occurrence of respiration deficient mutants in baker's yeast cultivated anaerobically

OCCURRENCE O F RESPIRATION DEFICIENT MUTANTS

IN BAKER'S YEAST CULTIVATED ANAEROBICALLY

MORGAN HARRIS l Laboratoire de Ge'nitique, Paculte' des Sciences

Universite' dej Paris, Paris, France

TWO FIGURES

INTRODUCTION

The analysis of respiratory function in yeast by Ephrussi and co-workers (see general reviews by Ephrussi, '52; Slon- imski, '53) indicates that the formation of respiratory enzymes is mediated by a complex involving genes, cytoplasmic particles, and environmental factors. This picture stems largely from the study of respiration-deficient mutants which occur spontaneously or may be induced in populations of Sac- charomyces cerevisiae. Such mutants are easily detected by small colony size in the presence of limiting concentrations of sugar, and by characteristic differences in the cytochrome spectrum. The variants constitute approximately 1% of the cell population in the majority of yeast strains studied. Respi- ration in mutant cells is essentially lacking owing to the loss of cytochrome oxidase and other associated enzymes although fermentation is unimpaired. Mutants of this type arise con- tinuously during vegetative reproduction of either haploid or diploid stains and have accordingly been termed vegetative littles.

'Merck Senior Postdoctoral Fellow in the Natural Sciences of the National Research Council, 1953-54. Present address : Department of Zoology, University of California, Berkeley 4, California.

95

96 MORGAN HARRIS

The origin of vegetative littles is not readily accounted for in mendelian terms as a gene mutation. The character in question disappears in the progeny of hybrids between normal and mutant cells, and is not recovered despite repeated back- crosses to the mutant strain. These facts suggest the occurrence of a cytoplasmic rather than a nuclear mutation. The explanation advanced by Ephrussi postulates the existence of cytoplasmic units endowed with genetic continuity and essential for the synthesis of respiratory enzymes in normal cells. On this basis the origin of vegetative littles would represent an irreversible loss or inactivation of the particles concerned.

The mechanism through which mutant littles are produced from normal cells is still obscure. The present investigation is concerned with one aspect of this problem, viz. whether production of vegetative littles occurs in anaerobic as well as aerobic cultures. If an aerobic process is essential to the cytoplasmic change, mutation should cease in the absence of oxygen. On the other hand, if production of littles is independent of the presence of oxygen, no difference would be expected under anaerobic conditions. I n practice an experimental answer to this question is complicated by the fact that normal cells and vegetative littles do not grow at the same rate (Ephrussi, L'HQritier et Hottinguer, '49), so that selection as well as mutation must be considered in population analyses.

The experiments to be described deal with cultures of normal yeast growing for long periods in rigorous anaerobiosis. For this work the apparatus of Stier, Scalf and Brockmann ( '50) has been used, which permits continuous cultivation of yeast under nitrogen for weeks or months. The incidence of vegetative littles has been determined in these cultures and the results interpreted on the basis of growth rates measured on normal strains of yeast, mutant strains, and mixtures of the two.

MUTANTS IN ANAEROBIC YEAST 97

MATERIAL AND METHODS

All experiments were performed with Succharomyces cere- visiue, strain “Yeast Foam,” obtained originally from Profes- sor 0. Winge in Copenhagen. Aerobic cultures were grown at 25” C. under conditions of maximum aeration (10 ml medium in 100ml Erlenmeyer flask with continuous agitation on a reciprocal shaker : 110 complete strokes/min with an amplitude of 4-6 em). Anaerobic cultures were grown at 28” C. with continuous stirring in an all-glass apparatus constructed according to the specifications of Stier et al. (’50). Slight modifications were made for convenient removal of daily yeast crops. The gas phase was high purity dry nitrogen furnished by the Soci6t6 “Air Liquide,” Paris, with an oxygen content, according to the producer, of less than 0.005%. The standard medium utilized in the present experiments contained 7% yeast extract and 15% glucose (Scalf and Stier, ’50), and was fortified with ergosterol and Tween 80 following the observation of Andreasen and Stier ( ’53 ) that yeast cells in anaerobiosis may require sterols and unsaturated fatty acids f o r optimal growth. The exact composition of the medium used was as follows:

