Received for publication June 18, 1952 are terminal, while in raffinose, which is fructoseglucose-galactose, the glucose is internal.

Transcription

1 A SNGLE ADAPTVE ENZYME N SACCHAROMYCES ELCTED BY SEVERLL RELATED SUBSTRATES1 NORBERTO J. PAERONam CARL C. LNDEGREN Southern llinois University, Carbondale, llinois. The adaptive elicitation of aipha-glueomeleziase by maltose, sucrose, or alpha-meta,yl glucoside. Heterologous adaptation. Hestrin and Lindegren (1951) demonstrated that interactions occur between the two nonallelic genes: MA, which controls the fermentation of maltose, and MG, which controls the fermentation of alpha-methyl glucoside. They concluded that each gene controlled the production of a distinct and separate enzyme, one, a hydrolase of maltose, the other a hydrolase of alpha-methyl glucoside. When the genes are present singly, each responds to the presence of its homologous exciter (maltose or alpha-methyl glucoside) by producing maltase or alpha-methyl glucosidase, respectively. When the cells which contain both the gene controlling the hydrolase of maltose and the gene controlling the hydrolase of alpha-methyl glucoside are grown on maltose, maltase and, in addition, alphamethyl glucosidase are produced. The phenomenon was called heterologous adaptation and was inferred to result from the conversion of maltase into alpha-methyl glucosidase by the MG gene. Lindegren and Lindegren (1953) introduced the gene MZ into their breeding stock (Lindegren, 1949). The present paper shows that MZ controls the adaptive production of alpha-gluco-melezitase which breaks down maltose, turanose, melezitose, alpha-methyl glucoside, and sucrose, but is incapable of acting on raffinose. This enzyme acts on alpha-gluco-saccharides by attaching to the free glucose end of the molecule. n melezitose, which is glucose-fructose-glucose, both glucoses 1 This work has been supported by research grants from Southern llinois University, the National Cancer nstitute of the National nstitutes of Health, U. S. Public Health Service, and Anheuser-Busch, nc. 2 Rotary nternational Fellow. Present address: Department of Microbiology, University of CUYO, Mendoza, Argentina. Received for publication June 18, 1952 are terminal, while in raffinose, which is fructoseglucose-galactose, the glucose is internal. Since the MZ gene (in its most potent condition) responds adaptively to at least five different exciters, maltose, sucrose, turanose, melezitose, or alpha-methyl glucoside, with the production of a single enzyme which acts on all five substrates, adaptation to maltose adapts the yeast heterologously to the fermentation of sucrose, turanose, melezitose, and alpha-methyl glucoside. The enzyme is adaptive; unadapted MZ cells do not contain alpha-gluco-melezitase. The heterologous adaptation effected by MZ differs from that described by Hestrin and Lindegren (1951) since the MZ gene controls the production of only a single enzyme which is induced adaptively by five different substrates. The type of heterologous adaptation described in this paper may be similar to that described by Rhoades (1941) although the genotypes of his cultures were indeterminate. MATERALS AND METHODS Purification of reagent grade saccharides. n analyzing the phenomenon of homologous and heterologous adaptation to different alpha-glucosides, the purity of the sugars must be examined critically. For this purpose the presence of glucose in maltose can be estimated quantitatively by exposing a hexose-specific yeast (one which can utilize glucose but not maltose) to a solution of reagent grade maltose under anerobic conditions, and observing the amount of CO2 produced (Hestrin and Lindegren, 1951). Both the maltose and the alpha-methyl glucoside were recrystallized from water and obtained in a purified state. The other saccharides (sucrose, melezitose, raffinose, and turanose) were found to be free of substances fermentable by unadapted yeast under anaerobic conditions. The solutions were purified of alcohol (which may or may not have been present) by boiling under vacuum distillation (Palleroni, Sheffner, and Lindegren, 1952). 122

