JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 1064-1073Copyright © 1975 American Society for Microbiology

Vol. 121, No. 3Printed in U.S.A.

Kinetics of Induced and Repressed Enzyme Synthesis inSaccharomyces cerevisiae

ROBERT P. LAWTHER AND TERRANCE G. COOPER*

Department of Biochemistry, Faculty of Arts and Sciences, University of Pittsburgh, Pittsburgh,Pennsylvania 15261

Received for publication 10 October 1974

Our previous work has shown that both induction, after addition of inducer,and loss of' ability to produce allophanate hydrolase, after removal of' inducer,proceed more rapidly than expected from the reported half-life of messengerribonucleic acid in Saccharomyces cerevisiae. As a basis of rectifying theseobservations, we have characterized induction and repression of allophanatehydrolase synthesis and find that: (i) induction of the hydrolase beginsimmediately upon addition of inducer, (ii) once induction has been initiatedremoval of inducer does not result in immediate loss of synthetic capacity, (iii)induction of the capacity to produce hydrolase can occur in the absence of proteinsynthesis, (iv) the half'-life of hydrolase synthetic capacity increases if proteinsynthesis is inhibited, (v) allophanate hydrolase itself is not degraded uponremoval of inducer, and (vi) induction and repression of allophanate hydrolasesynthetic capacity likely occurs at the level of transcription.

Two major avenues of investigation have beenfollowed in quest of the molecular events sur-rounding gene expression. One avenue involvesmonitoring the metabolism of collective mes-senger (m) ribonucleic acid (RNA); the otherassay of specific gene products. Our currentunderstanding of poly(A) and its participationin processing of eucaryotic transcripts (5-7, 11,15) represents a major achievement of the firstapproach. However, a detailed account of geneexpression and its control ultimately requiresconcentration upon a specific well defined set ofgenes.

In Saccharomyces cerevisiae, studies of col-lective mRNA metabolism have yielded anmRNA half-life of 20 min. Hutchison et al. (10)observed polysome decay with a half-life of 23min after shift of a temperature-sensitive mu-tant, defective in an unidentified reaction ofRNA metabolism or transport, to the nonper-missive temperature. In support of thisTonnesen and Friesen reported loss of ['IC ]leu-cine incorporation into trichloroacetic acid-precipitable material with a half-life of 21 + 4min after addition of' ethidium bromide ordaunomycin to their cultures (18). Monitoringspecif'ic gene products, Kuo et al. (13) observedthat lomofungin, an RNA polymerase inhibitor,inhibited formation of invertase, a-glucosidase,and acid phosphatase only after a 30-min lag;an observation consistent with a synthetic ca-pacity half-life of approximately 20 min. How-

ever, Lawther and Cooper (14) have recentlyobserved that: (i) only 3 min elapsed betweenaddition of inducer to a culture and appearanceof allophanate hydrolase activity, and (ii) thecapacity of induced cultures to continue pro-ducing allophanate hydrolase decayed with ahalf-lif'e of 3 min after inducer removal.These observations raise the possibilities that

the allantoin degradative system is regulated ata level other than transcription of mRNAspecies of S. cerevisiae are subject to widelyvarying rates of metabolism (17). The informa-tion needed to distinguish between these possi-bilities can be obtained by careful definition ofallantoin system induction. The data reportedhere result from studies of the characteristicsand requirements of allophanate hydrolase in-duction and repression using the methods for-mulated by Hartwell and Magasanik and byKepes (9, 12).

MATERIALS AND METHODSCulture conditions. Wild-type strain M25 was

grown in minimal medium as previously described(2). Cell density measurements were made using aKlett Summerson colorimeter (500 to 570 nm bandpass filter). One-hundred Klett units is approxi-mately equivalent to 3 x 107 cells per ml of culture.Urea was added to cultures to bring about inductionof allophanate hydrolase, because of the rapidity withwhich it both enters the cell when added exogenouslyand is lost when cells are resuspended in urea-freemedium.

1064

INDUCED ENZYME SYNTHESIS IN SACCHAROMYCES

Concentration of cultures. Cultures to be concen-trated were grown in the cell densities indicated andcollected by centrifugation (3 min at 3,000 rpm in anSS-34 rotor or 1,085 x g) at room temperature.Harvested cells were resuspended in the indicatedvolumes of prewarmed, preaerated medium and al-lowed to equilibrate for 2 to 5 min before initiation ofthe experiment. Kinetics of induction in concentratedcultures were compared to those observed in normaldilute cultures and were found to be exactly the samein every respect.

Transfer of cels from one medium to another. Ina number of the subsequent experiments it wasnecessary to transfer cell samples from one medium toanother. The total time elapsing between removal ofcells from the first medium and their resuspension inthe second was 20 to 30 s and is indicated on eachfigure as the space between two vertical lines whichinterrupt the experimental curves. All of the glass-ware used for cell harvest by filtration (membranefilters 0.45-Am pore size; Millipore Corp.) was pre-warmed to 30 C to avoid temperature shock of thecells. The extent of cell loss during this procedure wasdetermined using radioactively labeled cells and wasfound to be negligible.Enzyme assays. Allophanate hydrolase was as-

sayed as previously described by Whitney and Cooper(21). Samples to be used for assay were, in all cases,transferred from the experimental culture to cold testtubes containing cycloheximide (10 Ag/ml final con-centration).

