cycle(generation time or Gj) was determined from therate of nuclear increase in the coenocytic germ-lings (Fig. 5, Table 1). We found that both thetime of the first nuclear division and the Gtvaried with temperature (Fig. 5). At 32, 37, and44°C the Gt was 75, 100, and 120 min, and thetime of the first nuclear division was at 4.5, 3.8,

o NO HU* 15 mM HU

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158 BERGEN AND MORRIS

o FIRST CELL CYCLE* SECOND CELL CYCLE

0

0

U 30 60 90

TIME OF RECOVERY (min)FIG. 3. Percentage of cells completing the cell cy-

cle after release of the HU block. Spores of the nimT-containing strain (0) were germinated at 44°C for 6 hand then shifted to 32°C in the presence of 15 mM HUfor 2 h, and spores of FGSC187 (0) were germinatedwith 15 mM HU at 32°C for 6 h, at which time the HUwas removed from both cultures and the cells wereallowed to recover. Samples were fixed with glutar-aldehyde, stained with mithramycin or 2,4-diamidino-2-phenylindole, and analyzed for the number of nucleiin each cell. The percentage of tetranucleate nimTcells or binucleate FGSC187 are weighted such that120 min equals 100lo. The nimT analysis was furtherweighted by removing the percentage of the cells thatwere uninucleate at to.

and 4.8 h, respectively. The time of the firstdivision is also a function of the age of thespores. To eliminate error that might be causedby differences in the spore age, the three experi-ments presented in Fig. 5 were done from thesame batch of freshly harvested spores. Bysubtracting the duration of S, G2, and M fromthe generation time, we calculated the durationof G1. In nimT cells at 32°C the duration of G1was 10 min. The mitotic index (percent mitoticfigures found in hyphal tips) for all temperaturestested was 3 to 4% (10; data not shown). There-fore the duration of mitosis for all temperaturesstudied was 3 to 5 min. This is rounded to 5 minat all temperatures in Table 1.

Since the S phase is defined by the synthesisof DNA and is inhibited specifically by HU, wedetermined the length of S as the duration ofsensitivity to HU during recovery from the drug,that is, the time required for cells to recoversuch that the reintroduction of HU would nolonger inhibit the next mitosis. Cells containingthe nimT mutation were synchronized by thetwo-step procedure described above, and com-pletion of the second cell cycle was monitoredby the appearance of tetranucleate cells (Fig. 6).We found that at 32°C, 50% of the germlingscompleted S within 50 min of removing the HU.By considering the lag time for reinitiation ofDNA synthesis after removal of HU (10 min),we estimated the duration of the S phase to beapproximately 40 min. From these separate de-terminations for the lengths of S, M, the entirecell cycle, and the aggregate duration of S, G2,

FIG. 4. Fluorescence micrograph of FGSC187germlings stained with 2,4-diamidino-2-phenylindole.Young germlings (6 h at 37°C) were fixed with glutar-aldehyde and stained as described in the text. Thenumber of nuclei in each cell can be counted; thosecontaining two or four nuclei have completed one ortwo cell cycles, respectively. Magnification, 1,300x.

and M, we deduced the durations of G1 and G2by subtraction (Table 1).Our original reason for using the nimT muta-

tion to analyze the kinetics of the second cellcycle was to be certain that our cells weresynchronized at the start of the S phase. Sincegermination in the presence of HU yields uninu-cleate cells we had concluded that dormantconidia are naturally growth arrested at somestage of the cell cycle before completion of S;however, we did not know whether these spores

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FIG. 5. Rate of nuclear doubling at 32, 37, and44°C. Conidia of FGSC187 were germinated in YG at32-C (0), 37°C (0), or 44°C (E), and samples wereremoved every 15 min, fixed, and prepared for nuclearstaining as described in the text. The sum of nuclei andthe sum of cells analyzed were converted to theaverage number of divisions per cell using the equa-tion, divisions per cell = log (N/C)/log 2; where N isthe number of nuclei, and C is the number of cellsanalyzed. We analyzed 100 to 200 cells for each timepoint. The generation times derived from the slopes ofthese lines are presented in Table 1.

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KINETICS OF A. NIDULANS NUCLEAR DIVISION CYCLE 159

TABLE 1. Estimated duration of cell cycle phasesaDuration of cell phase (min)

