JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1501-15070021-9193/78/0133-1501$02.00/0Copyright ( 1978 American Society for Microbiology

Vol. 133, No. 3

Printed in U.S.A.

Reaction Order of Saccharomyces cerevisiae Alpha-Factor-Mediated Cell Cycle Arrest and Mating Inhibition

MARK M. UDDENt AND DAVID B. FINKELSTEIN*

Department of Biochemistry, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235

Received for publication 10 November 1977

Alpha-factor-mediated cell cycle arrest and mating inhibition of a mating-typecells of Saccharomyces cerevisiae have been examined in liquid cultures. Cellcycle arrest may be monitored unambiguously by the appearance of morpholog-ically abnormal cells after administration of alpha factor, whereas mating inhibi-tion is determined by comparing the mating efficiency in the absence or presenceof added alpha factor. For both cell cycle arrest and mating inhibition, a dose-dependent response may be observed at limiting concentrations of the pheromone.If cell cycle arrest and mating inhibition require a small number of alpha-factormolecules, one might expect that responsive/nonresponsive cells = K(alphafactor)N where N is the order of dependence of cell cycle arrest (or matinginhibition) on alpha-factor concentration. The value ofN has been determined tobe 0.98 ± 0.18 (standard error of the mean) for cell cycle arrest and 1.08 ± 0.32 formating inhibition. These results support the notion that saturation of a single siteby alpha factor is sufficient to cause cell cycle arrest or mating inhibition of amating-type cells.

The yeast Saccharomyces cerevisiae can existin either the haploid or diploid state. Haploidcells of opposite mating type can conjugate toform a diploid zygote that, by mitotic division,will give rise to a clone of stable diploid cellswhich are heterozygous for mating type (a/a).The conjugation process of yeast appears torequire a mutual arrest of haploid cells early inthe Gl phase of their cell cycles as a prelude tosuccessful mating (6). This mutual arrest in themating mixture is accomplished by the mutualsecretion of diffusible factors by the haploid cellsthat are capable of causing arrest of cells of theopposite mating type at the "start" point of theircell cycles (3, 14). The arrest of a mating-typecells is accomplished in the mating mixture bya small polypeptide "alpha factor," which isexcreted by cells of the a mating type (4). Whilea cells are themselves immune to the effects ofalpha factor, a mating-type cells are arrested asunbudded mononucleate cells at the same pointin the cell cycle as arrest by Hartwell's cdc28mutant (7). As arrest at this point in the cellcycle by alpha factor continues, one sees ap-pearance of morphological alterations in the cellwall (8) that manifest as mycelial-type processes.The arrest ofa mating-type cells is a reversible

process. After removal of alpha factor from thegrowth medium, the cells immediately begin a

t Present address: Department of Internal Medicine, Bay-lor College of Medicine, Houston, TX 77030.

synchronous round of division. The time courseof alpha-factor arrest is related to the concentra-tion of alpha factor in the medium: low concen-trations of alpha factor cause only transient cellcycle arrest (2, 13).As the cell wall of the parent cell remains

morphologically altered during the recoveryfrom alpha-factor arrest, one has an unambigu-ous method of scoring whether or not an individ-ual cell has been arrested by alpha factor. Thispossibility of examining the response of individ-ual cells to alpha factor should allow one todetermine the dose response of submaximal lev-els of alpha factor in order to discern the reactionorder of alpha-factor arrest. We report resultsthat are consistent with the notion that satura-tion of a single target by alpha factor is sufficientfor the cell cycle arrest of an a mating-type cell.

MATERIALS AND METHODSStrains. The haploid grande strain of S. cerevisiae

X2180-1B a gal2 (obtained from the Yeast GeneticStock Center, Berkeley, Calif.) was used for the prep-aration of alpha factor. The tester strain for alphafactor was 55-R5-3C a ura. For mating experiments,55R5-3C was crossed with 650-2C a his trp. All cultureswere maintained and routinely subcultured on YPDagar plates containing 1% yeast extract (Difco Labo-ratories, Detroit, Mich.), 2% peptone (Difco), 2% glu-cose, and 2% agar (Difco). All cultures were routinelygrown at 30°C in a Lab Line Orbital shaker in flasksfilled to less than 20% of their stated capacity in eitherYPD or on modified G medium (5).