Inhibitors. VE-76 is a preparation containing prin-cipally verrucarin A. However, small amounts of otherverrucarin species were also present. That this prepa-ration inhibits translational initiation in S. cerevisiaewas shown using techniques similar to those reported(19; C. S. McLaughlin, personal communication).Lomofungin was prepared and used according to theprocedures described by Lawther and Cooper (Arch.Biochem. Biophys., in press).

RESULTSEnzyme production in the presence and

absence of inducer. Figure 1 shows the time-dependent appearance of allophanate hydrolaseafter a pulse of induction. The experiment wasinitiated at zero time by addition of urea (10 mMfinal concentration) to a growing culture. Afterremoving samples at 0, 1, 2, and 3 min forenzyme assay, the culture was filtered andresuspended in urea-free, minimal medium(total time required for transfer of cells to ureafree medium was 20 to 30 s) and additionalsamples were taken as indicated. As shown,allophanate hydrolase activity continued to in-crease in the absence of exogenous inducerreaching a constant level 15 min after itsremoval. These data may be accounted for intwo ways. A burst of enzyme production canoccur in the absence of inducer once inductionhas been set into progress, or alternatively, the

endogenous level of inducer requires 15 min tofall below the threshold amount needed tosupport continued enzyme production. To dis-tinguish between these two possibilities, thecellular accumulation and loss of urea wasfollowed. As shown in Fig. 2, a large amount ofurea is observed within cells very shortly afterits addition to the medium. At the concentra-tions used in this experiment, Cooper andSumrada (3) have shown that the predominantmode of entry is diffusion. Two to 3 min afteraccumulation begins, there is a drastic disap-pearance of radioactive material from the cells;a steady state level is attained at 9 to 10 min.Loss of urea coincides closely with the firstappearance of active urea amido-lyase afterinduction and may be a result of its degradativeaction. This steady state level of urea reflectsthe amount of inducer needed to maintain thesystem in its fully induced state. In a secondexperiment, cells were permitted to accumulate[14C Jurea for 1 min. At this time, the cells wereharvested by filtration, resuspended in urea-free medium, and sampled at short times there-after. The observed level of urea dropped to anundetectable amount before the first sample

4.0-j

0

o3.60

1-1

3.2

Z 2.8

LWJ 2.4 NO UREA ADDEDOi UREA

4 8 12 16 20 24 28

MINUTES

FIG. 1. Pulse induction of allophanate hydrolase.A 240-mi culture of strain M25 (cell density of 40Klett units) was concentrated to 35 ml as described.Six milliliters of cells was removed to serve as anuninduced control (the four points indicated as "nourea added"). Urea (10 mM final concentration) wasadded at zero time to the remainder of the culture (29ml) and 1.0 ml samples were removed for assay at 0, 1,2, and 3 min. The 24 ml of culture remaining afterremoval of the 3-min sample were collected by filtra-tion, washed with 2 volumes of prewarmed, pre-areated medium, and resuspended in 250 ml of pre-warmed, preaerated, urea-free medium. Samples (10ml) were removed at the indicated times and trans-ferred to cold tubes containing cycloheximide. Allsamples were assayed for allophanate hydrolase.

VOL. 121, 1975 1065

LAWTHER AND COOPER

40

rn 20 00

I0

7

4 _ O2 4 6 8 10 12

MINUTESFIG. 2. Intracellular urea concentration during in-

duction of the allantoin degradative system. Theexperiment was initiated at zero time by addition ofurea (10 mM final concentration at a specific activityof 0.75 mCi/mmol). Thereafter, at the times indi-cated, 1.0-ml samples were removed from the culture,collected on glass fiber or membrane filters (MilliporeCorp.) and washed with 8.0 ml of cold minimalmedium containing 10 mM urea and 0.1 M KCN. Thewashed filters were immediately placed, without dry-ing, in alkaline aquasol for radioactivity determina-tion. A thorough discussion of these procedures andtheir limitations appears elsewhere (3).

could be taken (i.e., by 30 s after removal fromradioactive medium). Collectively these dataargue against the second possibility raisedabove and suggest that enzyme production cancontinue beyond the point of inducer exhaus-tion.