Yeast extract (Difco) 70 gm

Glucose 150 gm

Ergosterol 0.05 gm Tween 80 8.0 ml Ethanol 12.0 ml Distilled water to 1000 ml

(control no. 413224)

KH,PO, 5 gm

To facilitate preparation of the medium a stock solution was first prepared by dissolving ergosterol in a boiling mixture of ethanol and Tween 80. The yeast extract and phosphate were then dissolved in water and the resulting preparation appropriately supplemented with the stock solution of ergosterol. Glucose was dissolved separately in a second solution and both sterilized at 120” C. f o r 20 minutes.

Growth was estimated principally from measurements of optical density made with a Meunier electrophotometer. Tav-

98 MORGAN HARRIS

litzki ( '49) and Slonimski ( '53 ) have demonstrated that cell number and dry weight are proportional within limits to the opacity of yeast cell suspensions. In addition, cells were enumerated directly with a Levy counting chamber, and protein determinations were performed by a biuret method based on the procedure of Stickland (,51).

The percentage of vegetative littles in the populations studied was estimated by plating diluted aliquots in Petri dishes containing agar with 0.5% yeast extract and 0.5% glucose. The proportion of small colonies was determined by counting after 6 days incubation at 25°C. Mutant strains were obtained by subculture of small colonies and in all cases the characteristic deficiency in the cytochrome system verified by examination with a Hartridge reversion spectro- scope (Slonimski, ' 5 3 ) .

For manometric work standard Warburg techniques were employed. Measurements of oxygen consumption, fermentation, and cytochrome oxidase were performed according to procedures outlined by Slonimski ( '53, '54). Details will be described in the appropriate experimental sections.

RESULTS

Characteristics of cultures growing in continuous anaerobiosis

In a typical operation, the Stier apparatus was assembled in a sterile room with 51 of medium in the reservoir flask and 30ml inoculum in the culture flask. The inoculum contained cells in the stationary phase after two previous aerobic transfers in the medium to be used. The culture was diluted to 230ml by addition of medium from the reservoir, giving an estimated initial optical density of 1800 (approximately 28 x lo6 cells/ml). Flushing with nitrogen was then initiated to establish anaerobic conditions in the entire apparatus. The flow rate was maintained at 50ml/minute for the first two days and reduced to approximately 2 ml/minute thereafter. The culture was maintained in an even suspension at all


times by the continuous operation of a magnetic stirrer (Stier et al., ,SO).

As a routine the progress of anaerobic cultures was followed by draining the culture flask every 24 hours. Each sample was removed under positive pressure to maintain anaerobiosis in the apparatus. A fresh growth cycle was then initiated by diluting the residue in the culture flask to a final volume of 230 ml. With this schedule daily samples consistently showed an optical density of about 25,000 units (approximately 380 X lo6 cells/ml). This limiting population was maintained through successive growth cycles for many weeks without change. No infections were observed at any time in the Stier apparatus.

The degree of anaerobiosis actually obtained in these cultures was studied in several experiments inasmuch as this point is clearly critical for the question of mutation in anaerobic cells. Anaerobic yeast is characterized by an in- ability to respire, owing to the rapid loss of cytochromes a, b, and c as well as cytochrome oxidase. An adaptive synthesis of these enzymes occurs, however, if the cells are exposed to oxygen and this process may be followed by measurements of oxygen uptake (Ephrussi and Slonimski, '50 ; Slonimski, '53). Following these observations, therefore, cytochrome oxidase was determined in several samples from a Stier culture over a 28 day period. The cells were removed from the apparatus, washed immediately in ice-cold M/5 phosphate buffer, pH 6.5, and exposed to ultrasonic treatment for 40 minutes in air. An apparatus manufactured by the SociBtB de Condensation et d 'Applications MQcaniques, Paris, was used for this purpose, giving a frequency of 960 kilocycles/sec. with an output of 10 watts/cm2 at the surface of the crystal. The extract obtained after treatment, still iced, was centri- fuged twice for 10 minutes each time at 3000RPM in a refrigerated International Centrifuge. The pellets were dis- carded and the final supernatant dialyzed overnight against M/50 phosphate buffer pH 7.3, before assay. The results (table 1) indicate that cytochrome oxidase falls to a low

