Inhibition by sulfanilamide of sporulation in Saccharomyces cerevisiae WILLIAM J. COLONNA, JAMESM. GENTILE,'A N D P. T. MAGEE Deprrrtt7zer1tof Hrrrrrrrr~G e r ~ e t i c Yrrle ~ , Ur711~ertrt)l S c l ~ o oof l Metlicine. 333 Cetlor Street, N P WHtnzrrr, C T , U . S . A . 06510

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Accepted F e b r ~ ~ a r16, y 1977 , P. T. MAGEE. 1977. Inhibition by sulfi~nilamideof C O L O N N AW. , J . , J. M. G E N T I L Eand sporulation in Strc~clrtrronrycescere~,isitre.Can. J . Microbiol. 23: 659-671. The antimetabolite s~~lfanilamide inhibits sporulation in Strccl1trrun7ycr~,rcc,re~'isitrcstrain API. Cells exposed to sulfanilamide at various times during the sporulation process become progressively insensitive to the d r ~ ~agl t,h o ~ ~ gaccum~llation h of sulfanilamide by the cells increases with time. Vegetative growth of API is practically ~lnaffectedby sulfanilamide: pregrowth of the cells in the presence of the drug does not prevent sporulation. Thus, inhibition is confined to the meiotic phase of the cell cycle. Sensitivity to sulfanilamide is independent of pH. Increasing the time cells are exposed to sulfanilamide results in a progressive reduction of ascus formation; however, the inhibition is reversible since sporulation can o c c ~ in~ rcells exposed to the d r ~ for ~g >24 h. The drug arrests the cells at a point before commitment to sporulation, since yeast cells exposed to sulfanilamide for 12 h do not complete the s p o r ~ ~ l a t i oprocess n when returned to vegetative medium, but resume mitotic growth instead. Meiotic nuclear division is largely prevented by sulfanilamide, and synthesis of RNA and protein is severely retarded. DNA synthesis is inhibited up to 50%; glycogen synthesis is -90% inhibited. Other yeast strains showed varying sensitivity to sulfanilamide; homothallic strains were generally less affected. C O L O N N AW. , J., J . M. G E N T I L Eet P. T. MAGEE. 1977. Inhibition by sulfanilamide of sporulation in Socclrtrr~or~ryces c.c~re~~i.sitre. Can. J . Microbiol. 23: 659-671. Le sulfanilamide, un antimetabolite, inhibe la sporulation chez la souche API de Strcchtrromyces cerevisitrc,. Les cellules exposees au sulfanilamide a diverses pkriodes du processus de spon~lationdeviennent graduellment insensibles acette substance toxique, bien que I'accumulation de sulfanilamide augmente dans les cellules avec le temps. La croissance vegktative d'API n'est pratiquement pas affectee par le sulfanilamide; la presence de la drogue aLI stade preparatoire h la croissance des cellules n'empiche pas la sporulation. Donc, l'inhibition est confinke a la phase meiotique du cycle cellulaire. La sensibilite au sulfanilamide est independante du pH. L'augmentation du temps d'exposition des cellules au sulfanilamide conduit i une reduction progressive de la formation d'asques; toutefois, I'inhibition est reversible puisque la sporulation peut prendre place chez les cellules qui sont exposees a la drogue pour plus de 24 h. Cette substance fige les cellules h un stade anterieur B I'initiation de la spo~ulation,vu que le processus de sporulation n'est pas complete lorsque les cellules de levure est exposee au sulfanilamide pour 12 h et qu'elle est transferee sur milieu vegetatif: au contraire, la croissance mitotique se fait a nouveau. Le sulfanilamide empiche fortement la division nucleaire meiotique, e t la synthese d'ARN et de proteine est grandement retardee. La synthtse d'ADN est inhibee jusqu'850%; celle duglycogene I'estjusqu'i environ 90%. D'autres souches d e levure prksentent une sensibilite variable au sulfanilamide; les souches homothalliques sont generalement moins affectees. [Traduit par le journal]

Introduction Developinental pathways in rnicroorganis~ns have long been recognized as convenient experimental systems for the study of general regulatory events. The process of meiosis and ascospore formation in Sacclia~~omycesc u e visiae is such a pathway and has been the object of considerable study in recent years (11). As a result, much information about sporulation has been obtained; however, many fundamental questions remain unanswered. 'Current address: Department of Biology, H o p e College, Holland, MI, U.S.A. 49423.

Diploid yeast cells become committed to sporulation at about the time of the first meiotic nuclear division (28). Comrnitme~lt is preceded by a period of adjustment to the sporulation medium and a period of relatively intense metabolic activity, including a high rate of respiration (4), synthesis of macromolecules (deoxyribo~~ucleic acid (DNA), ribonucleic acid (RNA), protein, and glycogen) (4, 8, 14), and recombination (7). At about the time of the first meiotic division, the rates of rnacromolecular synthesis and respiration decline. Soon after meiosis I1 spore walls begin to form around each of the four haploid nuclei; incipient

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660

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ascospores soon become visible in the light microscope. Protein synthesis is apparently required throughout the sporulation process since cycloheximide blocks ascospore formation if added as late as 2 h before the asci are due to appear (8, 18). Attempts have been made to examine the interdependence of events during sporulation using temperature-sensitive mutants. The best characterized ones seen1 disrupted in the coordination of nuclear division and wall formation, but others seem to be deficient in adjustment to the medium or in D N A synthesis (5, 6, 22, 23). Specific inhibitors have also been used in these studies. Hydroxyurea seems to block D N A synthesis specifically and inhibits recombination at levels which correlate with tlie time of addition of tlie drug (27). Serious effects on other parameters were not apparent although a detailed study was not made. It has been shown (15, 17, 30) that D N A synthesis by yeast cells in media with glucose as tlie sole carbon source can be blocked by a combination of sulfanilamide and aniinopterin. These drugs apparently inhibit tetrahydrofolate dehydrogenase (EC 1.5.1.3), an enzyme essential for the synthesis of thylnidine monophosphate. It was of interest to us to examine the effect of these drugs on sporulating cells. We found an inhibition by sulfanilamide on meiotic development which is not potentiated by aminopterin. This comniunication describes the physiological changes in sporulating cells inhibited by the addition of sulfanilamide.

Materials and Methods Yeclsi Sirnitls The standard test strain ~ ~ s eind this study was API (previo~~slyreferred to as API a / u (14)) a n adenine auxotroph derived from thc two haploids A364A (a, acle I, ncle2, rtr.o I, l l i ~ 7 ,1 . ~ ~ iy.1, 2 , go1 I) and u , 13 1-20 (u, crcle2 [noncomplementary with ode2 from A364AI rirn3, ql112, cotll, Ierrl) (14). Other strains occasionally used were Y-55, C-21, M D 9 x I I , A-169, PAR-100, D-7A, D-10. Stock c i ~ l t ~ l r ewere s D-4, SC-68, and M D S maintained at 4°C o n slants containing 1% yeast extract, 17, peptone, 3 z dcxtrose (YPD), a n d 1.77, agar.