Protein degradation contributes significantlyto the regulation of some eucaryotic systems(16). To determine whether or not this type ofregulation is operative in the case of allopha-nate hydrolase, the level of hydrolase activitywas monitored for two generations after removalof inducer from the culture medium. As shownin Fig. 3A, the amount of enzyme activityobserved per unit volume of cells remainedreasonably constant suggesting that no enzymedegradation occurred during the two genera-tions monitored. However, the amount of activ-ity per cell decreased logarithmically (Fig. 3Aand B) with a half-life of 165 min or somewhatlonger than the 143-min doubling time of theculture. This discrepancy is accounted for by abasal rate of hydrolase production in the ab-sence of added inducer (note the slight increasein total activity in Fig. 3A).The data in Fig. 1 and those reported earlier

(14) demonstrate that induction can be divided

into at least two phases. During the first period(3 min at 30 C) no increase in enzyme synthesiscould be detected; thereafter the rate of synthe-sis increased, becoming constant at 5 to 6 min.In view of similar observations in Escherichiacoli (12) and Aspergillus nidulans (4), it may bereasoned that the synthetic capacity to produceenzyme begins to accumulate during the initial3-min period. This was shown to be the case bythe following experiment which was initiated atzero time by adding inducer to an ammonia-grown culture. Thereafter samples were trans-ferred to inducer-free medium for 30 min addi-tional incubation to permit expression of what-ever synthetic capacity accumulated during theperiod of inducer presence. As shown in Fig. 4A(filled circles), induction began immediatelyupon addition of inducer. However, the initial

120

ioo

0

zD

w-iy

LL)

-nz

M.

(f)LLJ

-i

02CL

150

100

80

60

40

100

80

60

40

201

O

r-men)cZ

4_3

2 -Lr-

m

60 120 8 24MINU TES

FIG. 3. Disposition of allophanate hydrolase activ-ity after removal of inducer. A culture of strain M25was grown overnight on minimal ammonia medium.At a cell density of 5 Klett units urea was added to a

final concentration of 10 mM. At 15 Klett units (zerotime), the cells were collected by filtration, washedwith 2 volumes of prewarmed, preaerated minimalmedium and resuspended in an equivalent volume ofprewarmed, preaerated minimal ammonia mediumdevoid of urea. Samples (10 ml) were removed at theindicated times for assay of allophanate hydrolase.

1066 J. BACTERIOL.

A

I

I, 1,40 80 120 160 200 240

B-

80

60

40

20

z

J-i

INDUCED ENZYME SYNTHESIS INSACCHAROMYCES1

complete cessation of synthesis requiring 3 min.A < B 40. On the other hand, addition of cycloheximide,

202 an elongation inhibitor or trichodermin, a ter-> mination inhibitor (19), abruptly halted any

7 further increases in enzyme activity. These data4\-5x argue that protein synthesis is necessary for anS3 increase in the enzyme level and raise the/_2 question of whether or not it is a prerequisite forI4

I, I,2 4

the induction process. To address this question,4 8 12 16 20 0 2 4 6 8

urea and trichodermin were added simultane-MINUTES

FIG. 4. Time-dependent accumulation of allopha-nate hydrolase synthetic capacity and activity incultures of S. cerevisiae. A 250-ml culture of strainM25 (cell density of 40 Klett units) was concentratedto 30 ml as described. Urea was added at zero time. Atthe times indicated, 1.0-ml samples were either trans-ferred to tubes containing 2 ml of cycloheximide (10Ag/ml) (0) or collected by filtration (0), washed with5.0 ml of prewarmed, preaerated medium, and placedin 10 ml of prewarmed, preaerated, urea-free mediumfor 30 min. At the conclusion of incubation, 5.0-mlsamples were removed and, along with the othersamples, assayed for allophanate hydrolase activity.

rate of synthetic capacity accumulation washigher than that observed after 1 to 3 min(range of values observed from experiment toexperiment; compare Fig. 4, 9, and 13).Whether this transient high rate of accumula-tion reflects physiological events or is an artifactof sample preparation is presently undecided.The difference between the enzyme contentobserved after inducer removal (closed circles)and that observed upon addition of cyclohexi-mide (open circles) is the residual syntheticcapacity. Its maximum value is represented bythe difference between total accumulated syn-thetic capacity (closed circle curve) and theasymptote of the amount expressed at cessationof protein synthesis (open circles). A semiloga-rithmic plot of the value "x" (Fig. 4A) isdepicted in Fig. 4B and has a half-life value of2.9 min. This value is interpreted by Hartwelland Magasanik and Kepes (9, 12) as represent-ing synthetic capacity half-life. As such, it isgood agreement with the 3.2-min value wereported earlier (14).Requirement of protein synthesis for ex-

pression of allophanate hydrolase syntheticcapacity. A requirement of protein synthesis forproduction of allophanate hydrolase was estab-lished by adding an initiation, elongation, ortermination inhibitor of translation to a culturesynthesizing allophanate hydrolase at a linearrate. As shown in Fig. 5A, addition of VE-76, aninhibitor of initiation (20), immediately de-creased the rate of enzyme production with

8

6

LU

0

CK

w

z

iC,)w

0

5

4

6

5

4

3

6

5

4

3

4 8 12 16 20 24 28

MINUTESFIG. 5. Effect of protein synthesis inhibitors upon

induction of allophanate hydrolase. A culture of strainM25 was grown to a cell density indicated below andurea was added to 10 mM final concentration at zerotimes. At the indicated time, an inhibitor of proteinsynthesis was added. (A) Cell density of 45 Klettunits; VE-76 to a final concentration of 40 Ag/ml. (B)Cell density of 25 Klett units; cycloheximide to a finalconcentration of 200 gg/ml (the same results, how-ever, is also observed at 10 gg/ml). (C) A 280-mlculture of strain M25 (25 Klett units) was concen-trated to 30 ml as described. Trichodermin added to afinal concentration of 30 ,g/ml.