100 MORGAN HARRIS

level by 4 days and remains without significant change during subsequent culture. It may be pointed out that the concentra- tion of cytochrome c used in the assay was saturating for the enzyme in anaerobic preparations but not for extracts from the initial aerobic inoculum. The actual differences between aerobic and anaerobic values are therefore greater than shown in the table (Slonimski, '53).

TABLE 1

Cytochrome oxidase activity in ultrasonic extracts made from yeast cultivated anaerobically

eyt. e oS (protein) cyt. c DAYS IH CULTURE or ( d . w . )

Aerobic inoculum 4 8

14 20

16.1 0.9 0.5 0.1 0.3

57.4 4.3 4.4

Standard conditions fo r determinations : Cytochrome c = 6.0 x M, ascorbate = 0.017 M, phosphate = 0.064 M,

NaCl = 0.011 M, pH = 6.9, t = 28°C. Q Cyt.

Oa

R b:t. (protein) = pl 0, consumed/hr/mg protein in ultrasonic extract

(d.w.) = p1 0, consumed/hr/mg dry weight of original cell suspension

before ultrasonic treatment.

In a second group of experiments, adaptation to oxygen was measured on cell suspensions taken from the Stier apparatus at various intervals, using the procedures of Slonimski ( '54). Measurements over an 8-hour period were used to determine initial and final rates of oxygen consumption ( -u$L) as well as the acceleration of oxygen up-

summarized in table 2. A marked decrease in initial oxygen consumption is apparent for samples of 4 days incubation or more, with a prominent adaptive rise over the 8-hour period of measurement. The data of tables 1 and 2 are thus in agreement with the typical picture for anaerobic yeast as outlined by Slonimski ( '54). It is interesting to find that

take ( _%'< dt2 ) during this interval. The values obtained are

MUTANTS I N ANAEROBIC YEAST 101

the characteristics of adaptation to oxygen are substantially similar in the two investigations in spite of differences in media, growth rates, and duration of anaerobiosis. On the other hand, the spectral changes observed f o r anaerobic yeast in the present experiments showed certain differences from those observed previously (Ephrussi and Slonimski, '50 ; Slonimski, '53), in particular the persistence of a very weak cytochrome c a-band and the absence of a strong cytochrome b, component. It may be emphasized, however, that the

TABLE 2

Respiratory adaptation of yeast taben from an anaerobic czllture

* ( p l O2 x h-% x mg d.w.-I) d tz

S ( p l Oa x h-I x mg d.w.-I) dt

DAYS IN CULTURE initial mean at 0 hours a t 7 hours (between (between

1-3 hrs.) 1-7 hrs.) ~~

Aerobic inoculum 85.3 122.4 9.1 4.5 1 18.5 66.5 12.0 5.8 4 8.3 57.9 11.1 6.4 8 10.9 62.6 11.3 6.6

15 2.3 67.5 13.5 8.6

Standard conditions for determinations :

90 mg glucose and 0.5 dry weight yeast in each flask. KOH in center well. 0.2 M phosphate, p H = 6.5, t = 28°C. Glucose added 30 min. before zero time; value at 0 hour obtained by

extrapolation (Slonimski, '54).

cytochrome oxidase values obtained here are of the same order of magnitude as those found in the earlier investigations. The spectral differences in question thus do not connote a lesser degree of anaerobiosis in the present experiments.

