THE RELATION OF SPORULATION AND THE RANGE OF VARIATIONOF THE HAPLOPHASE TO POPULATIONAL ADAPTATION'

S. SPIEGELMAN AND C. C. LINDEGREN2Department of Bacteriology and Immunology, Washington University School of Medicine

and the Henry Shaw School of Botany, Washington University, St. Louis, Missouri

Received for publication September 29, 1944

INTRODUCTION

In previous publications (Spiegelman, Lindegren, and Hedgecock, 1944;Spiegelman and Lindegren, 1944) the authors have shown that in certain haploidstrains populational adaptation to galactose fermentation may involve muta-tion and subsequent selection of the adaptable type. Using two diploid strainsof Saccharomyces cerevisiae they established that in other strains adaptationcould be effected by a direct interaction between the substrate and the cyto-plasm of the existent cells. These experiments emphasized the importantpoint that the particular biological mechanism involved in the production ofa given enzyme or enzyme system in a population of cells is a characteristic ofthe strain being examined rather than of the enzyme system itself. It becameapparent that to solve the problem of the biological mechanisms involved inany particular case of adaptation reference must be made to the genetic back-ground and stability of the population being studied.These same experiments presented the opportunity of analyzing the problem

of unadaptability. From the earliest investigations by Armstrong (1905)and Kluyver (1914) the existence of unadaptable yeasts was noted. Subsequentinvestigations have uncovered many more. As may be seen from a perusal ofStelling-Dekker's monograph on the sporogenous yeasts (1931), examples ofnonfermenters of galactose exist in all the genera. As a matter of fact, Stelling-Dekker has used fermentability with respect to galactose and several othersugars as the basis of identification within several of the yeast genera. Anexamination of the mechanisms whereby nonfermenting types gain or lose thischaracter is of some importance in evaluating the phylogenetic significance ofthese properties.The question has in addition taken on some practical significance because ot

the tendency in recent years to use various yeasts as tools in the analysis ofsugar mixtures. Recently, for example, Wise and Appling (1944) publisheda method for determining galactose in mixtures with other hexoses. They usedSaccharomyces carlsbergensis, which could ferment galactose, in conjunctionwith Saccharomyces bayanas, which could not. They also reported that certaingalactose-fermenting strains of S. cerevisiae had apparently lost that ability,at least partially, within a 5-year period. Our earlier experiments suggest that

1 This investigation was aided by a grant from the Penrose Fund of the American Philo-sophical Society.

2Supported by a grant from Anheuser-Busch, Inc.257

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the retention by a nonfermenter of this character after a long period of contactwith galactose might be due to its genetic stability. Thus, a nonsporulatingpopulation of diploids, or one in which some other mechanism existed for thesuppression of the haplophase, would be unlikely to gain a new character orlose an old one by mutation. This suggested the possibility of attempting toadapt strains which had been previously labeled as nonfermenters by encourag-ing the production of haploids and thus disturbing the genetic stability of thepopulation. The same type of experiments could also be used to investigatethe conditions for the reverse phenomenon, that is, the loss of adaptability byadaptable strains. The basis for the method of inducing haploidy within adiploid population consisted essentially in stimulating sporulation, the detailsof which are described in the section on methods.

It is the purpose of the present paper to describe the experiments which leadto the adaptation to galactose fermentation of a previously unadaptable form.This will be compared with the negative results obtained with the same methodson other forms, in which additional mechanisms for suppression of the haplo-phase exist, which tended to minimize the effect of heavy sporulation. Experi-ments will also be discussed in which unadaptable progenies were isolated from-adaptable parents. The latter experiments indicate that the range of bio-chemical variation of mutant types may become limited as compared with theparent types from which they arose.