2 SNGLE ADAPTVE ENZYME N SACCHAROMYCES 123 Cultivation of cells. Cells usually were cultured by inoculating 50 ml of nutrient medium in 250 ml Erlenmeyer flasks, allowing to grow for 24 hours at 30 C; the flasks were shaken by hand approximately once every 8 hours. The medium contained: Bacto peptone (Difco), 2 g; KH2PO4, 2 g; MgSO4, 0.5 g; liquid yeast extract, 2 ml per liter. The designated sugar was added at 2 per cent concentration. n some experiments comparable results were obtained using cells washed from agar slants. After 24 hours the cells were centrifuged and washed twice with M/15 KH2PO4. Manometric analysis. The cells were resuspended and made up to the Klett values (green filter) indicated in the graphs, and 1.5 ml of washed cells were placed in each Warburg vessel. The classical two vessel direct method was used; one vessel contained 0.2 ml of 10 per cent KOH in the center cup; the gas phase was air. Experiments were always preceded by a period of about 2j/ to 3 hours of endogenous respiration until equilibrium was established before 0.5 ml of saccharide was added from the side arm. Dried cell preparations. For the dried cell preparations the yeast was grown in 1 liter of medium containing 2 per cent of the sugar in question in a 3 liter Erlenmeyer flask for 48 hours at 30 C with occasional shaking. The cells were harvested by centrifugation, washed twice with distilled water, and dried overnight in vacuo over Hydralo dessicant. The dried cells were ground to a fine powder and passed through a 100 mesh sieve. Ten grams of cells were mixed with 2 ml of toluene and ground in a mortar. Ten ml of water were added and the suspension left at room temperature for 24 hours. This preparation was centrifuged at 5,000 rpm, decanted, and centrifuged again. Demonstration of hydrolytic activity of the dried cell preparation. A suspension of cells (500 Klett) of a hexose-specific yeast in 1.5 ml of 1.5 per cent of the saccharide in question was placed in paired Warburg vessels. After endogenous equilibration a 1 :10 dilution of the dried cell preparation was dumped from the side arm. Hydrolysis of the saccharide by the preparation produced hexoses which were fermented by the living hexose-specific cells. Controls were run to show that the mixture of the saccharide with the dried cell preparation yielded no CO2. Heterologous adaptation of culture by growth on maltose. Culture is of genotype ma MZ mg su (table 7, Lindegren and Lindegren, 1953). t can ferment or grow on maltose, sucrose, melezitose, turanose, and alpha-methyl glucoside ,' U # t~ Culture /3765,,,,,, ocp bwfa oaro"/ 1100 CO2 loo A -Erdagerocs of uaodapted celbs -Ltopted with pure altose C llose-odap ed cek on ethyl 0/gk o-adapted ceokn uncross / / 900 E atoe-adapted cfl on matoe 8 50~~~~~~~~~ 400~~~~~~~~ P d O~~~~~~/ 0 0o /~~~~~~~~O NWfsoV*zfu PC /~~~~~~~~~~~~ 30C ibfl #>~ ,,/D L B.l HOUNRS cd Figure 1. Comparison of fermentative ability of unadapted (glucose grown) and maltose adapted cells of culture Maltose adapted cells obtained by growth in 2 per cent maltose liquid medium; the unadapted cells were from glucoseagar slants. 1.5 ml of washed cell suspension (200 Klett) in each Warburg vessel. A: Endogenous of unadapted cells. B: Unadapted cells on maltose. 0.5 ml of 6 per cent purified maltose dumped from side arm. C: Maltose adapted cells on alpha-methyl glu- -coside. 0.5 ml of 6 per cent alpha-methyl glucoside. D: Maltose adapted cells on pure sucrose. 0.5 ml of 6 per cent sucrose. E: Maltose adapted cells on pure maltose. 0.5 ml of 6 per cent maltose. The endogenous activity of adapted cells is greater than of the unadapted cells. The arrow indicates the time at which the saccharide was dumped from the side arm. Open circles represent C02 produced and full circles 02 uptake. t is raffinose negative. When cells of this culture are exposed to purified maltose, turanose, sucrose, 6