(r

-J 10

8E

w 6

z

W 2-i

- A

+ VE- 76

4 8 12 16 20 24 28

- B +CYCLOHEXIMIDE_/_- ,/_

4 8 12 16 20 24 28

+ TRICHODERMIN

i-

I x

1067VOL. 121, 1975

LAWTHER AND COOPER

ously to an uninduced culture of Sac-charomyces. After a short (6 min) incubationperiod, the culture was divided into two por-tions and rapidly harvested by filtration. Thetwo portions were resuspended in either induc-er-free medium containing trichodermin or in-ducer-free, trichodermin-free medium. Asshown in Fig. 6A, enzyme was synthesizednormally in the sample transferred to inducer-and inhibitor-free medium, indicating that in-

AH 8

70

, 6

z 5

w 4

0

30. *\

I3I X8 16 24 32 40 48

6

4

2

LAJ

0.8

0.2

8 16 24 32 40 48

MINUTESFIG. 6. Requirement of protein synthesis for induc-

tion of allophanate hydrolase. A 240-ml culture ofstrain M25 (cell density of 45 Klett units) wasconcentrated to 29 ml as described. At zero time, ureaand trichodermin were added simultaneously to finalconcentrations of 10 mM and 15 ug/ml, respectively.Samples (1.0 ml) were removed at 0, 2, 4, and 6 minfor assay. At 6 min, the remaining cells were collectedby filtration, washed with 2 volumes of prewarmed,preaerated minimal medium, resuspended in 250 mlof prewarmed, preaerated, urea-free medium, anddivided into two parts. A 100-ml portion of culturereceived 15 A.g of trichodermin per ml (0), whereasthe remainder (0) received no further additions.Samples (10 ml) were removed at the times indicatedand assayed for allophanate hydrolase.

duction may be set in progress in the absence ofprotein synthesis. It should also be noted (seeFig. 6B) that this treatment decreased the rateof synthetic capacity loss in inducer-free me-dium by only 1.5 min (half-life is 4.5 min).However, as shown in Fig. 7, the rate of syn-thetic capacity loss can be greatly decreased iftranslation is inhibited by inclusion of tri-chodermin in the culture medium (half-life is 50min).The facts that (i) loss of protein synthesis

does not impair the induction process and (ii)synthetic capacity half-life greatly increases inthe absence of translation leads to the possibil-ity that the synthetic capacity to produce allo-phanate hydrolase may reach greater than nor-mal steady-state levels if induction is carried outin the absence of translation. This possibilitywas evaluated by monitoring the synthetic ca-pacity for allophanate hydrolase production inthe absence and presence of trichodermin (Fig.8). An uninduced culture was divided into twoportions; one-half received urea (open circles) atzero time and the other was pretreated withtrichodermin for 2 min before addition of ureaat zero time on the figure (closed circles). At thetimes indicated, samples were transferred toinducer-free, trichodermin-free medium andpermitted to express whatever synthetic capac-ity had accumulated. As shown in this figure,both cultures initially accumulate hydrolasesynthetic capacity at a similar rate. However,contrary to the proposal above, hydrolase syn-thetic capacity does not accumulate to abnor-mally high values in the absence of proteinsynthesis. Rather, it levels off in about the sametime as observed in Fig. 6.

Kepes (12), in his studies of ,B-galactosidaseinduction, observed that induction of f3-galac-tosidase synthetic capacity was temperaturedependent, whereas expression of accumulatedsynthetic capacity appeared temperature inde-pendent. To ascertain the effects of temperatureupon the accumulation and expression of syn-thetic capacity to produce allophanate hydro-lase, a culture was divided into two equalportions, harvested, and resuspended in mediaat either 21 C (open and closed circles in Fig. 9)or 35 C (open and closed squares in Fig. 9). Theexperiment was initiated at zero time by addi-tion of urea to the cultures. At the timesindicated, samples of each culture were rapidlyharvested and transferred to inducer-free mediaat both 21 C (closed circles or squares) and 35 C(open circles or squares). As shown in Fig. 9,minimal differences were observed between cul-tures that expressed their synthetic capacity at

1068 J. BACTERIOL.

INDUCED ENZYME SYNTHESIS IN SACCHAROMYCES

D12

_ <(.210-

E 8

J_ _0

r22__

8 16 24 32 40 48 56

MINUTES

FIG. 7. Loss of synthetic capacity to produce allo-

phanate hydrolase in the absence ofprotein synthesis.A 300-ml culture of M25 (cell density of 50 Klettunits) was concentrated to 29 ml as described. At zero

time, urea and trichodermin were added simultane-ously to final concentrations of 10 mM and 15 jsg/ml,respectively. Samples (1.0 ml) were removed at 0, 2, 4,and 6 min for assay. The remaining 25 ml of culturewere then collected by filtration, washed with 2volumes of prewarmed, preaerated medium, andresuspended in 25 ml of prewarmed, preaerated,urea-free medium containing 15 Ag of trichoderminper ml. Samples (1.0 ml) were, at the indicated times,(0) placed in cycloheximide or (0) collected byfiltration, washed, and placed in 10 ml of freshprewarmed, preaerated, urea-free and trichodermin-free medium for 30 min additional incubation. At theend of incubation, 5.0-mi samples removed for allo-phanate hydrolase assay along with the samplesobtained earlier.