Incidence of Vegetative littles in amaerobic cultures

The proportion of vegetative littles in yeast populations under aerobic conditions represents a balance between uni- directional (or irreversible) mutation and selection since the mutants grow more slowly than normal cells (Ephrussi, L'HQritier and Hottinguer, '49). If under anaerobiosis the

102 MORGAN HARRIS

action of either factor is increased or decreased relative to the other, a change in proportion of mutants should result. This possibility has been examined by plating samples taken from an anaerobic culture over a period of 32 days. Table 3 gives the results of this experiment, which show clearly that the percentage of vegetative littles remains constant under anaerobiosis and does not differ significantly from the value for aerobic cultures (e.g. the original inoculum). The data also suggest that the proportion of mutants remains the same whether transfer is made from stationary phase or log phase to successive growth cycles.

TABLE 3

Incidence of vegetative littles in yeast cultured anaerobically in Stier apparatus

TOTAL OPTICAL ELAPSED DENSITY GENERATIOXS ~~~~~ I%;:& % DAYS I N CULTURE

~~~ ~

Aerobic inoculum . . . . . 0 789 7 0.9 4 25,200 15 942 4 0.4 8 24,800 l 31 2248 5 0.2

15 25,600 59 1177 10 0.9 22 25,000 87 1486 8 0.5 28 19,200 135 1511 4 0.3 32 16,800 a 173 684 7 1.0

Sample taken from stationary growth phase. * Sample removed before Stationary growth phase was reached.

These results permit alternative interpretations : either (1) mutation and selection are both unchanged under anaerobic as compared to aerobic conditions, o r (2) both are altered in the same direction and to the same degree. Stating the same problem in a somewhat more useful form, if one factor (e.g. selection) can be demonstrated to occur anaerobically, it follows that the other (mutation) must take place under the same conditions to maintain the constant proportion of mutants as documented in table 3. These considerations led to a study of growth rates in normal and mutant strains of yeast, under both aerobic and anaerobic conditions, and to the examination of population changes in synthetic mixtures of


the tu7o. The results obtained will be outlined in the following sections.

Growth rates of strains derived from normal cells and vegetative littles

A primary group of experiments was performed to compare growth rates under aerobic conditions of strains recently

TABLE 4

Aerobic generation times of normal and mutant strains of yeast in second fluid transfer f rom colonies on agar plates

GENERATION TIMES ( I N HOURS) IN SECOND €IoURS IN STRAIN FLUID TRANSFER

TRANSFER NO. TYPE FIRST

Successive determinations Averaee

G 1 1.68 1.49 1.62 1.6 G 2 G 3 Normal 24

1.56 1.53 1.56 1.73 1.42 1.40

1.6 1.5

6 4 1.72 1.45 1.50 1.6

P 1 4.51 4.02 . . . 4.3 P 2 4.10 4.45 . . . 4.3 P 3 2.81 2.67 . . . 2.7 P 4 4.60 5.78 . . 5.2

Little 120

Little

P 11 11.00 10.43 . . . 10.7 P 12 P 13

48 11.22 8.67 . . . 10.0 9.83 9.77 . . . 9.8

P 14 5.13 5.54 . . . 5.3

Little 24 P 31 16.84 16.08 . . . 16.5

P 101 32.01 15.45 . . . 23.7 Little 17 P 102 27.65 19.77 . . . 23.7

P 103 16.11 15.77 . . . 15.9

isolated from colonies on solid media. Accordingly normal or mutant colonies were selected from agar plates after 6 days of incubation and inoculated into 10 ml standard medium in a 100 ml Erlenmeyer flask. After a variable interval (see table 4) but before the stationary phase was reached, sub- tures of each strain were established in a second flask of the same medium for growth measurements. The initial optical density was adjusted to 50-200 units and changes determined

104 MORGAN HARRIS

over a period varying from 12 hours to 5 days. In order to compare the results obtained, mean generation times over successive intervals were calculated from the formula :

Time in hours log, O.D., - log, O.D.,

M.G.T. =

Table 4 summarizes these findings. It is strains established from normal colonies are

apparent that more rapid in

