JOURNAL OF BACTERIOLOGY, Jan. 1973, p. 203-211Copyright 0 1973 American Society for Microbiology

Vol. 113, No. 1Printed in U.S.A.

Effect of Growth Rate on the MacromolecularComposition of Prototheca zop ii, a Colorless

Alga Which Divides by Multiple FissionROBERT 0. POYTON'

Microbiology Group and Department of Botany, University of California, Berkeley, California 94720

Received for publication 29 June 1972

Prototheca zopfii, a eukaryote that divides by multiple fission, was investi-gated to determine how growth rate controls daughter cell number. Themacromolecular composition, cell size, and number of nuclei per cell were

determined in cultures during balanced growth in various media. Cellular mass,ribonucleic acid (RNA), deoxyribonucleic acid (DNA), carbohydrate, and nuclearnumber increased as positive linear functions of growth rate, whereas nuclearploidy remained constant with a value of 0.098 pg of DNA/nucleus. The ratios ofRNA to protein, protein to mass, and carbohydrate to mass were unaffected bygrowth rate, whereas the ratios of DNA to protein and RNA to DNA could beexpressed as curvilinear functions of growth rate, the former negative and thelatter positive. The dependency of normalized gene dosage (DNA/protein) on

growth rate appeared as a distinguishing feature of multiple fission. Determina-tion of the normalized rates of protein and RNA synthesis revealed that bothincrease linearly with growth rate. It is concluded that Prototheca zopfii mayexist in a number of physiological states which are characterized by a unique sizeand macromolecular composition and which are dictated by growth rate.

Since the classic work of Schaechter, Maaloe,and Kjeldgaard (29) showing the dependency ofthe macromolecular composition of Salmonellatyphimurium on growth rate, much study hasbeen devoted to the regulation of macromolecu-lar synthesis in bacteria (18). Similar studieswith eukaryotes have been slow in coming,probably because of the structural complexityand demanding growth requirements of thesecell types. Investigations with three eukaryoticmicroorganisms, Saccharomyces cerevisiae (19,35), Tetrahymena pyriformis (14), and Euglenagracilis (7), have confirmed in a general way thedependency, noted for several bacteria (23, 24,28, 29), of the relative levels of deoxyribonucleicacid (DNA), ribonucleic acid (RNA), and pro-tein on growth rate. Together, these studiesindicate that both prokaryotes and eukaryotesmay exist in one of a continuum of steady stateswhich are dictated by the growth rate. Thepresent study confirms the existence of thiscontinuum in yet another eukaryotic microor-ganism, the colorless alga Prototheca zopfii.

Prototheca differs from the organisms stud-'Present address: Section of Biochemistry and Molecular

Biology, Comell University, Ithaca, N.Y. 14850.

ied previously in its growth and division bymultiple fission. (Multiple fission is defined asthe complete cleavage of a multinucleate cellinto uninucleate daughters after a cell cycle inwhich there were two or more nuclear divisionswithout concomitant cell divisions.) In an ear-lier study (26), it was shown that the meandaughter cell number produced by Protothecaincreases with growth rate. It was suggestedthat this dependency of daughter cell numberon growth rate resulted from the existence ofdifferent physiological states at differentgrowth rates. The present study was initiated todefine these physiological states and investi-gate possible ways in which daughter cell num-ber may be controlled by growth rate.

MATERIALS AND METHODSCell strain and culture media. P. zopfii 68-16A,

kindly provided by C. B. van Niel, was maintained asdescribed previously (25). All media were prepared ina mineral base containing: K2HPO4, 5.3 g; KH2PO4,1.95 g; MgS04.7H20, 460 mg; CaCl2 .2H20, 70 mg;disodium ethylenediaminetetraacetic acid, 3.2 mg;FeSO4.7H20, 2.5 mg; ZnSO4 7H20, 0.5 mg;MnSO4.H20, 0.5 mg; CuSO4, 0.1 mg; Co(NO0)2.6H20, 0.1 mg; Na2B407.10H20, 0.1 mg; Na2MoO4.