MATERIALS AND METHODS

Strains of yeast. Three yeast species, Schizosaccharomyces pombe, Schizosac-charomyces octosporus, and Saccharomycodes ludwigii,3 were chosen among theknown nonfermenters of galactose. These particular ones were selected forthis study for several reasons. All three were included among the yeastsexamined by Armstrong (1905). He concluded from his study that they wereincapable of adaptation to galactose fermentation. In addition to the fact thatthey have been studied more thoroughly than other nonfermenters of galactose,they have another advantage of some importance from a comparative point ofview: S. pombe can without any difficulty exist in the haplophase; this is nottrue of S. octosporus, and still less so for Saccharomycodes ludwigii.Although S. octosporus sporulates with ease, the haploids which result from

the germination fuse rapidly to produce diploid cells. When four- and eight-spored asci from the stock culture were dissected and the spores planted, theyall grew, and every single-ascospore culture sporulated on the agar in less than48 hours. The further spore analysis of S. octosporus indicates that the diploidstock culture was completely homozygous and that, unlike Saccharomycescerevisiae, the production of viable spores apparently does not depend on thepre-existence of a heterozygous nucleus. The sporulation phenomenon of a

W 3 These as well as Saccharomyce8 carlsbergen48is were obtained from the yeast collectionof the Northern Regional Research Laboratory at Peoria, Illinois, through the courtesyof Dr. L. J. Wickerham.

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single-spore culture is not observed in the S. cerevisiae strains employed. Thefact that it does occur in S. octosporus is an indication of heavy diploidization,confirming the direct microscopic observations (see fig. 1, discussed below).This process of immediate fusion effectively suppresses the haplophase in anyculture in which it occurs.

This suppression mechanism is even more highly developed in the case ofSaccharomycodes ludwigii. Guilliermond (1903) reported that this yeast usually

r W

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FIG. 1. FIELD OF A SPORTJLATING CULTURE OF SCHIZOSACCHAROMYCES OCTOSPORUS (SEETEXT FOR DESCRIPTION)

forms four spores without previous conjugation. On germination, however,the spores conjugate within the mother cell two by two so that only two vege-tative cells emerge from each 4-spored ascus. Winge and Laustsen (1939)confirmed these observations and described the successful isolation of thehaplophase by a micromanipulative dissection of the ascospore and separationof the four spores before germination. As a result of their examination of thehaplophase cultures, they concluded that Saccharomycodes ludwigii was a bal-

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anced heterozygote. The net result of the germination mechanism is thealmost complete suppression of the haplophase under normal conditions.The adaptable strains from which unadaptable progenies were isolated

consisted of tw^o Saccharomyces cerevisiae strains (Db23B and Lk2G12) previ-ously studied in this laboratory (Spiegelman, Lindegren, and Hedgecock, 1944;Spiegelman and Lindegren, 1944) and S. carlsbergensis which was tested andfound to be rapidly (90 minutes) adaptable to galactose fermentation.

Media. One liter of the basic medium contained the following substances:2 g of autolyzed yeast extract powder, 3 g of bacto-peptone, 1 g of (NH4)2SO4,2 g of KH2PO4, 0.25 g of MgSO4, 0.13 g of CaCl2, 1 ml of 50 per cent sodiumlactate. To this wvas added the desired carbohydrate. The medium used foradaptation of mixed haploid and diploid populations contained, in additionto the above, 80 g of galactose and 20 g of glucose.

Carbohydrates. Reagent grade glucose and galactose were used in the mediawithout further purification. The galactose used in the manometric test foradaptation (see below) was further purified according to a method modifiedfrom that used by Stephenson and Yudkin (1936). The final solution obtainedwvith their method wvas evaporated under reduced pressure and the galactosewas recrystallized from 70 per cent alcohol. The crystals wvere then redissolvedand again recrystallized, and dried in vacuo at 60 C.

Tests for adaptation. All cultures were grown at 29 C. A sample of theculture to be tested was centrifuged from its medium and Avashed twice in largevolumes of M/15 KH2PO4. The cells were then resuspended in M/15 KH2PO4and made up to the desired density using a nephelometer. Samples wNere takenfor the determination of dry weights when necessary. A sample (1.8 ml) ofthis suspension was then placed in a Warburg cup with 0.2 ml of 20 per centpurified galactose in the side arm. After being flushed with nitrogen, the stop-cocks were closed and the galactose tipped in. The nitrogen used to displacethe air in measurements of anaerobic CO2 production was passed over hot cop-per to remove any traces of oxygen. All measurements were taken at 30.2 C,the vessels being shaken at a rate of 100 oscillations per minute over a 7-cmarc. A QNo2 of 100 or above with galactose wvas taken as adaptation. Inpractice lower values were not found; either the rate of CO2 evolution waszero and remained there or it could be made to reach 100 and exceed it bycontinued culturing in the presence of galactose.