growth than any of the mutant strains, with little variability between individual normal strains studied. On the other hand, a wide spectrum in generation time is observed for the vegetative littles. A clear correlation appears to exist between the duration of the initial culture and the mean generation time as measured in the succeeding transfer. These facts suggest that growth begins at a very slow rate following the initial isolation of vegetative littles in the standard fluid medium. The phase of acceleration is greatly extended and a logarithmic growth phase is reached only after a considerable period in culture. The irregularity of acceleration between individual strains similarly incubated suggests the selection of rapidly-growing variants rather than adaptive changes in the population as a whole, but the existing data are not adequate for a decision on this point. It may also be pointed out that the growth rates of recently isolated mutants were more rapid when measured in a medium containing 0.5% yeast extract and 3% glucose rather than in the more complex standard medium used generally in the present experiments, but did not in any case approach the low generation times of normal cell strains.

A more general comparison of mutant and normal strains was also carried out using a single little, P,, stabilized by 5 transfers under aerobic conditions in the standard experimental medium, and the stock strain “Yeast Foam.’, An anaerobic P, culture was established in the Stier apparatus and after 7 days a growth curve was obtained for the mutant strain by withdrawing samples at intervals during a single growth cycle. Simultaneous measurements were carried out


on an aerobic P, culture. Cell counts and protein determinations were made in addition to measurements of optical density. The data obtained are shown in table 5 and can be compared with similar information derived for anaerobic and aerobic Yeast Foam cultures. These data show that even after repeated transfers in the standard medium the mutant strain has a higher generation time and lower maximal population than the normal strain of origin. Secondly, the results indicate that Yeast Foam can grow at the same rate aerobically and anaerobically, whereas a considerable decrease in growth rate of normal strains under anaerobic conditions was observed by Tavlitzki ( '49) and Slonimski ( ' 5 3 ) . It may be emphasized, however, that the medium used in the present investigation was markedly different from those of the earlier experiments mentioned, and contained in particular ergosterol and Tween 80, shown by Andreasen and Stier ( '53 ) to be growth factors for yeast under anaerobic conditions. Table 5 on the other hand indicates that the mutant P, grew more slowly under anaerobiosis. The metabolic studies listed suggest that aerobic cultures of vegetative littles may have a higher rate of fermentation than the corresponding anaerobic cultures although the data on this point are restricted to the stationary phase and do not cover an entire growth cycle.

Population. chawges in mix tures of rzormal and mutant strairzs

On the basis of the growth studies outlined above, two experiments were carried out to follow population changes in known mixtures of normal cells and vegetative littles. The first experiment was performed under anaerobic conditions in the Stier apparatus with an inoculum consisting of 90% P3 vegetative littles and 10% G, normal cells. Anaerobiosis was established as usual and samples withdrawn daily for plating to determine the proportion of littles in the population. Subsequent counts showed that the percentage of littles remained close to the initial level for two o r three days in

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M U T A N T S I N ANAEROBIC YEAST 107

culture and then declined rapidly. Figure 1 compares the observed population shifts with a curve of expected changes based on relative growth rates and maximal populations for the two strains respectively. The two curves are approximately parallel in their latter parts but differ with respect to the initial phase. The simplest interpretation of these facts is that population shifts in the mixture are governed by relative growth rates but normal cells do not grow at a maximal rate for the first two or three days in anaerobiosis.

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Fig. 1 Population changes in mixtures of G, normal cells and P, littles, under anaerobic culture.

The possibility remained, however, that growth of the P3 littles was stimulated by the presence of normal cells in the mixture, if only temporarily. An interaction of this type, if present, should be more clearly evident in mixtures with a higher disparity in growth rates of the two components. A second experiment was therefore carried out using a freshly isolated vegetative little instead of the P3 strain described above.