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1502 UDDEN AND FINKELSTEIN

Partial purification ofalpha factor. Alpha factorwas purified from 60-liter cultures of X2180-1B by theprocedure of Duntze et al. (4) to the stage of neutral-ized eluent III. This material was then sterile filteredthrough a 0.2-jm porosity membrane filter (MilliporeCorp., Bedford, Mass.) and stored at 4°C. Alpha-factoractivity is expressed as units per milliliter, as definedby the diffusion assay of Duntze et al. (4) on YPD agarplates using strain 55-R5-3C as a tester.

Liquid assay for alpha factor. Liquid assays foralpha factor were performed in a volume of 200 jil inglass test tubes (12 by 75 mm), using cells at a densityof 5 x 10' to 1 x 107 cells/ml and a twofold serialdilution of alpha factor. The cultures were allowed toincubate at 23°C with occasional shaking. The reac-tions were terminated after 4 h by addition of NaN3 toa final concentration of 20 mM. Cells were counted ina hemacytometer, and cell morphology was scored.Unless stated otherwise, the following conventionswere used: all buds were counted as cells, and mor-phologically abnormal cells with a normal bud (re-covering cell) were scored as one abnormal cell plusone normal cell. In plotting the dose response of alpha-factor-mediated cell cycle arrest, a correction wasmade for the one-generation lag in the appearance ofmorphologically abnormal cells by dividing the num-ber of morphologically normal cells by two.The best-fit line and slope for log-log plots was

calculated by use of a linear regression analysis usinga Monroe calculator (model 1785).

Micrographs were taken by using a Leitz Diavertmicroscope equipped with Smith Differential Interfer-ence Contrast optics at an instrument magnificationof x600.

Mating. Cells were grown with shaking at 30°C tomid-log phase in liquid YPD to a density of 2 x 107cells/ml. One-half milliliter of each culture plus 1 mlof a suitable dilution of alpha factor (in water) werecombined in a conical centrifuge tube and centrifugedfor 5 min. at 800 x g at room temperature (ca. 22°C),using a Clay Adams bench-top centrifuge. The cellpellets were allowed to incubate at room temperaturefor 15 min. After a brief sonic treatment the tubeswere incubated on a gyratory shaker at 30°C for 4 h.The mating reaction was terminated by diluting themixture 1:1,000 with cold sterile water. Appropriatedilutions of this mating mixture were plated ontominimal medium, 0.67% yeast nitrogen base withoutamino acids [Difco]-2% glucose-2% agar) to score fordiploid cells by prototrophic selection. The matingefficiency (relative to the a cell input) varied from 2 to10% depending on the experiment.Mating inhibition due to alpha factor is expressed

relative to a control mating performed at the sametime without alpha factor taken as 100%.

RESULTSResponse to alpha factor as a function of

alpha-factor concentration. To study thedose response of a mating-type cells to alphafactor, it is necessary to have some unambiguousmethod of distinguishing a cell that has re-sponded to the pheromone. Whereas the mea-sure of the increase of unbudded cells in an

alpha-factor-treated culture realtive to an un-treated culture has been used by others to detectalpha-factor arrest (1, 2), this method suffersfrom the presence of a background of 20 to 40%unbudded cells in a normal asynchronous log-phase culture, thus making the detection of asmall number of arrested cells impossible. Wehave chosen instead to examine alpha-factorarrest by the examination of cell morphology.After arrest by alpha factor, cells of a mating-type become morphologically abnormal. Thismorphological alteration can be readily scoredmicroscopically.