3 124 N. J. PALLERON AND C. C. LNDEGREN [VOL. 65 melezitose, or alpha-methyl glucoside in the Warburg apparatus under aerobic conditions, production of CO2 or uptake of 02 does not occur within 6 hours, demonstrating that the enzymes involved are adaptive. The paired curves A in figure 1 show the 02 consumption and C02 production of the endogenous metabolism of culture 13765; the paired curves B show the 02 consumption and C02 production of the same cells when purified maltose is added after 3. hours of dissimilation. No action on maltose occurred in the subsequent 6 hours HOURS The rate of fermentation finally achieved is substantially equal to that occurring when the maltose adapted cells are exposed to maltose. Turanose was used in a limited number of experiments. The rate of fermentation of turanose by maltose adapted cells is slightly lower than that of maltose, but fermentation occurs immediately without lag. The immediate action on turanose suggests that it is hydrolyzed by the same enzyme which acts on maltose and sucrose. Conditions controlling adapt ion of culture to maltose. Since adaptation to maltose oc- Figure B. Action of sucrose adapted cells of culture on maltose and alpha-methyl glucoside. Cells of culture grown in 2 per cent sucrose liquid medium 24 hours. 1.5 ml of washed cell suspension (200 Klett). A: 0.5 ml of 6 per cent purified maltose added from side arm. B: 0.5 ml of 6 per cent purified alpha-methyl glucoside added from side arm. Culture was adapted to maltose by growth in complete medium containing reagent grade maltose. The cells were harvested after 24 hours, washed, and tested manometrically. Curves E (figure 1) show the reaction to purified maltose of the yeast adapted to maltose by growth in medium containing reagent grade maltose, and curves D show the action of the same cells to sucrose. Adaptation to maltose has rendered the cells capable of acting on both maltose and sucrose at substantially the same rate, suggesting that adaptation has produced a single enzyme, alpha-gluco-melezitase, which acts on both saccharides. Curves C (figure 1) show the characteristic reaction of the maltose adapted cells of culture to alpha-methyl glucoside. curred during growth in full nutrient containing maltose but failed to occur after 6 hours' exposure to purified maltose in buffer in the Warburg vesel, the conditions controlling adaptation were tested to determine whether mutation and selection had intervened in the adaptation achieved during growth in full nutrient. The conditions were assumed to parallel those demonstrated by Lindegren and Palleroni (1952) in galactose adaptation in which an external source of energy other than galactose was shown to be necessary. The addition of maltose to unadapted cells produces no increase over the endogenous respiration in 6 hours. Furthermore, the addition of ammonium (0.02 per cent (NH4)2S04) to the maltose buffer mixture also fails to effect adapta-

4 SNGLE ADAPTVE ENZYME N SACCHAROMYCES 125 tion to maltose. The addition of glucose plus maltose produces no more effect than the addition of glucose alone. When glucose plus maltose plus ammonium were added simultaneously to the unadapted cells in the Warburg apparatus in the same concentrations as were used in the preceding experiments, the glucose was consumed almost completely after 500 ul of CO2 had been evolved, but fermentation continued after that period, proving that utilization of maltose had begun and adaptation had been achieved. Heterologous adaptation of culture by growth on sucrose. Cells of culture adapted to maltose acted on both maltose and sucrose without significant difference. Similarly, cells of culture which had been adapted to sucrose acted on both maltose (figure 2A) and sucrose without lag at substantially the same rate. After adaptation to sucrose, the cells acted immediately on alpha-methyl glucoside (figure 2B), turanose, and melezitose. The identical behavior of sucrose and maltose adapted cells strongly suggests the view that each elicits the identical enzyme, alpha-gluco-melezitase. Heterologous adptaion by growth on alphamethyl glucoside. Cells of culture were adapted to alpha-methyl glucoside. When exposed to alpha-methyl glucoside in the Warburg apparatus, they fermented it immediately without lag. They also fermented maltose, sucrose, turanose, and melezitose without lag. The heterologous adaptation to the alpha-gluco-saccharides is interpreted to indicate that the same enzyme has been induced by each of these