108 MORGAN HARRIS

10,00(

o p t i c o dens i t !

1,00(

10( 5 10 20 30 40 50 60 70

Hours of i n c u b a t i o n

Fig. 2 Aerobic growth rates of P,,, G, and mixture (19% P, : 21% G2).

TABLE 6

Population changes in mixtures of G, normal cells with P,, vegetative littles recently isolated f rom colony on agar plate

VEGETATIVE LITTLES NORMAL COLONIES

NO. % N O . 70 TYPE O F HOURS O F CULTURE INCUBATION E$h$::

Iiiitinl mixture 0 586 438 78.8 118 21.2 Aiinerobie 21 870 7 0.8 863 99.2 Aerobic 24 956 6 0.6 950 99.4


The mutant selected ( P31, table 4) was removed from an agar plate after 6 days of incubation, suspended in 10 ml standard medium, and incubated aerobically on a shaker for 24 hours. A culture of G2 normal cells was simultaneously inoculated from an agar slant and incubated similarly. From these cultures a mixture was prepared consisting of 79% P,, littles and 21% G, normal cells as determined subsequently by plate counts. An aliquot of the mixture was placed in the Stier apparatus and incubation begun immediately with a rapid flow of nitrogen. A second aliquot of the mixture was incubated aerobically, along with cultures of the two components in separate flasks. Changes in the aerobic cultures were followed by measurements of optical density at appropriate intervals and the growth curves obtained are shown in figure 2. The wide difference in growth rates of normal and mutant cells is evident, and the net growth rate of the mixture shows a rapid acceleration to parallel that of the G, normal culture. These facts suggest the essential elimination of added P,, littles in a single aerobic growth cycle, and the results from plating (table 6) verify this conclusion. While the growth curve of the anaerobic mixture was not followed in detail, the facts available indicate a similar disappearance of the P,, mutants in the first 24 hours of incubation. The limiting optical density observed at this time was typical of normal rather than mutant strains, and plate counts (table 6) con- firmed the absence of added littles in the cell population. The experiments described thus provide no evidence for growth stimulation of vegetative littles by normal cells present in the same population.

DISCUSSION

The significance of the data from mixture experiments is based on the assumption that the growth rates measured are typical of mutant and normal cells in natural populations. On technical grounds this might be questioned since in the present investigation mutants were isolated on a solid medium of simple composition, and could conceivably require more

110 MORGAN HARRIS

adaptation than normal cells when subsequently introduced into the complex standard medium for growth measurement. That this objection is not valid, however, is indicated by the original experiments of Ephrussi, Hottinguer and Chimenes ('49). In this investigation the solid and liquid media used were identical in composition except for the presence of agar. Nevertheless freshly isolated mutants grew more slowly than normal colonies and accelerated in subsequent transfers. Similar observations have been made by Prevost ('53).

It thus seems apparent that f o r the standard medium used in the present experiments, selection operates against vegetative littles and is active in anaerobic as well as aerobic conditions. Since the proportion of spontaneous mutants remains constant in normal strains cultivated anaerobically, it appears that the production of vegetative littles must con- tinue in the absence of oxygen to balance the observed process of selection. The fact that the incidence of vegetative littles is the same under both aerobic and anaerobic conditions (table 3) suggests that (1) selection in either case is essentially complete under the conditions described so that prac- tically all the littles present in these populations at any one time are recently produced, and (2) the mechanics of the mutation process are independent of the presence of oxygen.