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2H20, 2.0 mg; and distilled water to a final volume of1.0 liter. The mineral base was prepared and auto-claved as two solutions: solution A contained thephosphate buffer and solution B contained all othersalts. Just prior to use, both solutions were mixedaseptically, and thiamine-HCl, sterilized by mem-brane filtration, and Tween 80, sterilized by auto-claving, were added to final concentrations of 200ug/liter and 0.02% (v/v), respectively. The carbon andnitrogen sources (Table 1) which had been sterilizedseparately, were added aseptically to the sterilemineral base, and the pH was adjusted to 7.0.Growth conditions. Cultures were grown in the

dark at 25 C in Erlenmeyer flasks, with side armswhich fit a Klett-Summerson colorimeter, and wereaerated on a rotary shaker (250 rev/min).

Balanced growth was achieved by growing the cellpopulation through 15 to 20 mass doubling times at alow cell density, maintained by periodic dilution(1: 10) with fresh growth medium. Cultures weresampled periodically for determination of turbidityand number. Culture turbidity was not allowed toexceed a Klett value of 50, which corresponds to a dryweight concentration of 170 jg/ml and a cell concen-tration of 0.4 x 10 to 2.1 x 10 cells/ml. Growth in allof the media used is exponential until the turbidityreaches a value of 220 Klett units or more. Underthese growth conditions, cultures are in balancedgrowth (26). Cell mass and number increase is paral-lel, and there is no lag in number or mass increase ondilution into fresh growth medium. The specificgrowth rate, k, was determined from the equation:log. X = log X. + kt by regression analysis asdescribed previously (26).

Determination of number, mass, and volume.Relative cell numbers used in the assessment ofgrowth rate were determined electronically with aCoulter counter model B (Coulter Electronics, Inc.)fitted with a 50-Mm aperture. Samples (2 ml) werewithdrawn from each culture, chilled to 0 to 4 C, andcounted within 2 hr. Prior to counting, cell clumps ineach sample were dispersed by sonic treatment for 5sec with a Branson Sonifier (W 185 D), equipped witha microtip, at an output of 20 w and a frequency of 20kc/sec. Samples were prepared for counting by dilut-ing to 104 to 4 x 104 cells/ml in the basal salts me-dium containing 0.02% (v/v) Tween 80. Absolute num-bers were taken from the average of four countsdetermined with a hemocytometer. Mass increasewas followed turbidimetrically on a Klett-Summer-son colorimeter with a blue (ca. 420 nm) filter. Stan-dard curves relating dry weight to Klett units wereestablished for different growth media, and turbiditydeterminations were taken on the linear portion ofthese curves. For dry weight determinations, cultureswere filtered through a membrane filter (0.45 ,m poresize; Millipore Corp.), washed twice with distilledwater, and dried at 60 C to a constant weight. Cellvolume spectra were determined with a Coultercounter model B equipped with a 50-Mgm aperture anda Size Distribution Plotter model J. Paper mulberrypollen, which was fixed for 3 hr in acid alcohol(concentrated HCl-95% ethanol, 1:1, v/v), was usedfor volume calibration. The mean pollen size (816.5

om8) used for volume calibration was determinedfrom measurements of 650 pollen grains with a lightmicroscope. The volume distribution generated fromdirect microscopic measurement was compared tothat generated with the Coulter counter by the X2 testas described by Harvey and Marr (10). The probabil-ity that the value of x2 obtained for the differencebetween the two distributions was due to chance was0.078.Number staining. Nuclei were stained with Azure

A as described by Levine and Folsome (16). All stepswere performed in a centrifuge tube. Cells harvestedfrom 5 ml of culture were fixed in Schaudinn'sfixative (saturated aqueous mercuric chloride-95%ethanol-glacial acetic acid, 66:33:5) for 60 to 90 min.After fixation, cells were hydrated by brief passage (2min in each solution) through 70, 50, and 30% ethanolinto water. Cells were rinsed twice with distilledwater and hydrolyzed in 1 N HCl at 60 C for 6 min.After hydrolysis, cells were stained for 60 min withfreshly prepared 1% Azure A (National Aniline Divi-sion, Allied Chemical Corp., New York, N.Y.) con-taining 0.15% (v/v) thionyl chloride. They were thenrinsed four times with tap water and dehydrated bypassage through 30, 50, 70, 90, 95, and 100% ethanol.Cells were cleared ovemight in xylene and "mountedin picolyte. Nuclear number per cell was determinedfor at least 400 cells per growth condition by use of aZeiss GFL 658 light microscope equipped with a 40xachromatic (Ph 40/0.65) objective. Nuclear numberusually fell into the 2n series. Numbers which did notfall into the 2" series were scored in the next highest2n class under the assumption that lower numbersresulted from the inability to resolve distinct nuclearbodies.