+

GI.OIIJ/~I o t ~ dS p o r r ~ l n i i oof~ ~Yeosi Yeast strains were pregrown in the acetate medium (PSPZ) of Roth and Halvorson (24) with the exceptions that yeast nitrogen base with amino acids was used, and that each litre of medium was s~lpplementedwith 4050 nlg of adenine sulfate. Spor~ilation medium (SP2) contained 3 g of potassium acetate and 0.2 g of raffinose per litre of distilled water (14). S ~ ~ l f a n i l a m i dmedia e were prepared by dissolving the required amount of the drug

in either PSP2 o r SP2 and sterilizing the solution by Millipore filtration. Aniinopterin solutions were sterilized in the same manner. Yeast strains were incubated with shaking overnight at 30°C in PSP2. After 12 to 18 h, the cells were harvested on Millipore filters (pore size 0.22 pin), washed twice with sterile distilled water, a n d then resuspended t o the desired cell density in either SPZ o r SP2 + sulfanilamide. The c ~ l l t i ~ was r e inc~lbatedwith shaking at 303C. After 24 to 48 h, the percentage of asci in the popillation was scored by counting a drop of the s p o r ~ ~ l a t i nc~lltilre g ~ l n d e ra inicroscope with an oil-immersion lens. A t least 250 cells were c o i ~ n t e d when ; sporillation was very low, LIP to 2400 cells were counted. Upinkc of Srrlfirt~ilcr~~~iclc Vegetative A P I cells were harvested as described, resuspended in SP2, and incubated with shaking a t 30°C. At various intervals, aliquots of cells were harvested a n d washed o n Millipore filters, resuspended in equal volumes of SPZ with 1.0-1.2% sulfanilamide, and returned t o shaking at 30 C for periods of either 2 or 50 h (the latter time to allow m a x i n i ~ ~ mpossible sporulation of all cultures). At the end of the incubation period, each sample was harvested on a Millipore filter, washed twice with I5 ml of ice cold distilled water, resuspended, a n d washed for 2-4 ~ n i nat room temperature in 2.5-5.0 rnl of distilled water, then recovered by centrifugation. The pellet was resuspended in 2.5-4.0 ml of distilled water and extracted for 15-30 min in a boiling water bath. The cellular residue was removed by centrifugation and the supernatants assayed for sulfanilamide according to the method of Bratton and Marshall (3) as described by Blanke (2). (It was f o ~ ~ nthat d if the residue froin t h e boiled cells was boiled for an additional 30 niin in 107, trichloroacetic acid (TCA) (a treatment which does not destroy sulfanilamide), n o m o r e sulfanilamide was extracted.) Sulfanilamide measurements were corrected for backgroilnd surface adsorption by subtracting values obtained from extracts of cells exposed to (he drug a t room temperati~refor 2- to 3-min periods, then harvested and processed a s described. Nrrcleor. Diui.siot~ Cells were fixed with 4% Formalin-saline a n d the nuclei s ~ ~ b s e q u e n t lstained y with Giemsa according t o Robinow and Marak (21) as modified by Hartwell (12). Glycoget~Cotric~t~i API cells were shifted (as described) into SP2 medium (with and without l . Z x si~lfanila~ilide) and incubated at 30°C. At intervals, 10-ml a l i q ~ l o t swere harvested o n Millipore filters, and washed twice with 10 nil of Or'C distilled water. The cells were resuspended in 2 mI o f l M K O H a n d extracted in a 100-C water bath for 140min. The samples were neutralized with glacial acetic acid, then centrifuged to remove insoluble matter. Glycogen was precipitated with 2 volumes of ice cold 95% ethanol, washed twice with 0°C 95% ethanol, then dried at 50-70°C. Three millilitres of 80 m M acetate buffer, p H 5.0, with 10 n i M N a C l were added to all samples. The glycogen samples were solubilized by heating in a 100°C water bath for 90 n ~ i n centrifuged , to remove insoluble matter, and then hydrolyzed for 18 h a t 30°C with 8 0 p g of Rlrizoprrs sp. amyloglucosidase (exo-l,4-a-glucosidase, E C 3.2.1.3) (10 units/mg; Sigma).

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C O L O N N A ET A L .

0

6 12 18" 21 H O U R S IN S P O R U L A T I O N M E D I U M

24

661

0 6 12 25 H O U R O F E X P O S U R E OF C E L L S TO S U L F A N I L A M I D E

FIG.l(n). Time course of sporulation by Soccl~oroti~j~ces strain API. Vegetative cells were shifted to sporulation medium and shaken at 30°C. At the indicated tinies, cell samples were fixed in Formalin and the percentage sporulation ( 8 ) determined (Materials and Methods). (6) Kinetics of pH change of SP2 during sporulation and inhibition of sporulation by sulfanilamide. Vegetative API cells were shifted to sporulation niediuni and shaken at 301C. At designated tinies, 0.5 nil of cells were transferred to a c i ~ l t ~tube ~ r e with 6 mg of solid sulfanilamide and returned to shaking at 30'C. Another I nil was transferred to a small test tube and placed in a refrigerator to pellet the cells by gravity. At the end of the experiment (48 h), the supernatants were recovered and the pH of each measured. The percentage sporulation of the sulfanilan~ide-treatedcells was measi~redas usual (Materials and Methods). ( a ) , Final percentage of sporulation at 48 h ; (O), pH. Glucose was measured with glucose oxidase (EC 1.1.3.4) (9; Sigma). Total carbohydrate was determined according to Badin r! ol. (1) with glucose as a standard.

DNA DNA synthesis was ~neasuredby uptake of specific label. Cells were shifted (as described above) into SP2 medium (with or without sulfanilamide) containing 0.35 pCi/ml ['"Cladenine (40-60 niCi/mn~ol)and 4.0 pCi/ 1111 [3H]~~racil ([3H]i~ra,20-30 Ci/nimol). At intervals, duplicate 2.5-nil aliquots were pipetted onto 0.22 nil of ice cold 1.5 N N a O H . All tubes were kept at 0°C until the sanlpling was complete, then renloved from the ice bath and left at room temperature. After 24 h, the samples were again chilled to 0°C and to each were added 0.2 n ~ lof ice cold 1009, TCA containing adenine and uracil (both 14.6 niM) and 7 5 x acetone. The sanlples were mixed and kept at 0°C. After 1 to 2 h, the cells were collected on Whatnlan G F / A glass filters, washed with 7.59, TCA containing adenine and uracil (both 1 n1M) and 259, acetone, then counted in a Becknlan liquid scintillation counter. Mroslrr.et??et~! of Proteitz otrd R N A Sj~tr!l~esis Duplicate samples (2.2 ml) were removed froni sporulation media (with or without sulfanilan~ide)at various intervals and harvested on Millipore filters. The cells l SP2 (either with or without were resuspended in 2.2 n ~ of sulfanilanlide) containing 0.5 pCi/ml ['"C]adenine ([lac]ade; 40-60 n~Ci/mmol) and 2.0 pCi/n~l [3H]glycine ([3H]gly; 5-15 Cilmmol) and incubated with shaking at 30°C. After 10 min, duplicate I-ml aliquots were pipetted into 1 ml of 209, TCA containing 259, acetone, 1 mM

adenine, and I mM glycine. Samples for nleasure~nent of protein synthesis were heated 15 niin at 90'C; samples for nleasurenient of RNA synthesis were kept at 4 C. The cells were collected on Whatman glass fiber filters, washed with 7 . 5 z TCA containing adenine and glycine (both I m M ) and 259, acetone, then counted in a Beckman liquid scintillation countel.. Cl~et~~icol.~ Ingredients for yeast culture media were obtained from Difco. All isotopes were purchased from New England Nuclcar Corporation. Aminopterin was supplied by Sigma; sulfanilamide was obtained from Sigma and Nutritional Biochemicals Corporation. All other chemicals used were rcagent grade.