saccharides through action of the MZ gene. Specificity of alpha-gluco-melezitse. Culture is raffinose negative by the Durham tube test. t also fails to adapt to raffinose. Although incapable of raffinose fermentation, it is a fermenter of sucrose. A dried cell preparation of cells grown on sucrose was tested for ability to act on raffinose. This preparation was extremely active and hydrolyzed sucrose, maltose, turanose, melezitose, and alpha-methyl glucoside at rates so high that measurement was not possible, but showed no action on raffinose. The inability of the preparation to act on raffinose indicates that the enzyme is not the standard invertase; the failure of the intact cells to act on raffinose cannot be ascribed to impermeability to raffinose. t is important in investigations of this kind to determine whether or not the adaptation involves a change in permeability and is especially pertinent to the present investigation since the enzymes were typical endoenzymes and the substrate must enter the cell before action can occur. This fact was established by a long series of experiments which it was considered unnecessary to report in detail.. Heterologous adaptation of cells carrying multiple alleles of MZ. Multiple alleles of the MZ gene. Lindegren and Lindegren (1953) showed that the MZ gene varies considerably in its manifestations. Culture carries the MZ gene in its most potent state being capable of growth on maltose, sucrose, turanose, melezitose, and alpha-methyl glucoside and producing alpha-gluco-melezitase by adaptation to these substrates. Culture can grow on maltose but is incapable of growth on either sucrose, melezitose, or alpha-methyl glucoside. Culture is related to other cultures capable of fermenting the wider spectrum of alpha-glucosides, and adaptation to maltose produces alpha-gluco-melezitase in its cells. Maltose, however, (turanose was not tested) is the only one of the four saccharides capable of producing adaptation in culture The adapted cells of culture can ferment maltose, sucrose, turanose, melezitose, and alpha-methyl glucoside suggesting that the allele in culture can respond only to maltose and not to sucrose, melezitose, or alpha-methyl glucoside, while the allele in culture can respond to maltose, sucrose, melezitose, or alpha-methyl glucoside. Although culture is Durham tube positive only on maltose and cannot grow on the other sugars, its cells contain alpha-glucomelezitase after growth on maltose and are capable of fermenting the other saccharides after adaptation to maltose. Furthermore, culture (like culture 13765) is incapable of adapting to pure maltose in the presence of buffer in the Warburg apparatus. Experiments on adaptation to pure maltose with other adjuvants gave substantially the same results as those described with culture Heterologous adaptation in culture induced by growth in maltose. Unadapted cells of culture taken from glucose agar slants show a very low endogenous activity which is not changed significantly by the addition of either buffer or pure maltose. The cells are incapable of adapting to pure maltose dissolved in buffer in

5 126 N. J. PALLERON AND C. C. LNDEGREN [vol. 65 the Warburg apparatus althoughtey are capable of growth and fermentation of maltose in the Durham tube. t is clear therefore that maltose is utilized by an adaptive mechanism. Figure 3 shows the oxygen consumption of maltose adapted cells in the Warburg vessel on sucrose, imelezitose, turanose, and alpha-methyl glucoside. t is obvious that cells of culture grown on -maltose are capable of immediate fermentation of sucrose, turanose, melezitose, and alpha-methyl glucoside. These data have led to the conclusion Co 4 -k AoA/ TarF - A nated as "slow" or "late" fermenters. Culture resembles culture except that it ferments sucrose slowly (after 3 days) by the Durham test. Extensive study of slow fermenting cultures has revealed that this condition may be due either (1) to mutation of a nonfermenter to the fermenter type with selection of the mutant leading to a shift in population and the eventual overgrowth of the mutant (Mundkur and Lindegren, 1949; Mundkur, 1949) or (2) to a genotype which is incapable of producing the Figure 3. Oxygen consumption of maltose adapted cells of culture A: Oxygen consumption of maltose adapted cells on sucrose. Maltose adapted cells by growth in 2 per cent maltose medium for 24 hours. 