Figure 1 presents the results of an experimentwhere log-phase cells were exposed to variousconcentrations of alpha factor for a period of 4h, and the cell morphology was scored for thepresence of abnormal cells. Figure 1A shows apicture of cells that were exposed to a dose of 64U of alpha factor per ml. It may be seen that theresult of this dose of alpha factor is that virtuallyall cells assume a distinctive morphology (cf.Fig. 1C, which represents a normal log-phaseculture untreated with alpha factor). Even atthis very high concentration of alpha factor onecan still detect an occasional normally buddingcell. In Fig. 1B a culture of cells has been exposedto 1 U of alpha factor per ml for 4 h. It may beseen that with this dose of pheromone at leastfour different cell morphologies are apparent: (i)normally growing budded cells, (ii) morphologi-cally abnormal unbudded cells (arrested cells),(iii) morphologically abnormal budded cells(cells which are recovering from alpha-factorarrest; D. Finkelstein and L. McAlister, unpub-lished observations), and (iv) unbudded appar-ently normal cells. This last morphology is some-what ambiguous since, as noted above, it couldrepresent either a normally growing cell or a cellwhich has become arrested but has not yet be-come morphologically abnormal. This ambiguityof unbudded cells becomes apparent if we ex-amine the time course of appearance of morpho-logically abnormal cells.

If cells were exposed to 64 U of alpha factorper ml and their morphology examined withtime, one obtained the results presented in Fig.2. Under the conditions of this experiment,greater than 90% of the cells became unbuddedby 2 h, yet the appearance of abnormal cells wasnot complete until 4 h. Hence, an apparent one-generation lag exists between the arrest of cellsin an unbudded state and the appearance ofmorphologically abnormal cells. This delay inthe appearance of detectable abnormal cell mor-phology complicates a quantitative study of al-pha-factor arrest. It leads to an underestimateof the degree of cell cycle arrest, as we can onlyscore cell cycle arrest one generation after the

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REACTION ORDER OF YEAST ALPHA FACTOR 1503

FIG. 1. Effect of alpha factor on growing yeast cells. Strain 55-R5-3C, actively growing on YPD at a celldensity of 5 X it'P cells/ml, was exposed to either 64 U (A), 1 U (B), or no alpha factor (C) per ml and allowedto continue growth for 4 h at 230C. After termnination ofgrowth by addition ofsodium azide to 0.02 M, the cellswere photographed under Smith Interference Optics. Normally growing budded cell (a), morphologicallyabnormal unbudded cell (b), morphologically abnormnal budded cell (c), morphologically normal unbuddedcell (d). Bar = 5 um.

arrest has occurred, whereas unarrested cells To quantitate the dose response of alpha-fac-have doubled during this timne period. It thus tor-mediated cell cycle arrest, cells were treatedbecomes necessary to correct the number of with alpha factor at concentrations varying overunarrested cells by one generation (the last point a 4,000-fold range, and the cultures were scoredat which a cell could become arrested and still for abnormnal morphology at 4 h (Fig. 3). Thoughbe detected as morphologically abnormnal in our it can be seen that the cell morphology changesassay). from predominantly normnal to predominantly

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FIG. 2. Time course of appearance of abnormalcells. An actively growing culture of 55-R5-3C at adensity of 5 x 10i cells/ml was exposed to 64 U ofalpha factorper ml and allowed to incubate at 23°C.At various times, samples were removed and growthwas inhibited by addition of sodium azide to 0.02 M.Morphologically abnormal cells were scored as de-scribed in the text.

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ALPHA FACTOR, UNITS/ML.

FIG. 3. Dose response of a mating-type cells toalpha factor. Actively growing cells were exposed totwofold serial dilutions of alpha factor of 4 h andtreated as described in the legend to Fig. 2. Abnormalcells (0), morphologically normal cells (corrected asdescribed in the text), (0) and corrected total cellcount (5).

abnormal over a relatively narrow range of al-pha-factor concentrations, it is important to notethat both normal and abnormal cells were ob-served over the entire range of alpha-factor con-centrations examined.Reaction order of alpha-factor arrest.