Results E f i c t of S~lynniln~niclfe or1 Spor~~lrtioll Strain APl, although requiring adenine for vegetative growth, sporulates normally in its absence. Figure 1 0 depicts the kinetics of sporulation for this yeast. As shown, asci begin to appear after 8 to 10 h of incubation and comprise -70x of the cell population after 18 to 24 h, when sporulation is complete. The effect of sulfanilamide addition (at hourly intervals) on sporulation is shown in Fig. Ib. Addition of the drug before 4 to 6 I1 completely blocks ascus formation; addition at later times severely reduces sporulation without entirely

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preventing the process. Rather, a period of diniinishing sensitivity (i.e., 'escape') is observed which extends until the 13th-14th h after which the cells are apparently unaffected. The appearance of asci in these cultures defines a period when the cells progress beyond the point of sensitivity to the drug. In Fig. 10 and b, comparison o f the curves obtained for tlie drugtreated and control cultures shows that the cells begin to become insensitive to sulfanilamide about 4 to 6 h before the appearance of the first asci.

60

r-----I

Uytalte of SuIjcinilaniic/e

It seenied reasonable that the escape from inhibition by sulfanilamide might be due to a failure of the drug to penetrate the cells after some stage in sporulation. T o test this possibility, uptake of sulfanilamide by, sporulating cells was measured (Materials and Methods). Figure 2 shows that sporulating yeast d o take up sulfanilamide and appear to accumulate the drug to a constant internal concentration beginning a t about 3 h. If added before this time, the drug does not seem to penetrate as effectively, even if exposure is continued for a long time. The results of short-term exposure of the cells to sulfanilamide (inset, Fig. 2) show that the ability of the cells to accumulate the drug increases (rather tlia~idiminishes) as a function of time in sporulation medium and becomes maxinial after 8 to 10 h of incubation. (In this experiment, the final intracellular concentration of sulfanilamide was calculated to be about 1.3 mg/ml of total cell volume.) Thus, the eventual insensitivity of the yeast to sulfanilamide is not due to a diminishing permeability of the cells to the drug. One problem with uptake experiments in sporulating yeast is that the population is heterogeneous. Thus, the uptake by sporulating cells cannot be measured independently of the uptake by the fraction of cells which d o not sporulate. Since it is impossible to separate the two populations until sporulation is complete, there seems no way to avoid this ambiguity. Lack of Synergisn? by Aminopterin Previous investigators (15, 17, 30) have shown that D N A synthesis in vegetative yeast is prevented by a combination of sulfanilamide and aminopterin (the latter an inhibitor of tetrahydrofolate dehydrogenase (EC 1.5.1.3), an enzyme required for synthesis of thymidine

0 6 12 24 HOUR O F E X P O S U R E TO S U L F A N I L A M I D E

FIG.2. Uptake of sulfanilamide by API cells during sporulation. Vegetative cells were shifted to SP2 a s usual and incubated at 30°C. At designated times, 10-ml cell aliquots were harvested, resuspended in 10 ml of SP2 with 1.2% sulfanilaniide, and returned to shaking a t 30°C. In order that all cell aliquots would be exposed t o sulfanilaniide for a niinimum of 24 h, all samples were harvested for measurement of sporulation and drug uptake at the 50th h after the initial shift to SP2. Sporulation was measured in the usual way; all subcultures were harvested, washed, and extracted and the extracts tested for sulfanilaniide (Materials a n d Methods). Inset: Uptake of sulfanilaniide by sporulating API cells during short-term exposure to the drug. Vegetative cells were shifted to SP2 as above. At indicated times, 10-ml aliquots were harvested, resuspended in SP2 with 1.0% sulfanilamide, and returned to shaking at 30°C. After 2 h, the cells were harvested and processed as above. T h e cell population exposed to sulfanilaniide at the 24th h contained 66% asci. (@), Percentage of sporulation; (O),,micrograms sulfanilamide taken i~p/mlcell suspenslon.

monophosphate). We therefore tested the effect of the two drugs, singly and in combination, o n sporulating yeast. Table 1 shows that the cells are totally insensitive to a~ninopterinin concentrations up to 0.1%. Indeed, sulfanilamide alone gave a sporulation profile identical with that observed in the presence of both drugs (results not shown). Thus, unlike the mitotic cell cycle in which, in A P I and other yeast strains, aminopterin and sulfanila~nide a c t synergistically (data not shown; see also 15, 17, 30), the sporulation process in yeast is very sensitive to tlie latter drug alone. Dose Response

When AP1 cells were incubated in SP2 containing varying amounts o f sulfanilamide, t h e relationship in Fig. 3 was obtained. Inhibition

COLONNA E T AL.

TABLE 1. EfTect of sulfanilamide and aminopterin on sporulation of strain API Expt. No.

None (control)

I

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Additions to spori~lationmedii~ni

0.1% A

% total sporulation" 69.9

71.9

OBased o n counts o r 2 0 0 to 600 cells. "AgnRlivl~TlO~s USED: S = si~lfnnilamide;A = nminopterm.

0

I

663

above 8.8, suggesting that sensitivity of the cells to sulfanilamide may be regulated by pH. However, the observation (Table 2) that sulfanilamide totally blocks sporulation even at elevated pH values (at which ascus formation can occur normally in the absence of the drug) indicates that the inhibitory action of the drug is not a pH-dependent phenomenon. Vegetative Gro~vth The vegetative growth of strain API in PSP2 is largely unaffected by sulfanilamide (data not shown). The growth profiles observed during log phase are virtually identical for control and drug-treated cultures and only a minor reduction in overall growth yield was observed at stationary phase in sulfanilamide-treated cells. Furthermore, when cells from both cultures were shifted to SP2 medium without drug, both cell populations sporulated to the same extent, suggesting that the sulfanilamide-sensitive inetabolic processes may be confined to the meiotic (sporulation) phase of the yeast cell cycle. However, since vegetative cells are less permeable to sulfanilainide than sporulating cells (results not shown), it is possible that the failure of sulfanilamide to inhibit mitotic growth may reflect an inability of the yeast to accumulate the drug to inhibitory concentrations.