Nucleic acid, protein, and carbohydrate deter-mination. After at least 15 doubling times of growthat low cell density, cultures of about 250 to 350 mg(wet weight) of cells were chilled to 4 C, harvested bycentrifugation for 10 min at 2,000 x g, and washedtwice with distilled water. Nucleic acids were ex-tracted by the Schneider procedure (30) as modifiedby Hutchison and Munro (11) and Munro and Fleck(22). The washed cell pellet was resuspended in 0.2 Nperchloric acid (PCA) and incubated at 0 to 4 C for 30min to remove cold acid-soluble material. The PCA-insoluble material was collected by centrifugation at25,000 x g for 5 min, washed once with 0.5 N PCA,and resuspended in 0.5 N PCA. It was hydrolyzed byheating at 70 C for 20 min. The hydrolysate wascooled to 0 C and centrifuged at 25,000 x g for 5 min;the supernatant fluid was retained. The cell pelletwas reextracted with 0.5 N PCA for 20 min at 70 C,and the two supematant fractions were pooled. ThePCA-insoluble residue was treated with 1 N NaOH at100 C for 10 min to solubilize protein (17). The DNAin the hot PCA extract was measured by the di-phenylamine reagent as described by Burton (4), withcalf thymus DNA (type 1; Sigma Chemical Co.) asstandard. The RNA in the hot acid extracts wasdetermined by measuring absorption at 260 nm (1optical density unit at 260 nm = 29.4 Mg of RNA/ml).Absorption values were corrected for polypeptidematerial present in the extract as described by Barker

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and Hollinshead (1). Protein was determined in thealkaline and hot acid extracts by the method of Lowryet al. (17). Crystalline bovine serum albumin servedas the standard. Carbohydrate was determined withthe anthrone reagent by the method of Roe (27), withglucose as standard.

RESULTS

Macromolecular composition at differentgrowth rates. The average mass and mac-romolecular composition of cells growing over a

2.6-fold range in specific growth rate are pre-

sented in Table 1. Under our growth conditionscarbohydrate accounts for most of the cell's dryweight, with values ranging from 59 to 70%(Table 3). This carbohydrate is present in cellwall material and storage products-presuma-bly glycogen (2)-which appear as highly refrac-tile iodine-positive granules in the cytoplasm.An estimate of how much of the carbohydrate ispresent in wall material was obtained by deter-mining the carbohydrate content of cells whichhad been starved for a carbon source (in 0.01 Msodium phosphate, pH 7.0) for 24 hr aftergrowth to mid-exponential phase in two repre-

sentative media, media 8 and 17. After 24 hr ofstarvation, the cytoplasm was completely de-void of the iodine-positive refractile granules,and carbohydrate accounted for 38 and 30% ofthe dry weight of cells grown on media 8 and 17,respectively. Therefore, it is concluded thatunder the growth conditions employed in thesestudies 50 to 60% of the total carbohydrate ispresent as cytoplasmic storage products and

the rest is present in wall material.The effect of growth rate on cell mass and

protein content is shown in Fig. 1A. Both cellmass and protein content increased linearlywith growth rate, with slopes of 25.4 and 39.0,respectively. These parameters are consideredmore reliable than volume as estimates of cell"size" for many eukaryotic cells because of theexistence in these cells of vacuoles which maytake up a large and variable portion of thecellular volume (21). Since no such vacuoleshave been observed in Prototheca either byphase-contrast microscopy (Poyton, unpub-lished data) or electron microscopy (34), volumemeasurements should reveal the same depend-ency of cell size on growth rate. Cell volumespectra for cells growing on three representativegrowth media are shown in Fig. 2. It can be seen

that mean cell volumes, as well as volumes ofthe largest cells (mothers) and smallest cells(daughters), increased with growth rate. Cellu-lar RNA and DNA contents also increasedlinearly with growth rate (Fig. 1B and C), withslopes of 100 and 13, respectively.

