56 BOTANY: RUESINK AND THIMANN PROC. N. A. S.

The mitochondrial jamming upstream from an obstruction readily accounts forthe accumulation of succinic acid dehydrogenase proximally to a nerve constric-tion,'° as that enzyme is intimately associated with mitochondria. It now seemsfeasible to harvest the solid mass of mitochondria collecting at the blind end of aproximal nerve stump (Fig. 7) for in vitro studies of live mitochondria.

* A preliminary communication was presented at the Annual Meeting of the National Academyof Sciences on April 25, 1962. Investigations supported by grant no. CA-6375 from the NationalCancer Institute (USPHS).

t New address: Dean, Graduate School of Biomedical Sciences, University of Texas atHouston.

1 Weiss, P., and H. B. Hiscoe, J. Exptl. Zool., 107, 315-395 (1948).2 Weiss, P., "The concept of perpetual neuronal growth and proximo-distal substance convec-

tion," in Regional Neurochemistry, ed. S. S. Kety and J. Elkes (Oxford: Pergamon Press, 1961),pp .220-242.

3Weiss, P., "Self-renewal and proximo-distal convection in nerve fibers," in Symposium on theEffect of Use and Disuse on Neuromuscular Functions. ed. E. Gutmann (Prague: CzechoslovakAcademy of Sciences, 1963).

4Causey, G., J. Anat., 82, 262-270 (1948).5Weiss, P., Anat. Record, 86, 491-522 (1943).6 Palay, S. L., and G. E. Palade, J. Biophys. Biochem. Cytol., 1, 69-88 (1955).7Webster, H. deF., J. Cell Biol., 12, 361-377 (1962).8 Taylor, A. C., J. Cell Biol., 15, 201-209 (1962).9 Pomerat, C. M., untitled film strip, courtesy of Pasadena Foundation for MLedical Research,

Pasadena, Cal.10 Friede, R. L., Exptl. Neurol., 1, 441 (1959).

PROTOPLASTS FROM THE AVENA COLEOPTILE

BY A. W. RUESINK* AND K. V. THIMANNtBIOLOGICAL LABORATORIES, HARVARD UNIVERSITY

Communicated May 21, 1965

Living protoplasts produced by removal of cell walls from higher plant tissueoffer good experimental material for attacking a number of long-standing problemssuch as water balance, wall formation, ion transport, and membrane structure.Until recently, such preparations could be obtained only by the laborious, low-yield-ing procedure of tearing apart strongly plasmolyzed tissue, usually from onion," 2or by using large, degenerating cells from ripe fruit which are losing their wallsnaturally.3 4 Cocking (1960)5 succeeded in preparing protoplasts from tomatoroot tissue by digestion with cellulase from Myrothecium verrucaria. In 1962 hereported that these protoplasts burst rapidly in 10-7 mg/liter indoleacetic acid(IAA) .6 The present paper describes the preparation, by a similar digestion tech-nique, of protoplasts from the Avena coleoptile, a tissue whose quantitative responseto IAA has been studied intensively and is much better understood. These cole-optile protoplasts appear to be in excellent physiological condition, and their be-havior on exposure to detergents and to various hydrolytic enzymes has been ob-served with a view to determining what macromolecules are essential to protoplast

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integrity. Some preliminary observations have also been made of their reaction toIAA.