I

Sensitivity of Other Yeasts Sttcrins To deterinine whether the inhibition by FIG.3. Dose response of sporillation to inhibition by sulfanilamide was a strain-dependent phesulfanilamide. Multiple aliquots (0.8 nil) of vegetative nomenon or a general effect among yeast, A P I cells were harvested by centrifugation and the cells several other yeast strains were examined for washed with 1 0 m l of sterile distilled water then resuspended in 5 nil of SP2 with either no siilfanilaniide o r sensitivity to the drug. In most cases, sensitivity to sulfanilamide was nearly complete (Table 3). with varying concentrations of the drug. One millilitre of each s ~ ~ s p e n s i owas n transferred to a sterile ci~lture However, in three of the strains tested, only a tube and shaken at 30°C. Percentage of spot.~~lation was partial inhibition was observed. The data inmeasured after 53 h of incubation. dicate that sensitivity of sporulation to sulby sulfanilamide is markedly concentration- fanilamide is a general phenomenon among dependent, with 7-8 ing/ml of drug being re- Sacckntonzyces, yet suggest that the degree of quired for total blockage; the half-maximal sensitivity is strain-dependent. The fact that the inhibitory concentration is about 3.5 mg/ml. least inhibited strains (C-21, 8'2-68, and Y-55) are all homothallic suggest that resistance to p H and Sensitivity to Sl~lfcrnilcrtlzide sulfanilamide may be correlated with this comIn agreement with previously reported data plex genetic system; partial impermeability of (4, 14, 19), the pH of the medium rises during these cells to the drug may also be responsible sporulation, the increase being most rapid in for this phenomenon. the first 5 h (Fig. lb). Cells exposed to sulfanilamide during this period are prevented from &ever.sibility of Ir~hibition The effect of short-term exposure to sulsporulating. A progressive insensitivity to sulfanilainide is observed in sporulating cells ex- fanilamide was examined. API cells were posed to the drug as the pH of the medium rises shifted to SP2 with 1.2% sulfanila~nideand in2 4 6 SULFANILAMIDE CONCENTRATION (rnq/rnl)

664

CAN. J.

MICROBIOL. VOL.

23. 1977

TABLE 2. Effect of pH on inhibition of sporulation by sulfanilamide Buffered with exogenous C03="

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Additions to sporulation medium None 1 .2% sulfanilamide None 1 .2% si~lfa~iilamide None 1.2% sulfanilaniide

No Yes

pH of sporulation medium at : b

Final

%

To

T 5

T2 3

sporulation

7.17 7.19 9.31 9.30 9.66 9.68

8.71 8.30 9.49 9.48 9.66 9.70

9.09 9.02 9.91 9.90 10.02 10.02

76.5 0 72.3 1.4 70.5 0.6

OSP2 buffered with KHC0,-K2C0,; [ C 0 3 = ]was 0.15 M. bCultures were s a m ~ l e da t t h e indicated hours. Cells were removed bv centrifurralion and oH of suoernatants was measured.

TABLE3. Effect of si~lfanilamide on sporulation in various strains of S. ce~.euisine" z

% reductio~i Yeast strain

of ascus formati011 by sulfatlilaniideh

> 98% >99z

API A- 169"

0 +

2

60

a 0 a VI

+

=

0 0

"Vegetative cells were har\'ested by ccntrif u g a t i o n , uashed I\\,ice with sterile distilled water, rcsuspcnded in SP2 \\!it11 and \\,ilhout sulfanilaniide, and shaken a t *Percentages dclcrrnined after 23 to 28 11 of' incubation rrom counts of 200-2400 cells from each strain. Except for PAR-100. MDS/D-10, and A-169, percenl?ges represent averages of lcast two deterniinat~ons. [A-I69 is diploid nl;~ting-type mulant which sporulates 20-30z, at cilher 23 or 32°C (13). ~iomolhallic

1.2Z

30'C.

strain.

cubated at 30°C. At hourly intervals, 5-ml aliquots were harvested, washed with sterile water, then resuspended in SP2 without drug, and incubated for an additional 20-47 11. At the end of the experiment, the percentage of asci in each cell aliquot was determined. Figure 4 sliows that little o r no reduction in overall ascus formation occul-s in cells exposed to sulfanilamide for up to 7 11. Beyond this, only a slight decline in total sporulation is apparent; furthermore, significant levels of sporulation are still observed in cells washed free of sulfanilamide even after a 27-h exposure to the drug. The reduction in ascus formation in the latter cells is due, at least partially, to loss of viability which, after 2 4 h of exposure to

5 10 27 H O U R S EXPOSED TO S U L F A N I L A M I D E

FIG.4. Effect of short-term exposure to sulfanilamide on sporulation by strain API. Vegetative cells were shifted to SP2 medium with 1 . 2 z si~anilamide(Materials and Methods) and incubated a t 30°C. At designated intervals, 5 ~ n of l cells were harvested by centrifugation, washed twice with 10 nil of sterile distilled water, resuspended in 5 ml of SPZ without drug, and retimed to shaking a t 30'C. Percentage of sporulation was measured when all ci~ltureshad been incubated for at least 43 11 after ren~oval of sulfanila~i~ide. (O),Two-spore asci; (O), four-spore asci; (*), total sporulation.

sulfanilamide, can exceed 50%, as measured by plate counts. However, it is clear that the inhibition of sporulation caused by sulfanilamide is reversible.

Nuclear Divi.rion In Fig. 5 the decline in total sporulation parallels the reduction in four-spored asci, suggesting that sulfanilamide blocks sporulation by preve~itingthe second meiotic nuclear division resulting in a preponderance of binucleate cells. T o examine this possibility, two identical populations of A P l cells were shifted to sporulation medium containing either no drug o r 1.2%

665

COLONNA ET AL.

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at 24 h. Thus, inhibition by sulfanilamide is apparently not due to preferential blockage of the second meiotic division.

0

6 12 H O U R S IN S P O R U L A T I O N MEDIUM

24

FIG. 5. Effect of s~~lfanilamicle o n meiosis in strain A P I . Vegetative API cells were shifted to SP2 medium with and witho~ltsulfanilamide and incubated at 30cC. At designated times, 5-rnl aliqilots were withclrawn from each c u l t ~ ~ and r e the cells harvested on Millipore filters, fixed overnight in 4% Formalin in 0.9% saline, then stained with Gienisa (Materials and Methods). The percentage of bin~lcleate( 0 ) and tetran~lcleate(@) cells was determined by counting i ~ n d e ra microscope with a n oil-ininiersion lens. Mature asci were not scored as tetranucleate cells. The percentages of spor~llationat 24 h in the control (o) and sulfanilamide-treated cilltures (b) were 69.2% and 0.8Y,, respectively.

sulfanilamide, then incubated at 30°C. A t hourly intervals, 5 in1 were harvested from each culture by Millipore filtration, fixed in 4% Forinalin in saline, and the nuclei subsequently stained with Gielnsa (Materials and Methods). The percentage of bi- and tetra-nucleate cells in each sample was determined by counting under a microscope. 111 the untreated cells (Fig. %I), the percentage of binucleate cells becomes maximal a t 8 h, then declines; this is followed by a marked increase in the number of tetranucleate cells which reaches a maximum at 9-1 1 h. In the sulfanilamide-treated culture (Fig. 5b), very few cells progress beyond the mononucleate stage. Furthermore, there is no accumulation of binucleate cells; instead, biand tetra-nucleate cells appear gradually and comprise the same proportion of the population

Sulfat~ilc~t~zide at~clCotnt?~itn~etlt to Sl~oritlc~tiotz Commitment to sporulation occurs 6-7 11 after the shift to sporulation medium and cells which reach this stage complete the sporulation process even when shifted back to PSP2 (28). Since the inhibition of sporulation by sulfanilamide is reversible, we asked whether cells blocked by the drug would, upon release and transfer to vegetative medium, revert to mitotic growth o r complete sporulation. Accordingly, A P l cells were incubated in SP2 with a n d without 1.2% sulfanilan~ide.After 12 h (when the control and sulfanilamide-treated cultures contained 25.1% and 0.4% asci, respectively), both cell populations were harvested, returned to PSP2 without drug, and incubated at 30°C. Periodically, both cultures were examined microscopically and turbidimetrically. Figure 6 shows that the control cells (which continued to sporulate in PSP2) revert to vegetative growth, but only after a prolonged lag period (because of the necessity of spore germination for re-

I

0

!