It is noteworthy that the linear functionsdescribing the dependency of each of the abovecellular constituents on growth rate cannot beextrapolated to positive values at a specificgrowth rate of zero. Since nongrowing cellsobviously cannot have negative values for theseconstituents, and since the values are bestfitted by a straight line, it is likely that thefunctions describing each of these parametersare biphasic, with small (possibly zero) slopes at

TABLE 1. Effect of growth rate on cell mass and compositiona

SpecificMas/Poen/Crhygrowth Medium Co .p.' Masse! Protein! Carbohy-a | RNA/cell DNA/cell Nuclei/rate, k no. nComositiongcell cell drate/cell (pg) (pg) cell(hr- ) (g n) (g

0.086 6 Ethanol, adenine .082 .010 .056 2.20 .117 -

0.097 5 Glucose, lysine .108 .013 .076 3.61 .134 1.360.098 8 Glycerol, adenine .078 .011 .052 2.77 .125 1.250.102 9 Glycerol, threonine .070 .012 .046 4.01 .117 -

0.109 10 Glucose, adenine .134 .021 .090 6.00 .157 1.650.118 11 Glucose, threonine .100 .026 .060 6.04 .150 -

0.138 12 Glycerol, NH4Cl .115 .021 .075 7.40 .206 2.110.154 13 Ethanol, NH4Cl .211 .040 .136 8.70 .192 -

0.162 14 Glucose, NH4Cl .231 .041 .151 12.01 .239 2.230.187 15 Propionate, NH4Cl .271 .049 .172 12.32 .240 -

0.210 25 Glucose, yeast extract, .370 .065 .218 13.54 .282 2.55NH4Cl

0.223 17 Glucose peptone, .410 .055 .255 15.61 .271 2.83NH4Cl

a Each value represents the mean of duplicate samples taken from two different cultures.° The concentrations of carbon and nitrogen sources are the same as those given in reference 26.c Mass expressed as dry weight.d Combined value of protein in hot acid and alkaline extracts.

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i Af j~~~~~* 00

Sp ific gowth rt (hrs")

B

D~~~~~~~~~~D-5

5~~~~~w 2

Specific growth rots (hrs-)

-W%M an Qsspsclflc g_ubf M (h-rs)

FIG. 1. Dependency of cellular mass, protein,RNA, DNA, and the number of nuclei per cell ongrowth rate. Specific growth rate was computed asdescribed previously (26). Each point represents theaverage of duplicate determinations on two separatecultures which had been maintained at a low celldensity (below 170 Ag of dry weight/mI) for at least 15mass doubling times.

low specific growth rates (less than 0.086 hr- l).Increase in nuclear number with growth

rate. The average number of nuclei per cellincreased linearly with growth rate (Fig. 1C)and with a slope (12.6) identical to that forDNA per cell. This increase in nuclear numberis due solely to an increase in the percentage ofmultinucleate mother cells in the population,since the uninucleate cell class, which presuma-bly consists of daughter cells, is most frequentat all growth rates (Table 2). An increase in thepercentage of multinucleate cells in the popula-tion reflects the increase in daughter cell num-ber with growth rate, as reported previously(26).Constancy of ploidy at all growth rates.

The parallel increase in DNA per cell andnumber of nuclei per cell in Fig. 1C indicatesthat the chromosomal ploidy does not vary withgrowth rate and allows a crude estimate of thegenome size of Prototheca. Our values for DNAper nucleus (Table 3) range from 0.090 to 0.105pg, with a mean value of 0.098 pg. This value isprobably a little high, because it fails to accountfor the presence of nuclei in the G2 phase of themitotic cycle, i.e., nuclei which had replicatedtheir DNA but which had not divided, in ourstained preparations. This value, however, is ingood agreement with values of 0.123 and 0.114pg reported for Chlamydomonas (5) andChlorella (33), close relatives of Prototheca.Effect of growth rate on the ratios of