The results of Fig. 3 demonstrate that the arrestof a mating-type cells by alpha factor, as de-tected by alterations in cell morphology, is re-lated to the dose of alpha factor administered.Since the arrest of cells by alpha factor is areversible reaction (2, 13; see also Fig. 1B), it ispossible to define alpha-factor-mediated cell cy-cle arrest by the following equation: unarrestedcell + N (alpha factor) arrested cell (1), whereN is the number of alpha-factor molecules re-quired to achieve cell cycle arrest. Under equi-librium conditions we may write: (arrestedcell)/(unarrested cell) (alpha factor)N = K (2).As shown above it may be seen that we can

determine the number of arrested cells by themeasurement of abnormal cell morphology.Thus, if equation 2 holds, it follows that a log-log plot of the ratio of abnormal to normal cellsversus alpha-factor concentration should give astraight line with a slope of N. If the data of Fig.3 is recast to give a log-log format, we get astraight line as shown in Fig. 4. The slope of thisline, as determined by a linear-regression anal-

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FIG. 4. Reaction order of alpha-factor-mediatedcell cycle arrest. The data is reduced from the exper-

imentpresented in Fig. 3. The line represents the bestfit by a linear-regression analysis. Slope is 1.07; cor-

relation coefficient is 0.97.

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REACTION ORDER OF YEAST ALPHA FACTOR 1505

ysis, is 1.07. The results of 10 such experimentsperformed under varying conditions are pre-sented in Table 1. It may be seen that under allgrowth conditions examined the value of N inequation 2 is very close to or equal to the integer1 (N = 0.98 + 0.18 standard error of the mean).As would be expected from the above equation,the experimentally determined value of N isindependent of cell density. This result supportsthe view that the saturation of a single target byalpha factor is required to arrest an a mating-type cell.Mating inhibition by alpha factor. In the

preceding experiments we showed that cell cyclearrest mediated by alpha factor is a first-orderreaction with respect to alpha factor. Sena et al.(12) have reported that the addition of exoge-nous alpha factor to a population ofmating yeastcells results in an inhibition of the mating reac-tion. If the mechanism of this mating inhibitionis the same as that causing the prolonged inhi-bition of the cell cycle, then one would expect tosee the same reaction order for mating inhibitionas for cell cycle arrest. However, it could also bepossible that newly formed zygotes are arrestedby alpha factor. Therefore, to test this hypoth-esis a mixture of a and a cells were permitted tomate, and, at various times during the matingreaction, samples were removed and plated onminimal medium and also on miniimal mediumcontaining alpha factor. (The concentration ofalpha factor used was sufficient to inhibit theappearance of a mating-type colonies on solidmedium.) The results of this experiment (Fig. 5)show that, once formed, zygotes can give rise toa normal diploid colony of cells even in thepresence of alpha factor and, therefore, must be

TABLE 1. Reaction order of alpha-factor-mediatedcell cycle arrest

GrowthGrowth phase denpitycl Correlationmedium of input (cens/ml) coefficient

cells (el/iYPD Stationaryb 5 x 106 0.72 0.995YPD Stationary 5 x 106 0.90 0.95G Stationary 5 x 106 1.05 0.990

YPD Stationary 1 x 107 0.48 0.97YPD Log 5 x 106 1.07 0.97YPD Log 1 x 107 1.33 0.94G Log 5 x 106 1.39 0.96

YPD Log 5 x 106 0.97 0.97YPD Stationary 5 x 105 0.92 0.97YPD Log 5 x 105 0.93 0.98a N, the reaction order of alpha-factor-mediated cell

cycle arrest, represents the slope of the line of log(abnormal cells/normal cells) = N log (alpha factor)+ log K.

b Stationary-phase cells were freshly diluted intonew growth medium.

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FIG. 5. Time course of mating. A mating reactionbetween 55-R5-3C and 650-2C was carried out as

described in the text. At various times, samples were

plated to select for diploid cells on either minimalmedium (0) or minimal medium containing 50 U ofalpha factor (0) per ml.

insensitive to cell cycle arrest by this phero-mone.