Preparation of Enzyme.-Since commercial sources of cellulase were found much too weak todissolve native plant cellulose, the following preparation was adapted from Whitaker et al.7 Cul-tures of Myrothecium verrucaria 460, obtained from the Quartermaster Labs, Natick, Mass., weregrown on potato dextrose agar slants8 until spore production followed good mycelial growth,usually about 8 days. Sterile distilled water was added, the mycelium broken up, and the contentspoured into 500 ml of Whitaker's salt9 medium containing 0.5 gm glucose and 5 gm cellulose(Munktell's Cellulose Powder, Grycksbo Pappersbruk, Grycksbo, Sweden). Vigorous reciprocalshaking at 220C for 14 days resulted in nearly maximal amounts of cellulase in the medium. Afterfiltration, the solution was evaporated to 40 ml under reduced pressure at 430C in 2 hr. Thisevaporation caused no loss in the cellulose activity as measured by the viscosity change in carboxy-methylcellulose7 (Table 1). Cellulase was precipitated by (NH4)2S04 at 20C, and the fractioncoming out at 35-70% of saturation was redissolved in 1-2 ml of distilled water (the smallestamount needed to get complete solution), and desalted with a 1.3 X 11-cm column of SephadexG-25 at 20C, using 0.1% NaCl to maintain ionic strength and prevent binding. The cellulaseactivity passed rapidly through the column in the front-running brown-pigmented band, ions andcertain pigments being retained. Three 1-ml fractions were usually collected; these were frozenand retained activity for as long as 6 months through repeated freezing and thawing. Table 1shows that virtually all of the initial activity reached this eluate.Procedure.-Husked seeds of Avena sativa, var. "Victory," were soaked for 2 hr, planted on wet

paper towels, and exposed to red light for 24 hr. They were then kept in the dark at 250C withintermittent dim red light until the coleoptiles were 25 mm long-about 70 hr after planting.To improve the penetration of cellulase, the epidermal cells were removed from each coleoptile in4-6 clean strips, peeling from the base to the tip with fine jeweler's forceps under dim red light.A 5-mm subapical section was then cut from the remaining tissue and the primary leaf removed.Two such sections were cut into l-mm2 pieces and placed in an enzyme-mannitol mixture consist-ing of 50 X of the enzyme solution prepared as above and 50 X of 1.0 M mannitol. In most casesno buffer was added, although 0.025 M NaH2PO4 at pH 6.5 was suitable when buffering was de-sired. After an hour in the dark at 250C, the tube was agitated gently to improve digestion.This and all subsequent manipulations were performed in dim green light. Digestion was termi-nated at 1-2 hr by adding 2 ml of 0.5 M man-nitol. After 10 min of allowing the protoplasts TABLE 1to settle, the solution was pipetted off the top to SEPARATION OF CELLULASE ACTIVITY FROMabout 0.1 ml, and the protoplasts were washed FUNGAL FILTRATEa second time in the same way. Specificactivity TotalTo determine that a given cell was releasing a (units/ml) activity

single protoplast and that all sizes of cells in the 480 ml filtrate 79 38,000coleoptile sections were producing protoplasts, 40 ml concentrate 1,000 40,000four peeled sections were split and half of each 0-35%S 2,000was plasmolyzed in 0.5M mannitol, the other half 35-70% 20,000 30,000being used to prepare protoplasts as above. Di- Supernatant 3,000ameters of 180 protoplasts were measured and the INlost active 1.0 ml ofvolumes computed by assuming the protoplasts 3 ml Sephadex eluate 13,000 13,000to be perfect spheres. At the same time, the Units are arbitrary and based on a viscometriclengths and diametersof the plasmolyzed contentsof 114 cells of the subepidermal layer from the other halves were measured and their volumescomputed by assuming the cells to be cylinders with slightly rounded ends. The length of eachcell wall was also measured so that the osmotic potential of the cells involved could be computedby the plasmometric method. loFor determinations of survival, 50-400 protoplasts were transferred with a 10-X pipette to flat-

bottomed depression slides, prepared by pressing 2-cm rings of parafilm four layers thick ontowarmed microscope slides. A thin ring of silicone grease was applied to the slide just inside thering to prevent liquid from being drawn under the parafilm by capillarity. After adding cells andcovering with a coverslip, the number of good protoplasts was determined by systematically scan-

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58 BOTANY: RUESINK AND THIMANN PROC. N. A. S.