macromolecular constituents. The ratios ofmacromolecular components in cells grown atdifferent growth rates are summarized in Table3. As the growth rate increased, there was aslight increase in the protein to mass ratio and aslight decrease in the carbohydrate to massratio. These changes probably result from adecrease in cellular surface to volume ratio withincreasing growth rate. The larger cells pro-duced by the faster growth rates would beexpected to have lower ratios of cell wall tocytoplasm than the smaller cells produced atslower growth rates. Variation in the ratios ofRNA to DNA and DNA to protein are shown inFig. 3. The dependencies of both ratios ongrowth rate are best represented as curvilinearfunctions.Normalized rates of RNA and protein

synthesis. The relationships RNA/DNA xgrowth rate (,u) and protein/RNA x uhave beenproposed to estimate the net rate of RNAsynthesis per DNA template (20) and the netrate of protein synthesis per unit weight ofRNA(13) in Escherichia coli. It can be seen from Fig.4 that both of these rates (with specific growthrate used in place of growth rate) for Prototheca

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VOL. 113, 1973 MACROMOLECULAR COMPOSITION OF PROTOTHECA ZOPFII

Ina)EI

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400 800 1200 1600 2000

Volume (ji3)FIG. 2. Frequency distributions of cell volume for cells growing at three different growth rates. The culture

growing at 0.098 hr-1 (dashed line) was grown in medium 8 (Table 1), the culture growing at 0.138 hr-1 (solidline) in medium 12, and the culture growing at 0.162 hr-1 in medium 14. For sizing, cells were diluted with themineral basal medium to a final concentration of 0.5 x 104 to 1.0 x 104 cells/mi.

TABLE 2. Distributions of number of nuclei per cell

Specifc Medium Nuclei/cell

thr ') no 1 2 4 8 16 32

.097 5 409 28 26 9 0 0 1.36

.098 8 443 31 17 6 0 0 1.25

.109 10 495 77 38 14 8 0 1.65

.138 12 450 100 54 50 8 0 2.11

.162 14 496 105 63 62 12 0 2.23

.210 25 440 73 65 76 16 0 2.55

.223 17 703 98 100 154 33 1 2.83

increased linearly with growth rate, the normal-ized rate of RNA synthesis with a slope of 62.4and the normalized rate of protein synthesiswith a slope of 3.6.

DISCUSSIONThe results presented above demonstrate

that there exists for P. zopfii a continuum ofphysiological states which are determined bygrowth rate and which are characterized byunique cell size and relative level of RNA,

DNA, and protein. Compared with the growthrate itself, the composition of the culture me-dium which supports each growth rate is oflittle consequence in determining levels of totalcellular RNA, DNA, and protein.

Differences in effect of growth rate onPrototheca and other microorganisms.When total protein and RNA contents areexpressed relative to total DNA, the proteincontent per genome for the bacteria E. coli,Aerobacter aerogenes, and S. typhimurium (24,28), the yeast S. cerevisiae (20), the protozoanT. pyriformis (14), and the alga E. gracilis (7) isnearly constant at all growth rates, whereas theratios of RNA to protein and RNA to DNA arepositive linear functions of growth rate. Theconstancy of DNA to protein at all growth ratesis one of the most intriguing relationships toemerge from these studies on cells which divideby binary fission. Along with similar findingsfrom studies on the effects of ploidy on cell"size" (6, 8, 9, 31), it suggests that the ratio ofDNA to protein for each organism is relativelyconstant and not subject to physiological con-trol or ploidy level. This is clearly not the case

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TABLE 3. Ratios between protein, carbohydrate, RNA, DNA, and mass for different growth rates

Specific growth Medium Protein/ Carbohydrate/ DNA/ DNA/ RNA/rate, k (hr-') no. massa massa protein nucleus protein RNA/DNA(pg)

0.086 6 0.121 0.681 .012 0.22 18.800.097 5 0.115 0.700 .011 .097 0.29 26.940.098 8 0.141 0.662 .011 .100 0.25 22.160.102 9 0.171 0.653 .010 0.33 34.270.109 10 0.156 0.670 .008 .105 0.29 38.220.118 11 0.260 0.601 .006 0.23 40.270.138 12 0.182 0.649 .010 .098 0.35 35.920.154 13 0.187 0.646 .005 0.22 45.310.162 14 0.177 0.652 .006 .095 0.29 50.250.187 15 0.180 0.633 .005 0.25 51.330.210 25 0.174 0.591 .004 .090 0.21 48.010.223 17 0.134 0.621 .005 .104 0.28 57.60

a Mass expressed as dry weight.