J

1

6 12 18 H O U R S OF I N C U B A T I O N

24 (30~~1

30

F I G . 6. Vegetative growth profiles of A P I cells after 12 h of exposure to SP2 and SP2 + l .2% sulfanilan~ide. Identical aliquots of vegetative cells were shifted in the ilsual way (Materials and Methods) to SP2 a n d SP2 1.2% sulfanilamide, and incubated with shaking a t 30°C. After 12 h, 4 nil of cells from each c u l t ~ l r ewere harvested by Millipore filtration. Each pellet w a s washed with 10 nil of sterile distilled water, then resuspended in 25 ml of PSP2 + adenine in a 250-ml sidearm flask, a n d incubated with shaking a t 30°C. At intervals, vegetative growth was monitored turbidimetrically o n a B & L Spectronic 20. N o asci appeared in the sulfanilamidetreated culture at times when asci were still present in the control. ( o ) , Control cells; (@), sulfanilamide-treated cells.

+

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sumption of mitotic growth). The sulfanilainidetreated cells also resume vegetative growth, though with a consistently shorter lag than the control cells. Furthermore, the former cells also do not complete sporulation in PSP2. Since cells from the same parent culture in SP2 + 1.2% sulfanilainide sporulated 63.1% when returned for -26 h to SP2 without drug, the absence of spores in the PSP2 culture was not due to a lingering effect of the drug. Rather, these observations indicate that treatment with sulfanilamide arrests the cells at a stage before the point of coininitinent to the sporulation process, a conclusion coilsisteilt with the finding (28) that commitment occurs 6-7 11 after the shift to SP2, after completion of D N A synthesis. Effect of S~~@niIc~r?~ide orz Mncror?~oleculeSyrlthesis h~rirlgSl~or~clntion ( i ) Glycogen Syntliesis Figure 7 shows the profile for glycogen synthesis and breakdown by sporulating AP1 cells. As shown previously (14, 16), synthesis begins soon after the shift to sporulation medium, becomes maximal a t 10 11 (when spores begin to appear), and is followed by a rapid breakdown during the spore-maturation period. In the sulfanilainide-treated cells, however, glycogen

-

HOURS I N SPORULATION MEDIUM

FIG.7. Glycogen synthesis and breakdown by sporulating API cells in the presence and absence of sulfanilamide. Vegetative API cells were shifted to SP2 medium, with and without 1.29, sulfanilamide, and incubated at 30°C. At intervals, 10-ml aliquots of each culture were harvested and washed on Millipore filters. Glycogen was isolated and enzymatically degraded, and the resulting glucose measured with glucose oxidase (Materials and Methods). Ascus formation was 64.59, and 09, in the control and sulfanilan~ide-treated cultures, respectively, at 234 h. (O), Control cells; (O), sulfanilamide-treated cells.

synthesis is almost entirely blocked, attaining a level of 0 1 1 1 ~ -9% of the control during the peak synthetic period. Furthermore, in the former cells, a period of glycogeil degradation (characteristic of sporulating cells; (14, 16)) is not observed; rather, a gradual accumulatioil of glycogen continues for the entire incubation period. It has been shown (14, 16) that asporogenous yeast strains synthesize glycogen when cultured under sporulation coilditions but fail to degrade it. Indeed, it has been suggested (16) that carbohydrate syilthesis during sporulation may be a response to the anitrogenous medium a n d not a sporulation-specific event. If so, then the absence of glycogen in sulfanilamide-treated cells may be due to a primary effect 011 glycogen biosynthesis and not a secondary event resulting from blockage of sporulation. (ii) DNA S y n t11esi.r D N A synthesis during sporulation of strain AP1 was measured by incorporation of [14C]ade and [3H]ura. The rationale was that if thymidine inonophosphate synthesis were blocked by sulfanilainide, the incorporation of exogenous uracil (as thymidine) into D N A would be more sensitive than the illcorporation of adenine. The labeling profile for untreated cells (Fig. 8) agrees well with the results of other investigators (4, 8, 14, 25) as to the timing and extent of DNA synthesis. The incorporation rates of [3H]ura and [14C]ade remain constant during the 2- to 8-h D N A synthetic period; after this time, incorporation of both isotopes continues (although a t sharply and equally diminished rates) as a result of ongoing mitochondria1 D N A synthesis (20). In sulfanilamide-treated cells, although the D N A synthetic period coincides closely with the control, there is a reduction of more than 5077, in incorporation of [3H]ura and [14C]ade into alkali-insoluble material. Incorporation of both isotopes shows a consistent decline beginning at about 2 h and continuing for the duration of the experiment. However, before conclusions could be drawn about possible drug-induced thymidine limitation, it was necessary t o ascertain whether the discrepancies in [3H]ura and [14C]ade incorporation were the result of alterations in uptake of the isotopes. Measurements of [14C]ade and [3H]ura in acid-soluble and alkaliinsoluble inaterial from control and drugtreated cells show that the [14C]ade pools are

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COLONNA ET AL.

TABLE 4. Effect of sulfanilamide on uptake of adenine and uracil by sporulati~lgAPI cells

Acid-soluble cpm

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Hours after transfer to SP2

8

Control cells

Sulfanilamide-treated cells

-

[14C]ade"

3549

-

[ 3 H ] ~ r a " , b [14C]ade/[3H]~ra

2229

1.59

[L4C]aden

298 1

[ 3 H ] ~ r a " * h [14C]ade/[3H]~ra

l20G

2.47

Alkali-insoluble cpm (DNA)c Control cells

24 60

[14C]ade

[3H]ura

[L4C]ade/[3H]ura

951 5 7445

11 163 9 623

0.85 0.77

Sulfanilamide-treated cells [I4C]ade [3H]ura [14C]ade/[3H]ura 5136 4614

4867 4623

1.06 1 .OO

2Z

a T r ~ c h l o r o a c e t ~acc ~ d(TCA) soluble cpm/O 2 In1 o f cell silspenslon Cells were s h ~ f t e d In , the usudl n a y , to SP2 w ~ t hand w ~ t l i o u t1 sulf a n ~ l a m ~ dplus e , 0 38 p C ~ / m [lAC]ade l and 6 p C ~ / m [l 3 H ] ~ r dand 1ncub.11ed dl 30'C At ~ndlc'lted tlmes cells were hdrvested on M ~ l l ~ p o filters, re washed 4 times w ~ t h5 ml of SP2, then eltracted for 15 mln a t 4°C with 10z, T C A T h e cells were removed by c e n t r ~ f u g a t ~ oann d the supernatants counted for r a d ~ o a c t ~ v ~ t y bCorrected for 1 1 4 z l A C spillover CCells were hydrolyzed for 18 h a t 30cC w ~ t h0 1 M NaOH, then collected and processed for s c ~ n t ~ l l d t ~counllng on a s d e s c r ~ b e d( M a t e r ~ a l s and Methods).