21 or 35 C (compare open and closed circles or

open and closed squares). However, the degreeof induction was moderately temperature de-pendent (compare open circles to open squaresor closed circles to closed squares).Requirement of RNA synthesis for accu-

mulation of allophanate hydrolase syntheticcapacity. A requirement of RNA synthesis forproduction of allophanate hydrolase was estab-lished by adding an inhibitor of RNA polymer-ase to a culture synthesizing allophanate hydro-lase at a linear rate. As shown in Fig. 10A, 8 to10 min elapsed between addition of lomofunginand cessation of hydrolase production. Thisvalue is consistent with losing hydrolase syn-thetic capacity with a half-life of slightly lessthan 3 min. The fact that hydrolase levelscontinue to slowly decline rather than remain-ing constant after reaching a plateau (Fig. 10A,closed circles after 20 min) is a secondary effectof lomofungin upon the enzyme. This effect hasbeen shown to occur in the absence of both RNAand protein synthesis by Lawther and Cooperin press). This complication should have littleeffect upon the observed time required to halt

hydrolase production via RNA synthesis inhi-bition, because the rate of the secondary drugeffect (90-min half-life) is so much smaller thanits primary effect (3-min half-life). If the aboveexperiment is repeated and gross protein syn-thesis is monitored (incorporation of [3H]leu-cine into hot trichloroacetic acid-precipitablematerial, Fig. 10B), 90 min elapse before syn-thesis stops. The half-life of this loss in capac-ity is 20 min, or seven times greater than thatobserved in the case of allophanate hydrolase.Since it has been shown above that protein syn-thesis is not required for synthetic capacity ac-cumulation but rather for its expression, it isreasonable to determine the effects of tran-scription upon these two processes. As shownin Fig. 11A, if lomofungin is added at the sametime as inducer no increase in hydrolase activityoccurs. However, in cells incubated with in-ducer and transferred to inducer-free mediumcontaining lomofungin, the transcription in-hibitor has no deleterious effects upon expres-sion of previously accumulated synthetic c pac-ity (Fig. 11B). On the contrary, cells trans-

12

C_j

~10

N8(0w

H

Z

N4

-LJ0-2

_10 20 30 40 50 60

MINUTES

FIG. 8. Steady-state levels of synthetic capacity inthe absence of protein synthesis. A 300-ml culture ofstrain M25 (cell density of 60 Klett units) wasconcentrated to 50 ml as described and divided intotwo 25-mi portions. One culture received trichoder-min to final concentration of 15 1sg/ml (0) and theother nothing (0). Two minutes later (zero time in thefigure), urea was added (final concentration of 10mM) to both cultures. Samples (1.0 ml) (0) and (0)were removed at the indicated times, collected byfiltration, washed with 5 ml ofprewarmed, preaeratedmedium, and resuspended in 10 ml of urea-free,trichodermin-free medium for 30 min. At the conclu-sion of incubation, 5.0-mi samples were taken forenzyme assay. Samples (1.0 ml) (U) were also takenfrom the trichodermin-containing cultures and trans-ferred to tubes containing 2 ml of cycloheximide (1.0mg/ml).

1069VOL. 121, 1975

I Q.

LAWTHER AND COOPER

w

202

or

20

0

4 8 12 16 20 24 28

MINUTE S

FIG. 9. Effect of temperature upon accumulationand expression of allophanate hydrolase syntheticcapacity. A 300-mi culture of strain M25 (cell densityof 60 Klett units) was concentrated to 30 ml asdescribed. This culture was divided into two 15-mIportions which were shifted from 30 to 21 C (0, 0) or35 C (U, 0), respectively, and allowed to equilibratefor 15 mmn. Thereafter, urea was added (10 mM finalconcentration) to each culture (this is zero time in thefigure) and 1.0-mI samples were removed at theindicated times, collected by filtration, washed with 5ml of prewarmed, preaerated medium, and placed in10 ml of urea-free medium at 21 C (0, *) or 35 C (0,0). At the conclusion of incubation, all of the sampleswere assayed for allophanate hydrolase activity.