for Prototheca, an organism which divides bymultiple fission.Despite similarities in the physiological con-

trol of macromolecular synthesis in these previ-ously studied microorganisms, a feature whichdistinguishes the prokaryotes from the eu-karyotes is the dependency of cellular massand protein on growth rate for the prokaryotesand the lack of such a dependency for the eu-karyotes. The "size" of Prototheca, as deter-mined from measurements of protein, dryweight and volume, is clearly dependent ongrowth rate. Prototheca is, therefore, the firstreported case of a eukaryote which behaveslike a prokaryote in this regard. It is importantto note here that the constancy of cell "size"(i.e., protein or mass per cell) cannot be in-ferred from cell volume measurements formany eukaryotes, such as yeast (19, 21), sinceany change in volume with growth rate may re-flect changes in intracellular vacuolar volumesand water contents, quite apart from changesin cellular mass or protein. The constancy ofcell "size" of Saccharomyces, Tetrahymena,and Euglena growing at different rates may beof important consequence to these cells, be-cause it makes it unnecessary for them to in-crease their ploidy or to become multinucleatein order to maintain a constant DNA to proteinratio at all growth rates. Apparently, the main-tenance of the uninucleate or haploid conditionis not as important for bacteria as it is forthese eukaryotic microorganisms, because withincreasing growth rate bacterial cells may be-come bi- and even tetranucleate (18). By sodoing, they are able to maintain a constantDNA to protein ratio even with a changing cell"size." Since there is a dissociation of nuclearand cytoplasmic division in multiple fission

(26), it might have been expected that Proto-theca cells could increase in "size" withgrowth rate and still maintain a constant DNAto protein ratio in a manner analogous to bac-teria, that is, by producing multinucleate orpolyploid daughter cells. This is clearly not thecase. Prototheca produces uninucleate daugh-ters (Table 2) and maintains a constant ploidyat all growth rates (Table 3). However, Proto-theca does increase the number of nuclei permother cell, and hence the number of daughtercells, with increasing growth rate (26). Thus, bymaking use of a somewhat novel form of celldivision, Prototheca behaves like the pre-viously studied eukaryotic cells, insofar as pre-serving a constant nuclear number and ploidyin its daughters at all growth rates.Similarities in growth rate effects on

Prototheca and other microorganisms.There is remarkable similarity betweenPrototheca and other eukaryotic microorga-nisms as regards the relative levels of mac-romolecular constituents and their response togrowth rate. For example, the magnitudes ofthe ratios RNA to DNA, DNA to protein, andRNA to protein in Prototheca, Saccharomyces(19), and Tetrahymena (14) are nearly identical.In addition, values for the ratio of DNA toprotein in each growing at its maximal growthrate fall almost exactly on a line relatingmaximal growth rate to this ratio for a largenumber of eukaryotic and prokaryotic microor-ganisms (15). Additional similarities betweenPrototheca and other microorganisms may befound in the normalized rates of piotein andRNA synthesis. Although early studies withbacterial cells growing in a state of balancedgrowth (13) or adjusting to a "shift-up" (12)suggested that bacterial ribosomes function

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FIG. 3. Dependency of ratios of DNA to protein and RNA to DNA on growth rate.

near maximal efficiency, as reflected by the rateof protein synthesis, at all growth rates, laterwork by Rosset, Julien, and Monier (28) re-vealed that the efficiency of ribosomes in pro-tein synthesis actually diminishes with decreas-ing growth rate. These studies have employedthe relationship protein/RNA x ,u in the assess-ment of ribosomal efficiency. It has been shownabove that protein/RNA x k increases withgrowth rate. Since ,u, growth rate, is related to k,specific growth rate, by a constant, protein/RNA x it also must increase with growth rate. Ifit is assumed that ribosomal RNA in Protothecarepresents a major portion of total RNA whichis nearly invariant with growth rate, as in

Saccharomyces (32), Tetrahymena (14) andbacteria (28), then we can conclude that riboso-mal efficiency in this eukaryote responds togrowth rate in the same manner as in prokary-otes. Although the normalized rates of proteinsynthesis in other eukaryotic microorganismshave not been published, a conclusion similar tothat arrived at here for Prothoteca can bereached for Tetrahymena by using the data ofLeick (14) in the above computation.The normalized rate of RNA synthesis, RNA/