0.5 ml of 6 per cent sucrose added from side arm. B: Oxygen consumption of maltose adapted cells on melezitose. 0.5 ml of 6 per cent melezitose added from side arm. C: Oxygen consumption of maltose adapted cells on turanose. 0.5 ml of 6 per cent turanose added from side arm. D: Oxygen consumption of maltose adapted cells on alpha-methyl glucoside. 0.5 ml of 6 per cent purified alpha-methyl glucoside added from side arm. F - 4DAPTED CELLS OF _ ~~CUL77rE /3777 A - Sucrose B - Melezitos -C - Turanoe D - Akha met"y glucceie f /-, 0~ A0 '.8a 41 o/ 0/ i---~ ~~~* T 6 HOURS that adaptation to maltose in culture leads to the production of alpha-gluco-melezitase which is capable of hydrolyzing maltose, sucrose, turanose, melezitose, and alpha-methyl glucoside, and that the heterologous adaptation in culture induced by growth on maltose is due to the possession of this enzyme by the adapted cells. "Weak" fermenter culture Culture which is Durham tube positive for maltose and negative for melezitose (but "late" for sucrose) also produces alpha-gluco-melezitase after growth on either maltose or sucrose. Many cultures do not ferment rapidly enough in the Durham tube test to produce gas in 24 hours. These are desigenzyme in sufficient quantities to register an immediate rapid fermentative activity (Winge and Roberts, 1948) but which on adaptation eventually achieves the regular rapid rate. Study of culture has revealed that its action on sucrose is slow because of some defect which is not corrected by prolonged contact with the adaptive substrate, thus comprising a third category. Heterologous adaptation in the "weak" fermenter culture Culture was distinguished in the Durham tube test by requiring more than three days to act on sucrose. Cells grown on maltose were tested for their ability to act on different saccharides in the Warburg apparatus

6 1953] SNGLE ADAPTVE ENZYME N SACCHAROMYCES 127 and were found to act at a much lower rate than either culture or culture The low rate was not substantially improvred by a second C,) lc r ( X lo 40C 30C 20C 1o BUFFER MALTOSE - ADAP/TD CELLS 0 CO B-, A MALTOSE - - v. - ~~~~~~~~ - L T ~~~~~~0~ ~B are relatively poorly adapted to the fermentation of maltose and which (figure 4C) acted even less rapidly on sucrose. Only slight improvement in '. - i k too too 30C _ 20C 10b OF Cut LfRE /3766 E SUJCOSE 11 A ELEZTOSE... TURAKOSE / ALUAMEHL -. 0 F - - H... t. o HOURS Figure 4. Fermentative activity of maltose adapted cells of culture A: Endogenous activity of unadapted cells. Cells grown in glucose agar slants 24 hours. 1.5 ml of washed cells suspension plus 0.5 buffer. B and C: Action of maltose adapted cells grown for 24 hours in 2 per cent maltose medium. B: Action of maltose adapted cells on pure maltose. 1.5 ml of washed cell suspension (200 Klett) plus 0.5 ml purified maltose 6 per cent. C: Action of maltose adapted cells on sucrose. 1.5 ml of washed cells suspension (200 Klett) plus 0.5 ml of 6 per cent sucrose. D, E, F, G, and H: Action of cells adapted to maltose by growth in 2 per cent maltose medium for 24 hours followed by a second transfer to maltose medium and a second growth period of 24 hours. 1.5 ml of washed cell suspension (200 Klett). D: 0.5 ml of 6 per cent purified maltose added from side arm. E: 0.5 ml of 6 per cent sucrose added from side arm. F: 0.5 ml of 6 per cent melezitose added from side arm. G: 0.5 ml of 6 per cent turanose added from side arm. H: 0.5 ml of 6 per cent purified alpha-methyl glucoside added from side arm. : Endogenous activity. transfer to maltose medium before testing in the ability to ferment maltose is achieved by a second Warburg. Figure 4A shows that cells of culture transplantation to maltose medium. Figures 4D, from a glucose slant have a very low rate E, F, G, and H show that cells adapted to maltose of endogenous respiration. Figure 4B shows that by two transfers on maltose are capable of fer- 24 hours' growth in maltose results in cells which menting maltose, sucrose, melezitose, turanose,