A further point of interest stems from the observation that littles and normal cells do not necessarily grow at the same rate in anaerobiosis (table 5) ; whereas the previous data of Slonimski ('53) appeared to show that no difference exists under these conditions. The evolution of growth rates here observed provides a simple explanation for this dis- crepancy, for it is apparent that a similar growth rate for mutant and normal cells is only to be expected after many serial passages of the mutant strain. The vegetative little studied by Slonimski had in fact been stabilized in this way by over 450 transfers before the measurements in question were made. In any event the fact that recently isolated littles do not grow as rapidly as normal cells even anaerobically suggests that the mutation process involves a broader defi-


ciency than the respiratory changes previously described. The nature of the additional deficiency remains as an interesting future problem.

ACKNOWLEDGMENTS

The author wishes to thank Professor Boris Ephrussi f o r suggesting the present problem and for the many courtesies received as a guest in his laboratory. The daily counsel and assistance of Dr. Piotr Slonimski as well as that of Professor Ephrussi was indispensable to the progress of the investigation and is gratefully acknowledged.

SUMMARY

1. Cultures of Saccharonayces cerevisiae, strain Yeast Foam, mere established in the apparatus devised by Stier, Scalf, and Brockmann f o r the continuous cultivation of yeast under anaerobic conditions. I n these cultures the proportion of respiration-deficient mutants (vegetative littles) remains constant over several weeks, and does not differ from values observed in aerobic cultures.

2. Comparison of normal and mutant strains indicates that vegetative littles are characterized by a low initial growth rate in the standard experimental medium, accelerating over successive transfers but remaining well below the growth rate typical f o r normal strains of yeast.

3. Normal and mutant strains were used to construct synthetic populations and the subsequent changes were followed under both aerobic and anaerobic conditions. I n either case the percentage of vegetative littles declines to the spontaneous level, over a period proportional to the difference in growth rates of the two components. 4. The occurrence of selective processes in anaerobic cul-

tures implies the continued production of vegetative littles to account f o r the constant percentage of spontaneous mutants observed in these populations. I t is concluded that mutation of normal cells to vegetative littles is independent of the presence of oxygen under the conditions of the present study.

112 MORGAN HARRIS

LITERATURE CITED

ANDREASEN, A. A., AND T. J. B. STIER Anaerobic nutrition of Saccharo- myces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J. Cell. and Comp. Physiol., 4 1 : 23-36.

The interplay of heredity and environment in the synthesis of respiratory enzymes in yeast. The Harvey Lectures, Series XLVl:

1949 Action de l’aeriflavine sur les levures. VI. Analyse quantitative de la transformation des populations. Ann. Inst. Past., 77: 64-83.

1949 Action de l’acriflavine sur les levures. I. L a mutation “petite colonie.” Ann. Inst. Past.,

EPHRUSSI, B., AND P. P. SLONIMSKI 1950 La synthhse adaptative des cytochromes chez le levure de boulangerie. Biochim. et Biophys. Acta, 6:

1953

EPHRUSSI, B. 1952

45-67. EPHRUSSI, B., PH. L’HBRITIER AND H. HOTTINGUER

EPHRUSSI, B., H. HOTTINGUER, AND A. CHIMENES

7 6 : 351-367.

256-267. PREVOST, G. 1953 Unpublished observations. SCALF, R. E., AND T. J. B. STIER 1950 Effect of commercial malt sprouts on

the anaerobic growth of distillers ’ yeast. Transact. Kentucky Acad. Sci., 13: 69-77.

L a formation des enzymes respiratoires chez la levure. Actualities Biochimiques So. 17, Masson, Paris, 204 pp.

1956 Mdcanisme de l’adaptation respiratoire chez le levure. I. Con- ditions et cindtique de la synthhse induite de la cytochrome oxidase en absence d ’azote exoghe. ( I n preparation.)

STICKLAND, L. H. The determination of small quantities of bacteria by means of the biuret reaction. J. Gen. Microbiol., 5: 698-703.

STIER, T. J. S., R. E. SCALF AND M. C. BROCKMANN 1950 An all-glass apparatus for the continuous cultivation of yeast under anaerobic conditions. J. Bact., 59: 45-49.

TAVLITZICI, J. 1949 Action de l’acriflavine sur les levnres. 111. Etude de la croissance des mutants “petite colonie.” Ann. Inst. Past., 76: 497-509.

SLONIMSKI, P. P. 1953

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occurrence of respiration deficient mutants in baker's yeast cultivated anaerobically

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