FIG. 10. Effect of lomofungin upon synthesis of (A)allophanate hydrolase and (B) gross cellular protein.(A) Urea (10 mM final concentration) was added to aculture of strain M25 (cell density of 35 Klett units) atzero time. Samples (10 ml) were removed for assay atthe times indicated. At 12.5 min after urea addition, aportion of the culture was transferred to a second flaskcontaining lomofungin (1 ug/ml) and sampling of bothflasks was continued as before. Lawther and Cooper(manuscript in preparation) have shown that at alomofungin concentration of 1 ug/ml, the rate of RNAand protein synthesis is inhibited 80 and 40%, respec-tively (these values are from measurements initiated30 min after addition of lomofungin). (B) The experi-ment was initiated at zero time by adding leucine (5iug/ml at a specific activity of 1 mCi/mM) to a cultureof strain M25 (cell density of 30 Klett units). Samples(0.2 ml) were taken as indicated. At 10 min, theculture was divided and manipulated as describedabove. Samples (in trichloroacetic acid) were heatedin a boiling water bath for 10 min and the precipitatedprotein was collected on glass fiber filters. The speci-ficity of lomofungin and its kinetics of RNA and grossprotein synthesis inhibition have been carefully stud-ied in the strains used in these experiments. Results ofthose studies will appear elsewhere (Lawther andCooper, accepted for publication).

ferred to lomofungin-containing medium pos-sess somewhat higher levels of hydrolase pro-duction that the control culture. Whether ornot this difference is physiologically significantis an open question.Level at which nitrogen repression occurs.

Bossinger et al. (1) observed that enzyme pro-duction ceased within 5 Klett units of cellgrowth after addition of a readily metabolizedamino acid to a fully induced culture. Thispreliminary indication of a fast repressive re-sponse prompted us to look carefully at thekinetics of enzyme synthesis during onset ofrepression. As shown in Fig. 12, 11 min arerequired to shift from the unrepressed to therepressed rate of enzyme synthesis. This isconsistent with a half-life loss of synthesizing

w A

-6

w

z

+I FNGIN

C,)w-J30

0 20 30 40 50 60

MINUTES

£~~~~~~~~~~~~~~~~~0

B60

00-~~~~~~~cr21200 0- + LOMOFUNGIN00 (I~~~~~~u~g /ml)z

r 80

z0 40

w

20 40 60 80 100MINUTES

1070 J. BACTERIOL.

INDUCED ENZYME SYNTHESIS IN SACCHAROMYCES

D3 A

H7

0

i

'-5

w

z 4

MINUTES

Cl4.03 + LOMOFUNGIN L/

n ~ ~~~(lgg/ml) >

L) 3.5_

-J-0

3v.0- /_

2c.5 Z

c 4 8 12 16 20 24 2

MINUTES20

w

FIG. 11. Induction of allophanate hydrolase andthe synthetic capacity to produce the enzyme in thepresence and absence of lomofungin. (A) A culture ofstrain M25 was grown to a cell density of 35 Klettunits and divided into two portions. One portionreceived urea (10 mM final concentration) and theother received lomofungin (I jug/ml final concentra-tion) followed I min later by addition of urea (10 mMfinal concentration). Zero time in this figure repre-sents the time of urea addition to both cultures. At theindicated times, 10-ml samples were removed fromboth cultures and assayed for allophanate hydrolaseactivity using standard procedures. (B) Accumulationof allophanate hydrolase synthetic capacity was mon-itored in the presence and absence of lomofungin (I,ug/ml final concentration). A culture of strain M25was grown to a cell density of 35 Klett units anddivided into two portions. One portion was concen-trated 10-fold- by centrifugation as described. Theexperiment was initiated (zero time in the figure) byaddition of urea (10 mM final concentration) to theconcentrated cells (-). Thereafter as indicated,1.0-ml samples of this culture were collected by

capacity in the area of 3 min (compare with Fig.1 and 10A). The 15% of initial hydrolase produc-tion rate observed after addition of the repres-sive agent may be caused by large intracellularpools of urea. However, there is presently noexperimental evidence to rectify this observa-tion to the fact that cells grown over night inmedium containing 0.1% serine have completelylost hydrolase activity.To decide whether repression is exerted at the

level of synthetic capacity accumulation orexpression of accumulated synthetic capacity,an experiment similar to that described in Fig.4A and 9 was performed. An uninduced log-phase culture was divided into three portions.At zero time, urea (10 mM final concentration)was added to two portions and urea plus 0.1%serine was added to the third. At the timesindicated in Fig. 13, cell samples from the firsttwo portions were harvested by filtration andtransferred to either urea-free medium contain-ing no serine (closed circles) or urea-free me-dium containing 0.1% serine (closed squares).Cell samples from the third portion were trans-ferred to urea-free and serine-free medium(open circles). After transfer, all samples wereincubated for 30 min to express whatever syn-thetic capacity they had accumulated. Asshown in Fig. 13, the presence of serine duringexpression of accumulated synthetic capacitydid not affect the rate of enzyme production(compare slopes for curves of closed circles andsquares). On the other hand, the presence ofserine during induction resulted in a signifi-cant decrease in the amount of hydrolase pro-duction (compare slopes for curves of open andclosed circles).

DISCUSSIONWe have demonstrated that the increase in

allophanate hydrolase levels after addition ofinducer results from at least two processes. Thefirst is accumulation of the potential to synthe-size the enzyme. This process begins immedi-ately after addition of inducer, is moderately

filtration and resuspended in 10 ml of prewarmed,preaerated, urea-free medium for 30 min additionalincubation. At the end of incubation, 5.0-ml sampleswere removed for enzyme assay. The second portion ofthe original culture (0) was not concentrated, butreceived urea (10 mM final concentration) at zerotime in the figure. Thereafter as indicated, 10-mlsamples were transferred to flasks containing lomo-fungin (I ug/ml final concentration) and incubationwas continued for 30 min. At the end of incubation,5.0-ml samples were removed for enzyme assay.