Strain 0cCellStrin OC cycle G, 5+ G2 o sG(min) + M GI S G2 M

nimT 32 Second 95 85 10 40 40 5FGSC187 32 First 100 85 15 40 40 5FGSC187 37 First 75 60 15 25 30 5FGSC187 44 First 120 70 50 25 40 5

a Durations of cell phases were determined as de-scribed in the text. The generation time (G,) wasdetermined from the rate of increase in the number ofnuclei per cell (Fig. 5). To analyze the kinetics of thesecond cell cycle, spores of the nimT-containing strainwere synchronized at the start of the second S phaseby first being germinated at 440C, which leads to theirbeing blocked in G2 and then being shifted to 320C inthe presence of 15 mM HU to give a synchronousblock at the beginning of the S phase. Spores ofFGSC187 were synchronized at the start of the firstcell cycle by being germinated at 320C in the presenceof 15 mM HU which, since they start in G1, also leadsto a block at the beginning of the S phase. Theaggregate length of S, G2, and M is the time requiredfor recovery from HU to the completion of the nextmitosis, as shown in Fig. 3, minus an estimated 10 minfor reinitiation of DNA synthesis. Subtraction of theaggregate time from the G, yields the approximateduration of G1. The S phase was defined as theduration ofHU sensitivity during recovery from priorHU inhibition at the beginning of S (Fig. 6). The lengthof mitosis was calculated from the mitotic index of log-phase cultures and the G,. By subtracting the values ofM and the S phase from the aggregate times weestimated the length of G2. All times were rounded tothe nearest 5 min.

were synchronously arrested at some specificpoint in the cell cycle. By germinating wild-typespores in medium containing HU and then ana-lyzing the time course of recovery, we could testthe position of this natural cell cycle block. Wewould predict the same values for the aggregatelength of S and G2 of the first cell cycle as wasfound for the second cell cycle only if thedormant spores were arrested at, or before, thebeginning of the S phase. However, if the sporeswere arrested in mid-S or were randomly distrib-uted, then these lengths would be different. Thespores of a wild-type strain (no ts markers,FGSC 187) were germinated in the presence of15 mM HU for 6 h and washed free of the drugas above, and the time course of nuclear division(as monitored by the arrearance of the binucle-ate cells) was recorded (Fig. 3). These data andthe analagous data for the second cell cycle canbe superimposed, which demonstrates that ger-mination in the presence of HU synchronizescells at the start of the first S phase. Similarly,we measured the duration of the first S phaseand found that these data are also superimpos-

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* SECOND CELL CYCLE0

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u 30 60 90 120TIME OF RE-ADDITION OF HU

FIG. 6. Duration of the S phase at 32°C. Sporeswere germinated in 15 mM HU for 6 h, at which timethe drug was washed out as described in the legend toFig. 3. At the times marked on the graph samples wereremoved from the recovering culture and reintroducedto HU (90 mM). All cultures were sampled and fixed att = 100 min and analyzed for percentage of binucleatecells. The lack of binucleate cells before 35 min andthe increasing percentage thereafter denotes the mini-mal time required to pass the HU-sensitive step (com-plete S phase). After considering the time required toinitiate DNA synthesis and then inhibit it with HU, wecalculate that the S phase requires 40 min (Table 1).

able with the data from the second cell cycle(Fig. 6). Together, these experiments show thatdormant conidia enter the cell cycle at, or be-fore, the start of S. Another reason for compar-ing the kinetics of the first and second cell cyclesis that it establishes the analytical method inde-pendent of temperature-sensitive mutations.The most direct application of this method is

to determine the durations of the various cellcycle phases at temperatures that are not amena-ble to analysis with the cell cycle mutants. Inaddition to this analysis at 32°C, we have alsostudied the kinetics of the first cell cycle at 37and 44°C (Table 1).

DISCUSSIONWe have demonstrated that HU is a specific

and reversible inhibitor of DNA synthesis in A.nidulans and with this drug have developed aparadigm for analyzing the kinetics of the cellcycle. We found that when cells not in the Sphase were incubated with HU they becamesynchronized at the start of the next S. Toassure that the starting cells were not in mid-Swhen the HU was introduced we used the tem-perature-sensitive cell cycle mutant nimT, whichspecifically and reversibly blocks G2. By germi-nating at restrictive temperature, spores of nimTwere synchronized in the first G2; upon shiftingto permissive temperature in the presence ofHUthese cells became binucleate and were blockedat the start of the second S phase. Similarly,when wild-type spores were incubated in mediaplus HU they germinated, but remained uninu-cleate and blocked at the start of the first S-phase. This was shown by the similarity in timecourses of recovery from HU to the completion

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160 BERGEN AND MORRIS

of the next mitosis, which was assayed by theappearance of binucleate (for the first cell cycle)or tetranucleate (for the second cell cycle) cells.This similarity strongly implies that the asexualspores enter the cell cycle at, or before, thebeginning of S.The time course of recovery from HU repre-

sents the aggregate lengths of S, G2, and M forthe first and second cell cycles. The total cellcycle length was determined by analyzing therate of nuclear increase in young germlings. TheS phase was defined by the duration of the HU-sensitive event during recovery from the drug-induced inhibition, and the duration of M wasdetermined from the mitotic index of log-phasemycelia and the total length of the cell cycle(generation time). The length of the S phase inA. nidulans has been determined directly byautoradiography of mycelia after a short pulse oflabeled DNA precursor (5). Kessel and Rosen-berger's published values of 19 to 23 min for theS phase at 37°C agree well with our value of 25min (Table 1). However, their study did notyield values for the duration of the other cellcycle phases.By subtracting the aggregate durations of S,

G2, and M from the length of the cell cycle wecalculated the length of G1. Similarly we calcu-lated the duration of G2 from the aggregatelength of S, G2, and M and the separatelyobtained values for S and M. We conclude-thatA. nidulans has a substantial G2 which is ap-proximately as long as the S phase.