0

6 12 HOURS I N SPORULATION MEDIUM

24

FIG. 8. Effect of sulfanilamide on incorporation of adenine and uracil (as thymine) into DNA by sporulating APl cells. Identical aliquots of vegetative cells were shifted to SP2 containing 0.35 pCi/ml [I4C]ade and 4.0 pCi/ml [3H]ura, with or without 1.2z sulfanilamide, and shaken at 30°C. At designated intervals, duplicate 2.5-ml aliquots from each culture were pipetted into 0.22 ml of 1.5 N NaOH and the tubes kept at 0°C until sampling was complete. All samples were processed for counting as described (Materials and Methods). Closed symbols ( 0 , V): [14C]ade cpm; open symbols (0, V): [3H]ura cpm. 0 , 0 :control cells; V, V : sulfanilamide-treated cells.

not significantly different in the two cultures (Table 4); however, in the sulfanilamidetreated yeast the [3H]ura level, although initially high, declines sharply and by 8 h measures only 54% of that in the control. On the basis of [14C]ade incorporation (Fig. 8 and Table 4), it is clear that sulfanilamide severely inhibits DNA synthesis during sporulation. However, since the drug also partially prevents entry of labeled uracil into the cell, it is not possible to ascertain whether the inhibition results from or is accompanied by blockage of the uracil to thymidine triphosphate conversion. (iii) Protein and R N A Syntlzesis T o study the effect of sulfanilamide on the relative rates of protein and RNA synthesis, AP1 cells were pulse-labeled at hourly intervals with [3H]gly and [14C]ade in SP2 plus o r minus drug. The labeling profiles of control AP1 cells (Fig. 9) agree with the results of others (14, 19) showing substantial uptake of label between 1 and 6 h into both RNA and protein. In contrast, the rates of protein and RNA synthesis, over the same interval, are both severely retarded in sulfanilamide-treated cells. The rates attained are only 44% and 56% of the controls for protein and RNA, respectively, during peak synthetic periods. Furthermore, in these cells, the period at which the rates of incorporation become maximal is delayed by

668

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I

I

I

0

5

VOL.

23. 1977 I

I

10 0 HOUR O F P U L S E

I

5

10

FIG.9. Pulse-labeling profiles of RNA ((I) and protein synthesis (b) by sporulating A P I cells in the presence and absence of sulfanilamide. Identical aliquots of vegetative cells were shifted to SPZ with or without 1.2% sulfanilamide, and incubated at 30°C. At designated times, duplicate 2.2-ml cell samples were removed from each culture, harvested on Millipore filters, then pulse labeled for 10 min at 30°C by shaking in 2.2 n ~ of l SP2 (with or without 1.2% sulfanilamide) containing 0.5 pCi/n?l ["Tlade and 2.0 pCi/ml [3H]gly. At the end of the pulse, duplicate 1-ml samples were pipetted into 1 ml of 20% TCA containing 25% acetone, 1 m M ade, and 1 m M gly. All samples were processed for counting as described (Materials and Methods). (a),Control cells; (O),sulfanilamide-treated cells.

as much as 2 h. The observation that incorporation of label from 7 to 10 h, though declining, is consistently and measurably higher in the sulfanilamide-treated cells, suggests that the mechanisms controlling shutoff of macromolecule synthesis are impaired, or that the development of these cells is blocked before this point. These effects could be due, however, to alteration of pool sizes by the drug; the observation that apparent rates are initially diminished and then later stimulated compared with control cultures would require a rather co~nplexeffect on the pools. However, since the specific activity of precursor pools depends upon both the rates of entry of label and the breakdown of preexisting macromolecules, this possibility cannot be entirely ruled out. Mills (19) has shown that apparently because of the increasing pH of the medium, sporulating cells become increasingly impermeable to isotope after 2 to 4 h. T o rule out the possibility that the shift to fresh medium affects incorporation, we have pulse-labelled control and sulfanilamidetreated cells without the shift to fresh medium. In agreement with Mills, we observed a sharply diminished incorporation of both adenine and glycine. The qualitative effect of sulfanilamide (reduced incorporation and change in the shape of the incorporation profile) remains unchanged

although the lowered number of counts incorporated makes exact interpretation difficult.

Discussion Grivell and Jackson (10) demonstrated that S. cerevisirre lacks thymidine kinase (EC 2.7.1.75). Consequently, this yeast is unable t o incorporate thymine or thymidine into D N A . It has been shown (29) that thymine-requiring E. coli mutants can be obtained by growing cells in the presence of thymine and the tetrahydrofolate dehydrogenase inhibitor trimethoprim. More recently, other investigators (15, 17, 30) used a combination of sulfanilamide and aminopterin to isolate mutants of S . cerevisiae capable of incorporating thymidine 5'-monophosphate (TMP) into DNA. When we tested sulfanilamide as an inhibitor of sporulation in S. cerevisirre, we discovered that the process was blocked by the antimetabolite. Aminopterin had no effect by itself nor did it potentiate the effect of sulfanilamide. Thus, the inhibition is caused by sulfanilamide alone. Attempts to reverse the inhibition with exogenous p-aminobenzoic acid (PABA), folic acid, methionine, adenine, and uracil were unsuccessful (results not shown). This is in agreement with the observation that the two drugs inhibit vegetative growth of yeast even in rich

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C O L O N N A ET A L .

medium (30); the inhibition is irreversible in vegetative cells as well. Sporulation in all the diploid yeasts tested (Table 3) was at least partially inhibited by sulfanilamide. The low sensitivity of three apparently unrelated hoinothallic strains is an intriguing observation. As yet, we have not investigated this phenomenon. Three lines of evidence suggest that a t least one major event sensitive to sulfanilamide occurs early in the sporulation process. Firstly, synthesis of DNA, RNA, and protein in sporulating cells is affected as early as 2 h after exposure to the drug. Furthermore, although not a sporulation-specific event, glycogen synthesis (which begins at the 2nd-3rd h during sporulation) is drastically inhibited in sulfanilamidetreated cells. Secondly, cells exposed to sulfanilamide fail to reach the state of coininitment to spore formation, an early developmental stage in the sporulation process (28). Finally, escape from sensitivity to the drug begins a t 5 to 6 h and is 50-75% complete at a time when asci normally begin to appear in control cultures. There seems no doubt, then, that the drug affects a t least one early event of sporulation. One possibly susceptible event could be the permeability change characteristic of sporulating yeast (19). If the cells normally become impermeable to sulfanilamide (as well as other compounds) at 5 to 6 h, the escape data would be explained although the first two arguments would not be affected. We find that API cells are permeable to sulfanilamide throughout the entire sporulation cycle, although the level of drug accumulated by the cells varies with time. During the first few hours after shift to SP2, only minute amounts of sulfanilamide are taken up. However, over the next 6-8 h uptake increases sharply, reaching a maximum level which remains constant for the duration of the sporulation cycle. In three separate experiments (results not presented in full) this final concentration ranged from 0.8 to 1.3 mg of sulfanilamide/ml of total cell volume. However, the minimum internal inhibitory concentration of the drug is actually much lower, since smaller quantities are accumulated during the 0 to 6-h period of acute sensitivity of the cells in SP2 (Figs. l b and 2). From our data (not presented in full), this concentration ranges from 0.1 1 t o 0.45 mg of sulfanilamide/ml of total cell volume for the 0 to 4-h period. As shown, sulfanilamide arrests