Sporulation. All the diploid strains employed in the present study sporulatedreadily if left standing over 7 days either in a fluid culture medium or on anagar slant. When particularly heavy sporulation was desired, a method in-volving inoculation on a presporulating medium and seeding suspensions fromthis on a Graham and Hastings (1941) gypsum block was used (Lindegren andLindegren, 1944).

Oxygen consumption and C02 production. The oxygen consumption (Qo2)and aerobic CO2 production (Q?o2) were measured by the Warburg manometrictechnique. Forty-eight-hour glucose-growin cultures washed twice in M/15KH2PO4 were always employed. The aerobic CO2 production was measured

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by the usual two-cup method. Inaccuracies owing to retention of the CO2were reduced to a minimum by using M/15 KH2PO4 as a suspending medium.All measurements were made at 30.2 C. The anaerobic CO2 production (Qo02)of unadapted cells was measured by the method already described.

EXPERIMENTAL RESULTS

Adaptation of Schizosaccharomyces pombe to galactose. Preliminary experi-ments indicated that the strain of Schizosaccharomyces pombe used sporulatedreadily if allowed to stand over 7 days in the culture medium. If the cultureswere transferred every other day, however, no spores were observed.To test the effect of sporulation on adaptation, several different kinds of

experiments were performed. Twelve flasks each containing 60 ml of 8 per centglucose liquid medium were inoculated and allowed to stand in the incubatorfor 20 days. At the end of this period, examination of the growth on the bottomof the flasks showed that in two of them 20 per cent or more of cells had sporu-lated. The supernatant medium was poured off from these two, and eachmixture of cells and spores was resuspended in 300 ml of 8 per cent galactoseand 2 per cent glucose adaptation medium and placed in the incubator. Twenty-ml samples were removed at two-day intervals, and the cells centrifuged downand washed in M/15 KH2PO4. After resuspension in the buffer fluid, they weretested for adaptation to galactose fermentation by the manometric methodpreviously described. One of the cultures so prepared adapted itself in 8 days,the other in 6. The 2 per cent glucose included in the galactose medium wasfound necessary after many unsuccessful attempts without its addition. Itserves the function of allowing the spores to germinate and of giving the haploidsan opportunity to divide rapidly and accumulate in large numbers. After theexhaustion of the glucose, the galactose which still remains can then selectfrom the heterogeneous population any existent fermenters.

In another experiment the growvth in 24-day agar slants containing between10 and 20 per cent sporulating cells was washed with sterile broth into theglucose galactose medium. Out of four attempts only one finally yielded anadaptable strain. Finally, the growths on gypsum blocks containing well over30 per cent of sporulating cells were heavily inoculated into the adapting medium.The results and time required for adaptation are given in table 1. All adaptedthemselves within 6 days.

While these experiments were being performed, two kinds of control serieswere run. Cultures originating from the same stock strain of Schizosaccharo-myces pombe were transferred every other day in the adapting medium. As waspreviously noted, sporulation does not occur under these conditions. Adapta-tion did not occur over a 3-month period during which these cultures weretransferred and observed. In another type of experiment the original culturewas inoculated into the basic medium containing 8 per cent of the purifiedgalactose and left standing without transfer. None of the 12 tubes so preparedshowed any evidence of adaptation to galactose fermentation at the end of78 days.

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Experiments with Schizosaccharomyces octosporus and Saccharomycodes lud-wigii. Experiments exactly like those described in the previous section werealso carried out with Schizosaccharomyces octosporus and Saccharomycodesludwigii but without success. S. octosporus is one of the best sporulating yeastsexamined in this laboratory, usually up to 80 per cent. Figure 1 is a representa-tive field of a sporulating culture of this strain. This same picture demon-strates why sporulation is not sufficient to disturb the genetic stability of theculture.