If mating inhibition by alpha factor involvesa small number of alpha-factor molecules, thenone can construct the following equation: matingcell + N(alpha factor) = nonmating cell (3).Since the mating efficiency in this cross is onlyabout 5 to 10% (relative to the a cell input), one

can neglect those cells that did not mate in theabsence of alpha factor. Thus, for a given doseof alpha factor, the nonmating cells are taken tobe equal to the number of mating cells in theabsence of alpha factor minus the number ofcells mating in the presence of alpha factor.Reasoning by analogy with equation 1 above

it may be seen that if equation 3 is valid, then itfollows that a log-log plot of the ratio of non-

mating cells to mating cells versus alpha-factorconcentration should give a straight line with a

slope of N. To test the validity of this relation-ship, matings were performed, using a gradedseries of alpha-factor concentrations as well asa control mating that did not receive exogenousalpha factor (Fig. 6). The slope of this best-fitline is 0.925. From four such experiments one

can see that mating inhibition by exogenousalpha factor is a first-order reaction (N = 1.08+ 0.32 standard error of the mean).

DISCUSSIONThe experiments reported in this paper were

designed to determine the reaction order of a

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ALPHA FACTOR, UMTS/ML.FIG. 6. Reaction order of alpha-factor inhibition

of mating. Strain 55-R5-3C and 650-2C were allowedto mate for 4 h in the presence of various concentra-tions of alpha factor. After plating, diploids werescored and the data was reduced as described in thetext. The line was fitted as for Fig. 4. The slope is0.925; correlation coefficient is 0.995.

mating-type cell cycle arrest by alpha factor.The two criteria we measured are the inhibitionof the mating reaction and the alterations in cellmorphology after alpha-factor arrest. Thoughneither parameter appears to be capable ofmeasuring a transient elongation ofthe Gl phaseof the cell cycle, both methods allow the advan-tage of low background measurement that ismuch more sensitive than the measure of thechange in the percentage of unbudded cells in acell culture exposed to alpha factor. Indeed,whereas the measurements of cell cycle arrest asmonitored by inhibition of DNA synthesis orarrest in the increase in cell density show no

apparent arrest at doses of alpha factor of lessthan 1 U/ml (Udden and Finkelstein, unpub-lished), examination of cellular morphology ormating inhibition allow one to obtain a measure-able response at these doses of pheromone.A first-order reaction for cell cycle arrest was

concluded from the measurement of the slope ofa log-log plot of responsive/nonresponsive cellsversus alpha-factor concentration. It should benoted that such a determination requires only a

knowledge of the relative concentration of alphafactor rather than the absolute concentration ofthe pheromone. Thus, though alpha factor isinactivated by proteolysis upon exposure to a

mating-type cells, this does not interfere withthe determination of the reaction order, as the

rate of alpha-factor inactivation is proportionalto the input of the pheromone over the concen-tration ranges used in these studies (D. Finkel-stein, unpublished observations).

It has been implied that the yeast matingpheromones are necessary to mutually synchro-nize cells as a prelude to a successful matingreaction (6), yet we as well as others (12) findthe apparently contradictory results that exog-enously added mating pheromones inhibit mat-ing. Presumably the optimal alpha-factor con-centration necessary for mating is well belowthat which produces major morphologicalchanges in a culture. From the data of Schereret al. (11), who examined the time course ofalpha factor-production by a mating-type cells,it is possible to estimate that the alpha-factorconcentration in a normal mating mixture wouldbe less than 0.1 U/ml, which would cause lessthan a 5% inhibition of mating due to prolongedcell cycle arrest (Fig. 6). Presumably, at this lowconcentration of alpha factor, one sees primarilya slight elongation in the G1 phase of the celldue to the reversible nature of alpha-factor ar-rest. Thus it would appear that as a transientresponse to alpha factor is necessary for mating,so the changes in the structure of the cell wall(or some other cell constituent) resulting fromprolonged cell cycle arrest are inhibitory to themating reaction of a mating-type cells.

First-order dependence of cell cycle arrest andmating inhibition yields information about themechanism of action of alpha factor. The pres-ence of a first-order reaction implies that thereis only a single target per cell that must besaturated for alpha-factor action. This wouldrule out any mechanism for alpha-factor actioninvolving the stoichiometric inhibition of an en-zyme or a species of tRNA, for example, as all ofthese molecules are present at more than onecopy per cell. It is interesting to note that thepossibility of alpha-factor arrest being mediatedvia a mechanism involving a single pheromonemolecule binding to a chromosome (or a chro-mosomal protein) to activate (or inactivate)transcription at a given site is not ruled out bya first-order-reaction mechanism, but alpha-fac-tor action in analogy with the inducer of theEscherichia coli lac operon would not be con-sistent with a first-order-reaction mechanism.The action of many mammalian polypeptide

hormones appears to involve the activation of amembrane-bound adenyl cyclase and the in-crease in the level of a second messenger, cyclicAMP (9). A first-order reaction of alpha factorwould be consistent with such a mechanism ifonly a single receptor needed to be occupied totrigger such a response. This would be consistent