669

yeast cells at a quasivegetative stage before the point of commitment to sporulation. Furthermore, DNA labeling studies (Table 4) show that sulfanilamide partially restricts uracil uptake. Therefore, it is not unreasonable t o speculate that the drug also arrests the changing ability of the cell to accumulate sulfanilamide. A secondary effect of the drug may therefore be an autoinhibition of its own entry into the cell, a conclusion consistent with the uptake profiles in Fig. 2. If one effect of sulfanilamide is to alter cellular permeability, this could explain the failure of PABA and other metabolites to reverse the inhibition. On the other hand, the irreversibility may reflect a physiologically normal inability of the cells to accumulate sufficient amounts of these compounds to compete with the drug. The fact that sulfanilamide and aminopterin block vegetative growth in very rich medium supports the second interpretation. Vegetative growth of API in PSP2 is practically unaffected by sulfanilamide alone in concentrations which prevent sporulation. We found that cells cultured in PSP2 d o take up sulfanilamide (results not shown), though to a lesser extent than d o sporulating cells. Thus, the insensitivity of vegetative cells may be due to an inability t o accumulate inhibitory levels of the drug under the growth conditions used. The mechanism by which sulfanilainide affects sporulation, or t o put it another way, the nature of the drug-sensitive event, remains unclear. In vegetative cells, the block in growth by sulfanilamide and aminopterin can be overcome by thymidine monophosphate in mutants able to accumulate this compound (15, 17, 30). This evidence that sulfanilamide affects the synthesis of thymidine 5'-triphosphate (TTP) forms the basis of our hypothesis that one primary effect of the drug is to decrease cellular pools of TTP. This hypothesis provides a possible explanation for the inhibition of the synthesis of both glycogen and D N A . If the cells enter the sporulation process with limited pools of TTP, and sulfanilamide acts to prevent sufficient further synthesis of this precursor, the effect upon D N A synthesis becomes understandable. Although the extent of inhibition (50%) is much less than the extent of inhibition of ascus formation (95-99%), the population is fairly homogeneous with respect t o D N A synthesis. Most cells will therefore fail to complete replication of the genome and will

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C A N . J. MICROBIOL. VOL. 23. 1977

be unable to undergo meiosis. The data on nuclear division (Fig. 56) indicate that in fact only about 9% of the cells complete meiosis 11, a result which is consistent with a population in which most but not all cells fail to complete DNA replication. The inhibition of glycogen synthesis could also be explained on the basis of TTP limitation if the precursor to glycogen in yeast is thymidine 5'-diphosphate (TDP) glucose. In fact, TDP-glucose has been found in vegetative yeast cells (along with uridine 5'diphosphate (UDP) glucose; (26)), so it is indeed a possible precursor. It is also possible that sulfanilamide inhibits some sporulationspecific glycosyl transferase reaction, although there seems to be no precedent for such an event. Our hypothesis that the primary effect of the drug is to limit TTP does not explain the changes in RNA and protein synthesis, since these processes should be independent of the TTP pools. Therefore, the alteration in the rates of synthesis of these macromolecules by sulfanilamide may reflect a general derangement of cellular metabolism, or it could also be due to an alteration of precursor pools. In the latter case, one would have to postulate a series of complex changes in the pools, since the rate of incorporation is lower than the controls at early times but higher after 6 to 7 h. An alternative hypothesis would be a primary effect 011 RNA or protein synthesis, leading to pleotropic inhibition of the other macromolecular processes. Cycloheximide has such an effect (8, 18). The biochemical mechanism by which such an inhibition might occur is hard to visualize, since sulfanilamide affects specifically folate metabolism, and sporulating yeast, being in anitrogenous medium, must derive their amino acids via protein breakdown. The concentrations of sulfanilamide used in this study (6-12 mg/ml, i.e., 35-70 mM) are high, but not excessively so in light of the general insensitivity of yeast to antimetabolites. Hydroxyurea, for example, inhibits DNA synthesis in yeast, but only at a concentration of 100 m M or higher. Furthermore, 35 m M sulfanilamide has been used in conjunction with aminopterin to inhibit vegetative yeast for selection of TMP uptake mutants (30). Therefore, although the concentrations of sulfanilamide used here may be high compared with other inhibitors, they

are not uniquely high; the high concentrations required are probably due to the relative impermeability of the cells, particularly during the sensitive period of the sporulation process. In experiments not reported, sporulation by APl was not diminished either by sulfanilic acid or sulfaceta~nide(both 70 mM), so the inhibition is quite specific. Furthermore, after our sulfanilamide was purified by crystallization from a saturated aqueous solution, it was still capable of preventing sporulation. Moreover, the inhibition was observed using sulfanilamide obtained from two suppliers. Therefore, the inhibition is due to the drug itself and not an impurity. Two questions about the state of the cells blocked by sulfanilamide remain unanswered. The first is whether they are blocked at a very specific point before commitment or whether they are arrested at a number of different stages. Release of the cells by transferring them to SP2 minus sulfanilamide gives no better synchrony than is found in an untreated culture, so there is no indication that drug treatment aligns them at some point in the developmental process. On the other hand, one would expect considerable heterogeneity in the TTP pools of an exponential vegetative culture, since synthesis of this precursor would be expected to be correlated with the DNA synthetic period. The second question concerns the 1-5% of the cells which manage to coinplete ascus formation in sulfanilamide-arrested cultures. Since in Fig. 5, asci were not counted as tetranucleate cells, and the final sporulation was 0.8%, in fact 9.2% of the cells appeared to complete meiosis 11, and 16% completed at least one nuclear division. It is not at all clear whether these results indicate that the cells progress randomly to points all through the cycle, or whether a small fraction completes DNA synthesis and then continues through at least one nuclear division but is partially blocked again between meiosis I1 and ascospore formation. If the latter hypothesis is true, a deficiency of glycogen may explain the late block, since this compound begins to be degraded about the time asci appear. Whatever the nature of the sensitive event (or events), the kinetics of escape from SUIfanilamide, being similar to and almost as synchronous as ascus formation, make sensitivity to the drug a useful landmark event during the sporulation process. In this regard, it would

COLONNA ET AL

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be verv i n t e r e s t i n " g to e x a m i n e t h e a v a i l a b l e t e m p e r a t u r e - s e n s i t i v e m u t a n t s (5, 6) to order t h e m w i t h respect to t h e s u l f a n i l a m i d e b l o c k . Acknowledgement hi^ investigation was supported by U.S.P.H.S. G r a n t A1 11730. The a u t h o r s a c k n o w l e d g e H. 0. H a l v o r s o n , A. K. H o p p e r , F. Z i i n m e r m a n , and P. R e d s h a w

14.