In addition to the heavy-walled spores contained in the asci, the majorityof the cells seen are the oblong, diploid cells. In the lower left-hand cornermay be seen 4 small, round haploids arranged in pairs. The upper pair hasalready begun the process of elongation to form the conjugation tubes. Anotherpair in the lower right-hand corner is much further along in the process. Iso-lated haploid cells are very rarely seen in these cultures. Since under theseconditions the haploids do not exist very long as such in the population, anygenetic instability they may have cannot express itself in terms of changing

TABLE 1Adaptation of Schizosaccharomyces pombe to galactose fermentation

ORIGIN OF HEAVILY SPORULATING PERIOD IN DAYS REQUIRED FOREXPERIMENT CULTURES (20% AND ABOVE) APPEARANCE OF ADAPTATION

1 20-day broth culture 82 63 24-day agar slant 124 Gypsum block 25 66 47 4

populational characteristics. As has been pointed out previously, this sup-pression of the haplophase is accentuated to an even greater degree in Saccharo-mycodes ludwigii, in which haploids rarely emerge as free cells from the ascus.On this basis, it is not surprising that methods which were successful in the caseof Schizosaccharomyces pombe, in which the mutational potentialities of thehaploids could be expressed, failed in the case of both S. octosporus and Sac-charomycodes ludwigii.

Isolation of unadaptable haploid strains from adaptable parent types. Under-lying the experiments on the relation of sporulation to adaptation was theimplicit assumption that all that was required to break down the genetic sta-bility of a diploid strain was the introduction of the haplophase. Although thevalidity of this assumption would in general not be questioned, it may be doubtedinsofar as variation in a particular direction is concerned. Thus it is con-ceivable that the haplophase of a particular strain might not contain withinits mutational potentialities the ability to mutate in the direction of, for ex-ample, galactose fermentation. To answer this question as well as certain

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others on possible origins of unadaptable types, experiments were undertakento see if unadaptable haploid populations could be isolated.The two Saccharomyces cerevisiae strains used were the objects of previous

study. Strain Lk2G12 is a stable diploid which adapts itself to galactose fer-mentation within 80 minutes by direct interaction. Strain Db23B is a haploidwhich adapts itself through mutation. The other diploid strain employed,S. carlsbergensis, adapts itself directly within 90 minutes.

In the case of both diploid strains, the haplophases produced from themcan exist independently and reproduce readily in the basic 8 per cent glucosemedium. The experimental method used for the two diploid strains was thesame. Heavy sporulation was induced by the methods already described.The mixture of spores and cells was heavily inoculated into 300 ml of basic 8per cent glucose broth medium. Samples were removed at intervals early inthe growth period and streaked on plates. The usual morphological andphysiological variants which accompany haplophase-containing cultures madetheir appearance. The different kinds of clones were then inoculated into the

TABLE 2Physiological characteristics of the three unadaptable strains

NQo2, Qco2, Qco2 are all calculated on the basis of gas consumed or evolved per mg dry

weight per hour. Each figure is the average of three determinations on 48-hour galac-tose-grown cultures.

WITHOUT GALACTOSE GALACTOSE ADDED

Strain Q02 Q0 QN R.Q. Qo2 Q0 QN R.Q.~C02 C0c2 Cg02 C02

L:\N 8.5 8.6 0.02 1.01 15.4 15.4 0.03 1.00CN 11.6 11.5 0.01 0.99 23.0 22.7 0.10 0.99DN 5.4 5.4 0.00 1.00 12.5 12.7 0.12 1.02

basic broth medium containing 8 per cent galactose. These were incubatedand their galactose fermentation characteristics followed. Some were obviousfermenters, as evidenced by copious gas evolution on shaking the culture flasks,and were discarded. Others, however, had to be examined manometricallyto be certain of the presence or absence of fermentation. Using this procedurethe authors examined 31 clones stemming from LK2G12 before a nonfermentingstrain (LN) was isolated. Two out of the 31 entirely failed to grow on galac-tose and were discarded since they could not be used to test the permanency ofthe galactose nonutilization character. The strain LN grew perfectly well ongalactose, apparently utilizing it by a purely aerobic mechanism. The R.Q.in three measurements averaged 1.01 and no galactose disappeared undernitrogen. The physiological characteristics of LN and the other unadaptablehaploid strains are summarized in table 2. The LN strain was carried on8 per cent galactose medium, transferred every 4 days for 5 months. It wastested weekly, and later twice weekly, for evidence of anaerobic CO2 productionfrom galactose, alw-ays with negative results. That it is a haploid strain is

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demonstrated by the predominant small, round cells observed in wet mountsand, more important, by its instability. The latter character is shown by figure2, which represents a streak plate made from one of the routine galactose brothcultures. Smooth, micro-type colonies may be observed. Occasionally, roughvariants may be noted. The very small colonies are physiological as well asmorphological variants. Their growth rates are much lower than those of theother haploids, and the individual cells are approximately 4 of the normal size.