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REACTION ORDER OF YEAST ALPHA FACTOR 1507

with the threshold model proposed by Rodbard(10). Unfortunately, there have been no experi-ments performed with hormone-stimulatedmammalian cells to demonstrate that a submax-imal response represents a complete response ofonly a portion of the cells. Though we havedemonstrated an all-or-none response for indi-vidual yeast cells arrested with alpha factor, itwill require future study to determine if there isa saturable, mating-type-specific, class of alpha-factor receptors on the yeast cell membrane.

ACKNOWLEDGMENTS

We thank Susan Strausberg for preparing the alpha factorused in these experiments and Robert 0. McAlister for allow-

ing us to use his interference microscope.This investigation was supported by grant PCM 76-17208

from the National Science Foundation and a Public HealthService, National Cancer Institute Specialized Cancer Centergrant (CA 17065) from the National Cancer Institute.

LITERATURE CITED

1. Bucking-Throm, E., W. Duntze, L. H. Hartwell, andT. R. Manney. 1973. Reversible arrest of haploid yeast-cells at the initiation of DNA synthesis by a diffusiblesex factor. Exp. Cell Res. 76:99-110.

2. Chan, R. K. 1977. Recovery of Saccharomyces cerevisiaemating type a cells from G1 arrest by a factor. J.Bacteriol. 130:766-774.

3. Duntze, W., V. MacKay, and T. R. Manney. 1970.Saccharomyces cerevisiae: a diffusible sex factor. Sci-

ence 168:1472-1473.4. Duntze, W., D. Stotzler, E. Bucking-Throm, and S.

Kalbitzer. 1973. Purification and partial characteriza-tion of a-factor, a mating-type specific inhibitor of cellreproduction from Saccharomyces cerevisiae. Eur. J.Biochem. 35:357-365.

5. Finkelstein, D. B., and R. A. Butow. 1976. DNA-bind-ing proteins in yeast. Effect of growth phase and mito-chondrial function. Arch. Biochem. Biophys. 174:52-65.

6. Hartwell, L. H. 1973. Synchronization of haploid yeastcell cycles, a prelude to conjugation. Exp. Cell Res.76:111-117.

7. Hereford, L. M., and L. H. Hartwell. 1974. Sequentialgene function in the initiation of Saccharomyces cere-

visiae DNA synthesis. J. Mol. Biol. 84:445-461.8. Lipke, P. N., A. Taylor, and C. E. Ballou. 1976. Mor-

phogenic effects of a-factor on Saccharomyces cerevi-siae a cells. J. Bacteriol. 127:610-618.

9. Robison, G. A., R. W. Butcher, and E. W. Sutherland.1971. Cyclic AMP, p. 17-47. Academic Press Inc., NewYork.

10. Rodbard, D. 1973. Theory of hormone-receptor interac-tion. III. The endocrine target cell as a quantal responseunit: a general control mechanism, p. 342-364. In B. W.O'Malley and A. R. Means (ed.), Receptors for repro-ductive hormones. Plenum Publishing Corp., New York.

11. Scherer, G., G. Haag, and W. Duntze. 1974. Mechanismof a factor biosynthesis in Saccharomyces cerevisiae. J.Bacteriol. 119:386-393.

12. Sena, E. P., D. N. Radin, and S. Fogel. 1973. Synchro-nous mating in yeast. Proc. Natl. Acad. Sci. U.S.A.70:1373-1377.

13. Throm, E., and W. Duntze. 1970. Mating-type-depend-ent inhibition of deoxyribonucleic acid synthesis in Sac-charomyces cerevisiae. J. Bacteriol. 104:1388-1390.

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