7 128 N. J. PALLERON AND C. C. LNDEGREN [VOL. 65 and alpha-methyl glucoside, but at much slower rates than cultures and t is clear, however, that although culture cannot grow in either melezitose or alpha-methyl glucoside, adaptation to maltose produces an enzyme capable of acting on these substrates and that the "weak" fermenter resembles culture in culture may be due to environmental conditions which can be altered to produce a "fast" culture. t is possible that components of the complete medium may be involved.. Competitive inhibition between substrates in the elicitation of alpha-gluco-melezitase %Mo u+02% Surs# -2.0%Mato"0+B%Sucr0s -4.0%Maltw*#s8%Sucose /2% Maltose HOURS Figure 5. Effects of increasing amounts of maltose in the growth medium (sucrose constant) on the synthesis of alpha-gluco-melezitase. Cells of culture grown in 0.8 per cent sucrose and and 4 per cent maltose, 24 hours, 1.5 ml washed cells suspension (230 Klett); 0.5 ml of 6 per cent sucrose was added from side arm. capacity for heterologous adaptation except for the depressed rate. Culture was adapted to maltose in the Warburg apparatus by suspending glucose grown cells in a mixture of maltose, glucose, and ammonium sulfate. After exhaustion of the glucose, fermentation of the maltose occurred at an extraordinarily increased rate in comparison with the activity of cells which have been adapted by growth in complete nutrient medium. This suggests that the low rate and the weakness of the c - 6 / 1% Maltose _/Z4% Maltose / 11 / 0 2% Maltose /,// / i/ri / f..-.4% Malto B$/ *f,, REAGENTS / S0EARM 4 /05 Maltose u -.M -- Oa 0.Co Culture is able to adapt to maltose with the production of alpha-gluco-melezitase, which hydrolyzes maltose, sucrose, melezitose, turanose, and alpha-methyl glucoside. Culture (like culture 13765) is capable of adapting to maltose only after the addition of a source of energy such as glucose and a small amount of ammonium sulfate. The addition of glucose alone to the maltose does not lead to the initiation of maltose fermentation within a period of 10 hours, while the addition of a nitrogen

8 19531 SNGLE ADAPTVE ENZYME N SACCHAROMYCES 129 source in small amount produces a definite adaptation within 4 hours. Since culture can produce alpha-glucomelezitase after growth on maltose but is incapable of growth on sucrose, an experiment was performed to determine if the presence of sucrose during adaptation to maltose inhibited the production of alpha-gluco-melezitase. t should be emphasized that use of this culture affords the unique opportunity of testing the ability of an organism to ferment and utilize a substrate to which it cannot adapt. When glucose, maltose, and ammonium sulfate are added simultaneously, adaptation occurs later than when maltose is added to the cell suspension before the glucose and the ammonium sulfate. Although glucose acts as a source of energy in adaptation, it also is an inhibiter of adaptation, but its inhibitory effect can be reduced by exposure to maltose previous to the introduction of glucose. When sucrose is added at the beginning of the experiment before the addition of glucose, maltose, and ammonium sulfate, the continued exposure to sucrose delays adaptation and causes fermentation to proceed at a much lower rate. t is inferred that sucrose acts as a competitive inhibitor of adaptation to maltose. The effect of competitive interaction between sucrose and maltose is shown in the experiment recorded in figure 5. The cells were adapted by growth in liquid medium containing 0.8 per cent sucrose and concentrations of maltose varying from 0.5 to 4.0 per cent. The cells were tested in the Warburg apparatus for their ability to ferment pure sucrose to which they are incapable of responding adaptively. They were capable of hydrolyzing sucrose in the Warburg apparatus but were incapable of increasing their content of alpha-gluco-melezitase while metabolizing sucrose. Adaptation to 0.5 per cent maltose was inhibited considerably by the presence of 0.8 per cent sucrose. A marked increase over the degree of adaptation occurs in the presence of 1 per cent maltose