FIG. 2. STREAK PLATE DEMONSTRATING GENETIC INSTABILITY OF LN, AN UNADAPTABLEHAPLOID STRAIN OF SACCHAROMYCES CELREVISIAE

In the case of Saccharomyces carlsbergensis, the examination of 22 clones ledto the isolation of a nonfermenting haploid strain (CN) which, on subsequentexamination by the same methods employed with LN, did not revert to thefermenting type. Table 2 gives its metabolic characteristics on galactose.None of the 22 clones failed to grow on galactose. Four of the 22 clones whenfirst isolated were nonfermenters when tested manometrically, but reverted

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within 2 weeks to the fermenting type when subcultured on galactose. StrainCN, however, has retained its nonfermenting character for 4 months, despiteits obvious colonial instability as evidenced by figure 3, which represents astreak plate from a routine galactose broth culture. Here a relatively smallrough type, a smooth type, and the micro-types are represented.

FIG. 3. STREAK PLATE DEMONSTRATING GENETIC INSTABILITY OF CN, AN UNADAPTABLEHAPLOID STRAIN OF SACCHAROMYCES CARLSBERGENSIS

In the experiments with Db23B the problem of getting variant types wassimpler since it was a haploid strain. It was seeded into the glucose medium,and streak plates were made from which clones were selected for testing. Thefifth clone tested (DN) behaved in the same way as both LN and CN. Oneof the 5 did not grow on galactose, whereas the other 3 could mutate to theadaptable type. Strain DN was carried in the usual manner in 8 per centgalactose for 4 months without showing any ability to produce CO2 anaerobic-ally from galactose (table 2). It is also an unstable strain as is shown by figure 4

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which represents a streak plate from a galactose culture. Here the rough andsmooth forms predominate, although a small rough form may also be seen.

DISCUSSION

The relative significance of the experiments reported may be best evaluatedin terms of the life cycle of yeasts as it is understood to date. Figure 5 is adiagrammatic representation of the life cycle and also indicates sources ofvariation at various stages. Starting with diploid cells, we find that thesemay sporulate and give rise to haploid segregants. These segregants may beand often are strikingly different from the parent types; the extent of the dif-ference would depend cn the degree of heterozygosis of the parent diploid.

FIG. 4. STREAK PLATE DEMONSTRATING GENETIC INSTABILITY OF DN, AN UNADAPTABLEHAPLOID STRAIN OF SACCHAROMYCES CEREVISIAE

Thus, in a recent investigation (Lindegren, Spiegelman, and Lindegren, 1944)into the mechanism of the inheritance of adaptation to melibiose fermentation,it was possible to demonstrate simple Mendelian segregation of the adaptablecharacter from a heterozygotic diploid parent.The haploid segregants originating from the ascus may, if of suitable mating

types, copulate to produce new diploid cells. These may or may not differ fromthe parent type depending on the nature of the recombination. On the otherhand, if the haploids do not copulate they can in some strains exist as such bycontinued vegetative divisions, during which they usually give rise to mutantsof various types. In general, it has been found that the mutational variants havelost the ability to copulate, probably owing to concomitant disturbances modify-

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ing the effects of the gene which differentiates the mating type. This resultsin the maintenance of the haplophase nature of the culture.From the point of view of the life cycle it seems clear why the experiments

reported here were successful in adapting Schizosaccharomyces pombe, whereasprevious ones had failed. Two conditions were satisfied in the present casethat were not satisfied simultaneously in earlier attempts. One is a massiveinoculation of heavily sporulating cultures which provides a source for a large

cn CELLS A COLONY

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SPORES

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number of various haploid segregants. The second was the inclusion of enoughglucose in the adapting medium to allow the asci to germinate and the haploidsto go through a relatively large number of divisions before intensive selectionfor the galactose fermenters took place. Under these conditions the hetero-geneity of the haplophase originating from the segregation is further increasedby the ability of the members to express their mutational potentialities. Thechances for obtaining mutants of the desired nature is thus greatly augmented.The experiments on the isolation of the three unadaptable variant haploid