ning the whole depression under phase contrast (150 X), scoring those showing spherical shapeand a smooth surface. A green interference filter was used on the microscope lamp. After deter-mining the initial number, 10 X of test solution was added carefully to the slide, and the protoplastswere counted at intervals up to 2 hr. Besides the above scoring, in some experiments a repre-sentative 25-50 protoplasts were inspected for organized streaming of cytoplasm at 300 X magni-fication. When it was desired to lower the osmotic potential of the solutions, 10 X of 0.2 M man-nitol was added to the solution on the slide to give a final concentration of 0.4 M.The effects of the following enzymes on protoplast survival were determined: trypsin-Wor-

thington, lyophilized, salt-free, crystalline; pronase-Calbiochem, B grade; wheat germ lipaseWorthington; pancreatic lipase-Worthington PLII; bacterial phospholipase C-WorthingtonPHL-C; cabbage phospholipase D-Koch-Light Laboratories; pancreatic ribonuclease-A-Sigma, 5 X recrystallized, type 1-A.

Formation of Protoplasts.-The cylindrical coleoptile cells gave rise without excep-tion to spherical nonrigid protoplasts (see Fig. 1). All contained either one largevacuole or a number of smaller ones, surrounded by cytoplasm which appeared quiteviscous with little apparent Brownian motion. In the viable protoplasts, vigorouscyclosis was usually apparent either as sheets of cytoplasm flowing around a largecentral vacuole or as narrow streams of cytoplasm slipping through the partitionsbetween smaller vacuoles. Large plastids were common and phase-dense granulesthe size of mitochondria were universally present. Some preparations included afew naked tonloplasts, easily distinguished by their lack of phase-dense cytoplasm.Figure 2 shows the relative distribution of the volumes of cytoplasm of plasmolyzedcells and of isolated protoplasts in 0.5 M mannitol. Note that protoplasts wereproduced from cells of a large range of sizes and that correlation in numbers wasvery good for the larger sizes. This correlation agrees well with the visual observa-tion that the cytoplasm in each plasmolyzed coleoptile cell was almost always seenas a single mass, which would be expected to produce a single protoplast. The ex-

FIG. 1 Left: View of a portion of the subepidermal cells of a peeled coleoptile, after 40 minexposure in 0.5 M mannitol. The plasmolytic treatment was followed by 1 min in toluidine-blueto enhance contrast. Right: Phase-contrast picture at the same magnification of some of theliving protoplasts, prepared from the other half of the coleoptile shown at left. Both X 630.

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cess number of very small protoplasts M50 _was probably a result of the occasion- 0 /ally observed reorganization from a /bursting protoplast of a very small 3spherical body (not included in usual 220protoplast counts), sometimes complete -0

with vacuole and apparently normal X''cyclosis. Unlike bacteria, coleoptiles a <- -2 2-3 3-4 6-7 -9of the age used show virtually no cell SIZE CATEGORES (yvxw7xj0-4)divisions.1' FIG. 2.-Comparison of the number of indi-

In the cells plasmolyzed in 0.5 M viduals having specified volumes of cell contents.Open circles, intact cells plasmolyzed in 0.5 M

mannitol, the ratio of protoplast volume mannitol; filled circles, protoplasts from theto cell volume was found to be 0.92. other halves of the same four sections.

It follows that the average osomotic potential of the cells in the intact coleopti]e was0.46 M or about 11 atmospheres.

Reactions of the Protoplasts to Treatment.-Exposure to 0.02 M NaOH or HC1 inthe usual osmoticum caused 50 per cent of the protoplasts to burst within 15 min.Reducing the osmotic concentration to 0.25 M mannitol caused 80 per cent burstingwithin 5 min. When 1 volume of a 0.5M solution of urea or glycerol was mixed withprotoplasts in an equal volume of 0.5 M mannitol on a slide, the percentage sur-vival after 1 hr was decreased to 50 per cent, indicating rapid entry of these solutes;with glucose the decrease was only 5-10 per cent. Sucrose stabilized the protoplastssatisfactorily but was inconvenient because its greater density caused the proto-plasts to float.Table 2 shows the survival of protoplasts and the cytoplasmic streaming observed