I5

16

f o r generously contributing yeast strains.

. .

.

.

.

. . . . . . . . . . . . . . . . . . . . . . .

. . . . . .. .. .. .. ... . . . . . . . . . . . .......... . . . . . . . .

-

.

1. B A D I NJ, . , C. JACKSON, and M. SCHUBERT. 1953. Improved method for determination of plasma polysaccharides with tryptophan. Proc. Soc. Exp. Biol. Med. 84: 288-291. 2. BLANKE, R. V. 1970. Toxicology. 117 Fundamentals of clinical chemistry. Edited by N. W. Tietz. W. B. Saunders Company, Philadelphia. London, and Toronto. pp. 833489. 3. BRATTON, A. C., and E. K . MARSHALL, JR. 1939. A new coupling reagent for sulfanilamide determination. J. Biol. Chem. 128: 537-550. 4. CROES,A. F. 1967. Induction of meiosis in yeast. I. Timing of cytological and biochemical events. Planta (Berlin), 76: 209-226. 5. ESPOSITO, M. S., and R. E. ESPOSITO.1969. The genetic control of sporulation in Socchtrrornyces. I. The isolation of temperat~rre-sensitivesporulationdeficient mutants. Genetics, 61: 79-89. 6. ESPOSITO,M. S., and R. E. ESPOSI-ro.1974. Genes controlling meiosis and spore formation in yeast. Genetics, 78: 215-225. 7. ESPOSITO,R. E., and M. S. ESPOSITO.1974. Genetic recombination and commitment to meiosis in Sotcl~rrronryccs. Proc. Natl. Acad. Sci. U.S.A. 71: 3172-3176. 8. ESPOSITO, M. S., R. E. ESPOSITO,M. ARNAUD, and H. 0 . HALVORSON. 1969. Acetate utilization and macromolecular synthesis during sporulation of yeast. J. Bacteriol. 100: 180-186. 9. GASCON, S . , and J. 0 . L A M P E N1968. . Purification of the internal invertase of yeast. J. Biol. Chem. 243: 1567-1572. 10. G R I V E L LA., R., and J . F. JACKSON. 1968. Thymidine kinase: evidence for its absence from N ~ r r r o s p o u r utrssn and some other micro-organisms, and the relevance of this to the specific labelling of deoxyribonucleic acid. J. Gen. Microbial. 54: 307-317. I I. HABER,J . , M. S . ESPOSITO,P. T. MAGEE,and R. E. ESPOSITO.1975. Current trends in the study of yeast sporulation. I n Spores VI. Edited by P. Gerhardt, R. N. Costilow, and H. L . Sadoff. pp. 132-137. 12. HARTWELL, L. H. 1970. Periodic density fluctuation during the yeast cell cycle and the selection of synchronous cultures. J. Bacterial. 104: 1280-1285. 13. HOPPER,A. K., and B. D. HALL.1975. Mating type and sporulation in yeast. I. Mutations which alter

17.

18. 19. 20. 21. 22.

23. 24. 25. 26.

27. 28.

29. 30.

67 1

mating-type control over sporulation. Genetics, 80: 41-59. HOPPER.A. K., P. T. MAGEE,S. K. W E L C H ,M. FRIEDMAN and . B. D. HALL. 1974. Macromolecule synthesis and breakdown in relation to sporulation and meiosisin yeast. J . Batter-iol. 119: 619428. J A N N S E NS, , I W I T I E .and R MEGNET 1973 MLItants for the speclfic labellrng of DNA In Sacthotot?~yceccerel IS IN^' B ~ o c h ~ rB~ophys. n Acta. 299. 68 1 4 8 5 K A N E ,S M ilnd R. R O I H 1974 Carbohyd~ate metabol~smd u l ~ n gascospole development In y e ~ ~J t . B~cterlol.118 8-14 L A S K O W S KW.. I , ilnd E. L E H M A N N - B K A U1974. NS. able to grow after inhibiMutants of Srrc~clrrr~~ur?q~cc.s tion of thymidine phosphate synthesis. Mol. Gen. Genet. 125: 275-277. MAGEE,P. T., and A. K. HOPPER.1974. Protein synthesis in relation to spor~rlalionand meiosis in yeast. J. Bacteriol. 119: 952-960. MILLS,D. 1972. Effect of pH on adenine and amino acid uptake during sporulation in Srrcchrrrotnyces c~rr12isitre.J . Bacteriol. 112: 5 19-526. PINON,R., Y. SALTS,and G. S I M C H E N 1974. . Nuclear and mitochondrial DNA synthesis during yeast S P O ~ L I lation. Exp. Cell Res. 83: 231-238. R O B I N O WC. , F., and J . MARAK.1966. A fiber appalatus in the nucle~rsof the yeast cell. J. Cell Biol. 29: 129-151. ROTH, R. 1973. Chromosome replication during meiosis. Identification of gene firnctions required for premeiotic DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 70: 3087-3091. ROTH,R., and S. FOGEL.1971. A selective system for yeast mutants deficient in meiotic recombination. Mol. Gen. Genet. 112: 295-305. ROTH,R., and H. 0 . HALVORSON. 1969. Sporulation of yeast harvested during logarithmic growth. J. Bacteriol. 98: 83 1-832. ROTH,R., and K . L U S N A K1970. . DNA synthesisduring yeast sporulalion: genetic control of an early developmental event. Science. 168: 493-494. SENTANDREU. R. S.. and J. 0 . LAMPEN.1970. Biosynthesis of yeast mannan: inhibition of synthesis of mannose acceptor by cycloheximide. FEBS Lett. 11: 95-99. SILVA-LOPEZ. E.. T . J. ZAMB,and R. ROTH. 1975. Role of premeiotic replication ingene conversion. Nature. 253: 212-214. SIMCHEN G., , R. PINON,and Y. SALTS.1972. Sporulation in Soc.c~htrr.ornycesccr.e~~isioe: premeiotic DNA synthesis. readiness and commitment. Exp. Cell Res. 75: 207-218. STACEY,K . A,. and E. SIMSON.1965. Improved method for the isolation of thymine-requiring mutants coli. J. Bacteriol. 90: 554555. of Esclrc~ricl~itr W I C K N E RR., B. 1974. Mutants of Saccl?trromyces ct>revi.sine that incorporate deoxythymidine-5'-monophosphate into deoxyribonuclei acid in vivo. J. Bacteriol. 117: 252-260.

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Inhibiton by sulfanilamide of sporulation in Saccharomyces cerevisiae.

Inhibition by sulfanilamide of sporulation in Saccharomyces cerevisiae WILLIAM J. COLONNA, JAMESM. GENTILE,'A N D P. T. MAGEE Deprrrtt7zer1tof Hrrrrrr...
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