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strains from adaptable parents suggests a possible explanation for Wise andAppling's (1944) finding on the loss of galactose fermentability by a strain ofSaccharomyces cerevisiae. Some cultures sporulate readily on agar slants, andit is probable that the variable haplophase was introduced from the stock strain.One interesting aspect of the three unadaptable strains is their inability, despitetheir demonstrated genetic instability, to mutate in the direction of galactosefermentation. While no absolute statement can be made, it must be recalledthat the generation time of these organisms is but a little over an hour. Conse-quently, these experiments have extended over 1,200 generations involvingbillions of individuals in each generation. All this has been going on underconditions where intense selection of the fermenting type is favored. Thus ifthe back mutation should occur, it either does so with a vanishingly small fre-quency, or it may be accompanied by secondary lethal effects.These experiments then would indicate that whereas sporulation is a neces-

sary condition for populational adaptation by mutation of a diploid strain,it may not be sufficient for two reasons: (1) a haplophase suppression mech-anism may exist as in Schizosaccharomyces octosporus and Saccharomycodesludwigii or (2) the haploids may not possess the capacity to mutate in thedirection sought.The wide variety of physiological variants which may be isolated from a

single strain once the haplophase is introduced makes it difficult to characterizeyeast types by their biochemical properties. In the present study closely re-lated forms stemming from the same parent may or may not possess the abilityto ferment galactose. Under these circumstances it is difficult to justify placingmuch weight on fermentation characteristics in classification schemes. Thisextreme heterogeneity is present in even the highly inbred strains as the bakingyeasts which have been intensely selected for high aerobic C02 production onsucrose over a number of years. A recent study (to be discussed elsewhere)of 12 baking strains has demonstrated that they all can give rise to progenieshaving physiological properties diametrically opposed to those for which theparent type was selected.

In any case it is hopeless to attempt to characterize a yeast strain by its bio-chemical or morphological properties unless it is a diploid and care is takento prevent its sporulation.

SUMMARY

Schizosaccharomyces pombe, previously reported as unadaptable, has beenadapted to galactose fermentation by inoculation with heavily sporulatingcultures into 2 per cent glucose 8 per cent galactose mixtures. The same meth-ods failed with both Schizosaccharomyces octosporus and Saccharomycodes ludu4ii.These results are interpreted in terms of the genetic instability of the haplo-

phase and its suppression by rapid copulation in the latter two strains.Unadaptable haploid strains have been isolated from adaptable parents.

The significance of this for an understanding of the range of biochemical vari-ation of mutant types is discussed.

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Research, 19, 251-256.GUILLIERMOND, A. 1903 Recherches sur la germination des spores dans le Saccharomyces

ludwigii (Hansen). Bull. Soc. Myc. France, 19,19-31.KLUYVER, A. J. 1914 Biochemische Suikerbepalingen. Dissertation. Technische Hoo-

geschoole, Delft.LINDEGREN, C. C., AND LINDEGREN, G. 1944 Sporulation in Saccharomyces cerevisiae.

Botan. Gaz., 105, 304-316.LINDEGREN, C. C., SPIEGELMAN, S., AND LINDEGREN, G. 1944 Mendelian inheritance

of adaptive enzymes in yeast. Proc. Nat. Acad. Sci. U. S. 30, 346-352.SPIEGELMAN, S., AND LINDEGREN, C. C. 1944 A comparison of the kinetics of adaptation

in genetically homogeneous and heterogeneous populations of yeast. Ann. MissouriBotan. Garden, 31, 219-233.

SPIEGELMAN, S., LINDEGREN, C. C., AND HEDGECOCK, L. 1944 Mechanisms of enzymaticadaptation in genetically controlled yeast populations. ProC. Nat. Acad. Sci. U. S.,30, 13-23.

STELLING-DEKKER, N. M. 1931 Die Sporogenen Hefen. Amsterdam, Holland. 547 p.

STEPHENSON, M., AND YUDKIN, J. 1936 Galactozymase considered as an adaptive en-

zyme. Biochem. J., 31, 1311-1315.WINGE, O., AND LAUSTSEN, 0. 1939 Saccharomycodes Ludwigii (Hansen), a balanced het-

erozygote. Compt. rend. trav. lab. Carlsberg, S6r. physiol., 22, 357-370.WISE, L. E., AND APPLING, J. W. 1944 Quantitative determination of d-galactose by

selective fermentation. Ind. Eng. Chem., Anal. Ed., 16, 28-32.

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