DNA x (specific) growth rate, increases withgrowth rate in Prototheca as it does in E. coli(20). When our data are expressed in micro-grams of RNA per microgram of DNA per

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protein as functions of growth rate.

second, they have values ranging from 4.5 x10-4 to 35.8 x 10-4 at specific growth ratesranging from 0.086 to 0.223 hr- '. These valuesand their dependency on growth compare favor-ably with the values of 3.0 x 10- 4to 24 x 10-4reported for E. coli (20) growing at specifi'cgrowth rates of 0.229 to 0.744 hr- 1. The fact thatthe net rates of RNA synthesis in E. coli, aprokaryote, and Prototheca, a eukaryote, are

nearly identical implies that, despite differ-ences in the structural organization of thechromosomes in these two cell types, the effi-ciencies of their DNA templates in RNA syn-thesis are similar. Recently, the same conclu-sion has been reached for Euglena, another eu-

karyote, from measurements of the chaingrowth rate of ribosomal RNA (3).Variable gene dosage as a distinguishing

feature of multiple fission. In pursuing theabove investigation, correlations were soughtbetween the effect of growth rate on the mac-romolecular composition of cells and the effectof growth rate on daughter cell number (26) inan effort to explain how an increasing growthrate is able to support an increasing mean

daughter cell number in multiple fission. Indoing so, it has been necessary to comparechanges in each physiological parameter withchanges in similar parameters for previouslystudied microorganisms which divide by binaryfission. From the above discussion, it is clearthat Prototheca cells respond to growth rate inmuch the same way as other eukaryotic andprokaryotic microorganisms. Prototheca does,however, differ from these other microorga-nisms in certain respects. It differs from othereukaryotic microorganisms in its failure tomaintain a constant "size" (i.e., protein or massper cell) at all growth rates. It differs fromprokaryotic microorganisms in its failure toproduce multinucleate daughter cells at rapidgrowth rates. And it differs from all previouslystudied microorganisms, which divide by bi-nary fission, in its failure to maintain a con-stant ratio of DNA to protein at all growthrates. This dependency of the ratio of DNA toprotein (normalized gene dosage) on growthrate appears to be the only distinguishingfeature of multiple fission. It is clearly anexception to what has previously been consid-ered a biological rule (6, 8, 9), albeit derivedonly from cells dividing by binary fission. Assuch, it may hold some interesting answers toquestions relating to basic differences betweenthe division mechanisms in cells dividing bybinary and multiple fission and to the means bywhich growth rate governs daughter cell num-ber in Prototheca.

ACKNOWLEDGMENTSI thank D. Branton for helpful suggestions during the

course of this work and assistance in preparing the manu-script. The excellent technical assistance of E. Crump wasgreatly appreciated.

This work was supported by National Science Foundationresearch grant GB 32024 to D. Branton, by an institutionalaward from the American Cancer Society, and by a predoc-toral fellowship from the National Institutes of Health (5 FO1GM43672-02).

LITERATURE CITED

1. Barker, G. R., and J. A. Hollinshead. 1964. Nucleotidemetabolism in germinating seeds. Biochem. J.93:78-83.

2. Barker, H. A. 1935. The metabolism of the colorless alga,Prototheca zopfii Kruger. J. Cell. Comp. Physiol.7:73-93.

3. Brown, R. D. and R. Haselkorn. 1971. Synthesis andmaturation of cytoplasmic ribosomal RNA in Euglenagracilis. J. Mol. Biol. 59:491-503.

4. Burton, K. 1956. A study of the conditions and mech-anisms of the diphenylamine reaction for the colori-metric estimation of deoxyribonucleic acid. Biochem.J. 62:315-323.

5. Chiang, K. S., and N. Sueoka. 1967. Replication ofchromosomal and cytoplasmic DNA during mitosisand meiosis in the eucaryote Chlamydomonas rein-hardi. J. Cell. Physiol. Suppl. 1 70:89-112.

6. Commoner, B. 1964. Roles of deoxyribonucleic acid in

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