VOL. 121, 1975 1071

LAWTHER AND COOPER

w

ELLJx

-J

D0

0

z

N

0

4 8 12 16 20 24 28

MINUTES

FIG. 12. Allophanate hydrolase production afterthe onset of repression. (A) A culture of strain M25was grown to a cell density of 30 Klett units. Theexperiment was initiated at zero time by addition ofurea (10 mM final concentration). Eighteen minutesafter urea addition, asparagine was added (0.1% finalconcentration). Samples removed as indicated were

assayed for allophanate hydrolase activity. The datain (B) was obtained in the same way except for thedifferences in the time of sample removal.

temperature dependent, requires RNA synthe-sis, and does not require protein synthesis. Thesecond process is expression of accumulatedpotential. This can occur in the absence ofinducer and RNA synthesis, is not apparentlytemperature dependent, but does require pro-

tein synthesis. It is likely that expression ofaccumulated synthetic capacity reflects trans-lation of an accumulated mRNA. However, thepossibility that observed increases in allopha-nate hydrolase level result from something otherthan de novo enzyme synthesis may be totallyeliminated only with techniques which immu-nochemically monitor enzyme protein levels.

If, in fact, "expression" is translation ofaccumulated hydrolase-specific mRNA, the ob-servations made in Fig. 5A require furthercomment. These data indicate that 3 min elapsebetween cessation of translational initiationand total inhibition of further enzyme produc-

tion. This is an estimate of the time required totranslate hydrolase-specific mRNA. Since it hasthe same duration as the period between in-ducer addition and appearance of active en-zyme, it could be argued that control of thissystem occurs at the initiation step of proteinsynthesis. However, it is more plausible that theobserved 3-min figure overestimates the trans-lation time due to a partial inhibition of proteinsynthesis elongation by this class of initiationinhibitors (20).Our data are consistant with the hypothesis

that accumulation of potential or syntheticcapacity to produce allophanate hydrolase uponaddition of inducer is likely accumulation ofhydrolase-specific mRNA and loss of syntheticcapacity resulting from inducer removal or theonset of repression reflects a loss of that mRNA.This implies that induction and repression ofthe allantoin degradative system occurs at thelevel of transcription. If this is true, the func-

1

w

-J

10

4 8 12 16 20

MINUTESFIG. 13. Effect of serine upon accumulation and

expression of allophanate hydrolase synthetic capac-ity. A 250-ml culture (cell density of 40 Klett units)was concentrated to 30 ml as described. The concen-trated culture was divided into three portions. Oneportion (0) received serine (0.1% final concentration).At zero time, urea (10 mM final concentration) wasadded to all three portions. Thereafter at the indi-cated times, samples were removed from the threeportions, and the cells were transferred to a secondmedium as described below and incubated in thesecond medium for 30 additional min. Samples fromthe portion incubated in the presence of serine (0)were transferred to serine-free and urea-free medium.The remaining two portions were transferred to eitherurea-free and serine-free medium (* or urea-freemedium containing serine (0.1% final concentration)(U). At the end of the second incubation, all of thesamples were assayed for allophanate hydrolase usingstandard procedures.

1072 J. BACTERIOL.

INDUCED ENZYME SYNTHESIS IN SACCHAROMYCES

tional half-life of hydrolase-specific mRNA in-creases in the absence of translation (see Fig. 7).This is in agreement with observations made inE. coli (12) and A. nidulans (4), but is atvariance -with the report of Hartwell et al. (8)that polysomes decay at the same rate whetheror not cycloheximide is present. However, theconflict may be more apparent than real be-cause their bulk mRNA measurements proba-bly did not include mRNA species with shorthalf-lives.Another significant implication of this con-

clusion is the clear possibility of mRNA specieswith different rates of metabolism. As citedearlier, the half-life for both cumulative mRNAand three specific synthetic capacities is 21 ± 3min. This must be contrasted with the presentresults suggesting that allophanate hydrolasesynthetic capacity decays with a half-life of 3min and those of Cybis and Weglenski (4) whoreport a 2.7-min half-life for arginase syntheticcapacity in A. nidulans. It is reasonable toquestion whether or not differences in the ap-parent rates at which specific mRNA species aremetabolized reflect differences in their paths ofbiosynthesis and degradation or differences intheir structure.

ACKNOWLEDGMENTSWe express our appreciation to Maurice Vaughan, Calvin

McLaughlin, W. 0. Godtfredsen, and Edel Kirstensen (LeoPharmaceutical Products, Ballerup, Denmark) for providingthe samples of trichodermin and VE-76 used in this work.

This work was supported by Public Health Service grantsGM-19386 and GM-20693 from the National Institute ofGeneral Medical Sciences and by a Brown Haxen grant-in-aid. Robert Lawther was supported by an Andrew MellonPredoctoral Fellowship Award.