TABLE 2PROTOPLAST SURVIVAL AND CYCLOSIS AFTER EXPOSURE FOR 1 HR TO VARIOUS ENZYMES

Per cent Per centNo. of expts. Treatment surviving streaming

1 0.05% Wheat germ lipase 96 921 0.05% Phospholipase D 86 941 Control pH 7.5 82 932 0.1% Pancreatic lipase 86 752 Control pH 7.5 97 743 0.05% Phospholipase C 90 582 Control pH 7.5 83 502* 0.5% Trypsin 72 502* Control 92 982 0.1% Pronase 74 622 Control pH 7.5 87 904 0.1% Trypsin and 0.1% phospholipase C 94 564 Control pH 7.5 85 722 0.07% Trypsin, 0.07% wheat germ lipase, and 67 43

0.07% phospholipase D2 Control pH 7.5 67 721 0.03% RNase 60 441 Control (unbuffered at pH 7.0) 87 G43 0.03% RNase and 0.1% trypsin 78 402 Control pH 7.5 88 672 0.03% RNase, 0.1% pancreatic lipase, and 0.1% 71 40

phospholipase C2 Control pH 7.5 87 58

* One with, one without buffer at pH 7.5.Where mixtures are indicated, trypsin was added last.

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following exposure to a series of hydrolytic enzymes. Survival was only slightlydecreased by 1-hr digestion in the proteolytic enzymes; the decreased streamingmay have been due to their penetration through the plasma membranes without deg-radation of these membranes. Surprisingly, no significant attack on the mem-branes was initiated by any of the lipases or phospholipases. In the thoughtthat enzymatic activity might be decreased by the osmoticum, the activities of thetrypsin and wheat germ lipase were assayed by the WillstAtter methods.'2 Thepresence of 0.5 M mannitol was found to decrease trypsin activity by only 17 percent and lipase activity by 30 per cent. These decreases are not enough to ex-plain the ineffectiveness of these enzymes, and it must be deduced that mem-brane integrity is not dependent on exposed protein or lipid.

In contrast to the enzymes, the anionic detergent, taurocholic acid, and the cat-ionic detergent, hexadecyltrimethyl ammonium bromide, caused rapid disruptionof the protoplasts. The cationic compound was over 30 times more effective thanthe anionic (Fig. 3). The basic proteins cytochrome c and protamine sulfate werealso effective, 50 per cent bursting being produced by concentrations of 200 and100 mg/liter, respectively.Ribonuclease (RNase) was the only enzyme which lysed a significant fraction of

the protoplasts in 1 hr. The concentration needed to disrupt 50 per cent of theprotoplasts was 0.01 per cent with EDTA present. As shown in Table 2, RNase didnot open up digestion sites for the other hydrolytic enzymes.A number of attempts were made to find an effect of auxin on protoplast survival.

The top two lines of Figure 4 show the results of incubating for 30 mm in variousconcentrations of IAA with and without buffer. No effect of IAA is apparent.Protoplasts were then prepared using 0.5 M sucrose as the osmoticum for both thedigestion and the auxin incubation. Here most of the protoplasts floated on thesurface and were more difficult to handle and count; some were lost by both burstingand sinking. As seen by the lowest line of Figure 4, no change in survival occurredafter 30 min in any IAA concentration. Survival was essentially the same afteronly 10 min incubation, indicating that the losses were due to handling problems.To determine whether IAA would influence survival under more drastic condi-

tions, protoplasts were incubated in IAA in 0.5 M mannitol for 50 min and then

CI0 I'0

F6 0.67 00.20 Mexo~decyI5trimhy Tossocholote

Q.4 .050 ammorium brode\\

< a RNase + P04

> .2 £ RNoseUnbuc_\ed00 RN e EDTA

10 100 1000 10 100 I000OONCENTRATION (pg/ml)

FIG. 3.-Left: Protoplast survival after 1 hr in RNase, expressed as the fractionof survival in the controls at 1 hr (which was 80-100%), alone and with 0.025 Mphosphate at pH 6.2 or 10-4M EDTA at pH 6.5. Fractions near the points indicatethe ratio of stable protoplasts showing streaming in RNase to those streaming inthe controls. Right: Similar representation of survival in a cationic and an anionicdetergent, both in 0.025 M phosphate at pH 7.5.