After establishing this experimental systemwe analyzed some cell cycle parameters, such asthe effect of temperature (Table 1) and theeffects of minimal media on the various phases.We found all phases of the cycle to changeuniformly in response to suboptimal tempera-ture. However, G1 alone was lengthened inresponse to superoptimal temperature. Similar-ly, G1 was prolonged by growth in minimalmedia, whereas the lengths of S and G2 were thesame for growth in both media (data not shown).This result is compatible with Kessel and Rosen-berger's finding that the S phase of myceliagrown on various carbon sources was not medi-um dependent, although the G, did vary.

In summary, we have developed the use ofHU as a DNA synthesis inhibitor for A. nidu-lans, and we have applied this inhibitor to ana-lyze and measure the phases of the cell cycle.With this method we have shown that there is asubstantial G2, and we have documented the

temperature dependence of the cell cyclephases. In doing so, we have found that thedormant asexual spore enters the cell cycle inG1. This kinetic method can be used to testwhether growth inhibitors specifically affect par-ticular cell cycle phases, and the most directapplication of this method will be to determinethe cell phases at which the temperature-sensi-tive cell cycle mutants are inhibited.

LITERATURE CITED

1. Byers, B., and L. Goedah. 1974. Duplication of spindleplaques and integration of the yeast cell cycle. ColdSpring Harbor Symp. Quant. Biol. 38:123-131.

2. Byers, G., and L. Goetsch. 1975. Behavior of spindles andspindle plaques in the cell cycle and conjugation ofSaccharomyces cerevisiae. J. Bacteriol. 124:511-523.

3. Grivell, A., and J. Jackson. 1968. Thymidine kinase-evidence for its absence from Neurospora crassa andsome other micro-organisms and the relevance of this tospecific labeling of DNA. J. Gen. Microbiol. 54:307-320.

4. Hereford, L., and L. H. Hartell. 1974. Sequential genefunction in the initiation of S. cerevisiae DNA synthesis.J. Mol. Biol. 84:445-461.

5. Kessel, M., and R. F. Rosenberger. 1968. Regulation andtiming of DNA synthesis in hyphae of Aspergillus nidu-lans. J. Bacteriol. 95:2275-2281.

6. Morris, N. R. 1976. Mitotic mutants of Aspergillus nidu-lans. Genet. Res. Camb. 26:237-254.

7. Morris, N. R., D. Kirsch, and B. Oakley. 1982. Molecularand genetic methods for studying mitosis and spindleproteins in Aspergillus nidulans. Methods Cell Biol.25:107-130.

8. Morris, N. R., M. Lal, and C. Oakley. 1979. Identificationof a gene for ca-tubulin in Aspergillus nidulans. Cell16:437-442.

9. Oakley, B., and N. R. Morris. 1980. Nuclear movement is,-tubulin dependent in Aspergillus nidulans. Cell 19:255-262.

10. Oakley, B., and N. R. Morris. 1981. A ,B-tubulin mutationin Aspergillus nidulans that blocks microtubule functionwithout blocking assembly. Cell. 24:8374845.

11. Oakley, B., and N. R. Morris. 1983. A mutation in Asper-gillus nidulans that blocks the transition from interphaseto prophase. J. Cell Biol. 96:1155-1158.

12. Orr, E., and R. Rosenberger. 1976. Initial characterizationof Aspergillus nidulans mutants blocked in the nuclearreplication cycle. J. Bacteriol. 126:895-902.

13. Roblnow, C., and C. Caten. 1969. Mitosis in Aspergillusnidulans. J. Cell Sci. 5:403-431.

14. Rosenberger, R., and M. Kessel. 1967. Synchrony ofnuclear replication in individual hyphae of Aspergillusnidulans. J. Bacteriol. 94:1461-1469.

15. Schindler, D., and J. Davies. 1975. Inhibitors of macromo-lecular synthesis in yeast. Methods Cell Biol. 12:17-38.

16. Sbeir-Nelss, G., M. Lai, and N. R. Morris. 1978. Identifi-cation of a gene for ,-tubulin in Aspergillus nidulans. Cell15:639-647.

17. Slater, M. 1973. Effects of reversible inhibition of DNAsynthesis on the yeast-cell cycle. J. Bacteriol. 113:263-270.

18. Walters, R. A., R. Tobey, and R. Ratlff. 1973. Cell cycledependent variation of deoxyribonucleic triphosphatepools in Chinese-hamster cells. Biochem. Biophys. Acta319:336-347.

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