LITERATURE CITED1. Bossinger, J., R. P. Lawther, and T. G. Cooper. 1974.

Nitrogen repression of the allantoin degradative en-zymes in Saccharomyces cerevisiae. J. Bacteriol.118:821-829.

2. Cooper, T. G., and R. P. Lawther. 1973. Induction of theallantoin degradative enzymes in Saccharomycescerevisiae by the last intermediate of the pathway.Proc. Natl. Acad. Sci. U.S.A. 70:2340-2344.

3. Cooper, T. G., and R. Sumrada. 1975. Urea transport inSaccharomyces cerevisiae. J. Bacteriol. 121:571-576.

4. Cybis, J., and P. Weglenski. 1972. Arginase induction inAspergillus nidulans. The appearance and decay of thecoding capacity of messenger. Eur. J. Biochem.30:262-268.

5. Darnell, J. E., R. Wall, and R. J. Tushinski. 1971. Anadenylic acid-rich sequence in messenger RNA of HeLacells and its possible relationship to reiterated sites inDNA. Proc. Natl. Acad. Sci. U.S.A. 68:1321-1325.

6. Edmonds, M., M. H. Vaughan, and H. Nakazato. 1971.Polyadenylic acid sequences in heterogeneous nuclearRNA and rapidly-labeled polyribosomal RNA of HeLacells: possible evidence for a precursor relationship.Proc. Natl. Acad. Sci. U.S.A. 68:1336-1340.

7. Greenberg, J. R., and R. P. Perry. 1972. Relative occur-rence of polyadenylic acid sequences in messenger andheterogeneous nuclear RNA of L cells as determined bypoly (U)-hydroxyl-apatite chromatography. J. Mol.Biol. 72:91-98.

8. Hartwell, L. H., H. T. Hutchison, T. M. Holland, and C.S. McLaughlin. 1970. The effect of cycloheximide uponpolyribosome stability in two yeast mutants defectiverespectively in the initiation of polypeptide chains andin messenger RNA synthesis. Mol. Gen. Genet.106:347-361.

9. Hartwell, L. H., and B. Magasanik. 1963. The molecularbasis of histidase induction in Bacillus subtilis. J. Mol.Biol. 7:401-420.

10. Hutchison, H. T., L. H. Hartwell, and C. S. McLaughlin.1969. Temperature-sensitive yeast mutant defective inribonucleic acid production. J. Bacteriol. 99:807-814.

11. Jacobson, A., R. A. Firtel, and H. F. Lodish. 1974.Transcription of polydeoxythymidylate sequences inthe genome of the cellular slime mold, Dictyosteliumdiscoideum. Proc. Natl. Acad. Sci. U.S.A.71:1607-1611.

12. Kepes, A. 1963. Kinetics of induced enzyme synthesisdetermination of the mean life of galactosidase-specificmessenger RNA. Biochim. Biophys. Acta 76:293-309.

13. Kuo, S. C., F. R. Cano, and J. 0. Lampen. 1973.Lomofungin, an inhibitor of ribonucleic acid synthesisin yeast protoplasts: its effect on enzyme formation.Antimicrob. Agents Chemother. 3:716-722.

14. Lawther, R. P., and T. G. Cooper. 1973. Effects of induceraddition and removal upon the level of allophanatehydrolase in Saccharomyces cerevisiae. Biochem. Bio-phys. Res. Commun. 55:1100-1104.

15. Lee, Y., J. Mendecki, and G. Brawerman. 1971. Apolynucleotide segment rich in adenylic acid in rapid-ly-labeled polyribosomal RNA component of mousesarcoma 180 ascites cells. Proc. Natl. Acad. Sci. U.S.A.68:1331-1335.

16. Schimke, R. T., and D. Doyle. 1970. Control of enzymelevels in animal tissues. Annu. Rev. Biochem.39:929-976.

17. Singer, R. H., and S. Penman. 1973. Messenger RNA inHeLa cells. Kinetics of formation and decay. J. Mol.Biol. 78:321-334.

18. T#nnesen, T., and J. D. Friesen. 1973. Inhibitors ofribonucleic acid synthesis in Saccharomyces cerevisiae:decay rate of messenger ribonucleic acid. J. Bacteriol.115:889-896.

19. Wei, C., B. S. Hansen, M. H. Vaughan, and C. S.McLaughlin. 1974. Mechanism of action of the myco-toxin trichodermin, a 12,13-epoxytrichothecene. Proc.Natl. Acad. Sci. U.S.A. 71:713-717.

20. Wei, C., and C. S. McLaughlin. 1974. Structure-functionrelationship in the 12,13-epoxytrichothecenes-novel in-hibitors of protein synthesis. Biochem. Biophys. Res.Commun. 57:838-844.

21. Whitney, P. A., and T. G. Cooper. 1972. Urea carboxylaseand allophanate hydrolase. Two components of adeno-sine triphosphate:urea amido-lyase in Saccharomycescerevis8iae. J. Biol. Chem. 247:1349-1353.

VOL. 121, 1975 1073