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VOL. 54, 1965 BOTANY: RUESINK AND THIMANAN 61

0.2 M mannitol was added to reduce 1.0the final concentration to 0.4 M.Table 3 shows that although the final 04

survival varied in different experiments, > 0.6 _there was no difference between proto- 5 4 MGM" p0H6.5

o 0.4 _ Unferd i'plast survival in each auxin concentra- < : UnZZd Sorroztion and that in the controls. OL2

Discussion.-Cellulase preparations 0 | | | ,are noted for their low activity in rela- 2 0 -I -2 -3 -4 -5 -6 -D

LOG CONCENTRATION IAA (mgr/Ute*tion to the cellulose-digesting activity of

FIG. 4.-The fraction of the initial number ofthe organism from which they are de- protoplasts remaining 30 min after the additionrived. This enzyme preparation was no of IAA. Average deviations were 6% with 4exception, and unless kept at maximum trials at pH 6.5, 3% with 2 trials in unbuffered

mannitol, and 12% with 3 trials in sucrose.concentration it was not active enoughto remove the walls within 2-3 hr. It was noted that although all of thetissue was softened during the digestion, the inner cell layers retained enoughcell wall to restrain their cell contents; the protoplasts were derived primarilyfrom the outer parenchyma layers. The method of fractionation probablyremoved much of the recently described hydrolyzing factors from Myrotheciumverrucaria filtrate, which are very active on cotton but inactive on carboxymethyl-cellulose.'3 On the other hand, the preparation contained many of the proteinspresent in the fungal filtrate, some of which may have been polysaccharidasesbesides cellulase and hence may have participated in the hydrolysis. Electro-phoresis on acrylamide gel at pH 9.4 showed, however, that the predominantamount of protein was in a single band, presumably the cellulase.The presence of organized cytoplasmic streaming in from 60 to 100 per cent of

the protoplasts was an indication that they were in good physiological condition.Some of those which were not streaming were clearly only vacuoles; some others hadno vacuole, and may have been the result of a rupture of the tonoplast leading to dis-organization of the cell contents. A third group were so completely filled with highlyrrefractile plastids that the smaller granules which make streaming observable couldnot be detected. Cocking reported "cyto- TABLE 3plasmic motion" which was probably cyc- RESISTANCE TO OSMOTIC STRESS AFTER Alosis in his protoplasts, but most previous 50-MIN EXPOSURE TO IAA IN 0.5 M

MANNITOLworkers used high ionic concentrations in Survival

after IAA Survival oftheir stabilizing solutions and failed to re- IAA concn. treatment controlsport the phenomenon, casting some doubt Gg/liter) M Mport ~~~~~~~~~~~500043 40

on the vitality of their material. 25,000 47 43The plasmalemma of plant cells is usu- 5,000 32 37

500 51 51ally visualized as a bimolecular layer con- 50 68 66sisting of protein and lipid components. 25 61 525 53 45Unless the vulnerable sites of such com- 0.5 46 52ponents were geometrically isolated from 0.05 46 60attack, one would have expected proteases Average of all 50.6 50.2and lipases to hydrolyze the membrane and The data show the percentage of protoplasts

intact after 10 min subsequent treatment in 0.4 Mcause cell lysis. Trypsin splits either mannitol.