together with 0.8 per cent sucrose. The highest value is obtained with the use of 2 per cent maltose. t is important to note that at a concentration of 4 per cent maltose the adaptation is diminished considerably indicating that an optimal concentration exists, and that an excess of maltose may act as an inhibitor of adaptation to maltose fermentation. n a supporting experiment in which the growth medium contained 0.8 per cent maltose and varying concentrations of sucrose, the inhibitory effect of sucrose on adaptation to maltose was confirmed. SUMMARY A culture of Saccharomyces able to ferment maltose, turanose, sucrose, melezitose, and alphamethyl glucoside, carrying the MZ gene, was found to produce the same enzyme (alpha-glucomelezitase) on adaptation to either maltose, sucrose, or alpha-methyl glucoside. t was capable of heterologous adaptation; growth on maltose resulted in ability to ferment either maltose, sucrose, or alpha-methyl glucoside. A culture carrying a multiple allele of MZ which had lost the ability to grow on alpha-methyl glucoside, melezitose, or sucrose gave negative fermentation tests in Durham tubes containing these sugars. t was also found to be capable of heterologous adaptation; growth on maltose resulted in ability to ferment sucrose, melezitose, or alpha-methyl glucoside. The MZ gene in its various manifestations appears to control the production of a single enzyme which is capable of breaking down maltose, turanose, sucrose, melezitose, and alpha-methyl glucoside (but not raffinose). This enzyme has been named alphagluco-melezitase because melezitose is the unique substrate by which the genotype can be determined. So far no member of the allelic series has been found which has lost the ability to ferment maltose. After growth on maltose, any cell carrying an allele of MZ is able to break down any one of the five alpha-glucosides. Culture 13777, which is capable of growth on maltose, is incapable of growth on sucrose. Cells of culture can ferment sucrose after growth on maltose although they cannot do so after growth on glucose. The growth of culture on maltose is limited and soon comes to an end. Hence a large transfer of maltose grown cells to a Durham tube containing sucrose results in a positive fermentation test, while a large transfer of glucose-grown cells results in a negative test. On the other hand, when only a few cells grown in maltose broth are inoculated into a Durham tube containing sucrose, a negative test results. This is due to the fact that an insufficient amount of enzyme is present in the smaller number of cells to break down enough sugar to cause gas to accumulate in the inverted insert. The phenotype

9 130 N. J. PALLERON AND C. C. LNDEGREN [VOL. 65 of cultures capable of heterologous adaptation, therefore, depends on the substrate upon which the culture is grown before the diagnostic test is made. The presence of sucrose during the adaptation of culture to maltose inhibits the production of alpha-gluco-melezitase. REFERENCES HESTRN, SHLOMO, AND LNDEGREN, C. C Gene function in the homologous and heterologous induction of a-glucosidases in yeast by a-glucosides. Nature, 168, 913. LNDEG(REN, C. C The yeast cell, its genetics and cytology. Educational Publishers, nc., St. Louis, pp LNDEGREN, C. C., AND LNDEGREN, GERTRUDE 1953 The genetics of melezitose fermentation in Saccharomyces. Genetics, in press. LNDEGREN, C. C., AND PALERON, N. J Absence of the preadaptive utilization of galactose by yeasts. Nature, 169, MUNDKUR, BALAJ D Long-term adaptation and non-mendelian inheritance in yeast. Nature, 164, 614. MUNDKUR, BALAJ D., AND LNDEGREN, C. C An analysis of the phenomenon of longterm adaptation to galactose by Saccharomyces. Am. J. Botany, 36, PALLERON, N. J., SHEFFNER, A. L., AND LNDE- GREN, C. C The absence of preadaptive oxidation of galactose by strains of Saccharomyces. Arch. Biochem. Biophys., 40, RHOADES, M. M The adaptive enzyme of certain strains of yeast. J. Bact., 42, WNGE, O., AND ROBERTS, C nheritance of enzymatic characters in yeasts and the phenomenon of long-term adaptation. Compt. rend. trav. lab. Carlsberg. Ser. physiol., 24, Downloaded from on April 16, 2019 by guest