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62 BOTANY: RIJESINK AND THIMANN PROC. N. A. S.

peptide or ester linkages of the carboxyl group of basic amino acids such as lysine andarginine; the observed resistance to tryptic attack shows that such common linkagesare not present at the surface of the protoplast envelope. The resistance of Avenaprotoplasts to both trypsin and lipase digestion was similar to that reported for yeastprotoplasts.'4 In contrast is the finding that protoplasts of Bacillus megaterium,normally much more stable than higher plant protoplasts, were rapidly lysed bythese enzymes."5 These findings indicate basic structural differences between themembranes surrounding bacterial cells and those of cells of higher plants.An alternative explanation might be that the cellulosic wall had been incompletely

removed. This is considered most unlikely for the following reasons: (1) some ofthe cellulase remained present throughout, yet the behavior of the protoplasts didnot change markedly with time, (2) the protoplasts were spherical with no angularmaterial of any sort, and were quite flexible when exposed to small currents on theslide, (3) no rigid shell was left on bursting, and (4) the protoplasts did disintegraterapidly in detergents and in ribonuclease. Furthermore, exposure of the proto-plasts to pectinase (Rohm and Haas Pectinol Concentrate 42E and/or 41P) or toa fresh concentrate of M11yrotheciumt cellulase for 1 hr caused no significant bursting.

Since these protoplasts are disintegrated by low concentrations of detergents,the integrity of their outer covering must depend upon noncovalent bonding, thatis, upon hydrogen bonding and van der Waals forces. The cationic detergent gives50 per cent bursting in 1 hr at a concentration of 3.3 X 10-5 M while 1.1 X 10-3 Manionic detergent is required. This 33-fold difference suggests that an anionicmoiety is being disorganized by the detergent, and is similar to results reported forbacterial protoplasts.'6The disintegration in RNase reported here was completely unexpected. The

enhanced sensitivity in EDTA probably reflects removal of heavy-metal inter-ference and has been previously reported for RNase acting on other systems. 7

Although the required RNase concentrations are rather high, a direct participa-tion by RNA in maintaining membrane integrity may well be suggested. Some ofthe earlier evidence suggesting the presence of functional RNA in the plasmalemma,unfortunately, could be ascribed to penetration of RNase into the cells. Thus,changes in ion uptake and respiration were observed only after 1 hr in 1 mg/mlRNase, and may therefore have been due to penetration.' RNase penetrated intobarley and onion root cells in 40 min,9 and it induced mitotic abnormalities in onionand lily roots within 2 hr.20 In Spirodela and in corn leaves it powerfully inhibitsanthocyanin formation, a reaction which is almost certainly not localized in theplasmalemma. 17 A change in calcium transport followed RNase treatment ofElodea leaves, but the site of this effect was unclear.2' However, Tanada showedthat in Mung bean roots 100 ,g/ml RNase produced an effect on ion uptake within10 min.22 Cells treated in RNase for 20 min were said to show decreased stainingby toluidine blue with some stain localized near the cell surfaces. Unfortunately,toluidine blue stains cell walls very intensely23 and is thus not a safe indicator ofRNA in cell membranes. Perhaps more significantly, Miasuda24 noted that RNasechanged the plasmolysis pattern of Avena coleoptiles from concave to convex, in-dicating a decreased binding of cytoplasm to wall. By two methods of ghost forma-tion and three types of RNA determination, membranes of B. rnegaterium, strainI, have been shown to contain 1-2 per cent RNA.2' Using a different method to

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VOL. 54, 1965 BOTANY: RUESINK AND THIMANN 63

form ghosts, strain KM of the same bacterium yielded membranes containing 11per cent RNA by orcinol determination.26 But even with their high RNA content,these latter membranes were stable in RNase although disintegrated by wheat germlipase. The present data would indicate that in higher plants, RNA, even if pres-ent only in small amount, functions as a structural component of the membraneof greater importance for its integrity than protein or phospholipid. An alternativeexplanation of the effect of RNase might be that it is acting as a base, much in thesame way as the cationic detergent. RNase is reported to have its isoelectric pointat pH 9.6, but of course the pH of the solutions was adjusted to about 6.5. Sup-porting such a view is the observation that two basic proteins also caused bursting;opposing it is the inactivity of trypsin, whose isoelectric point is 10.5. If this ex-planation were to prove correct, it would point to the critical presence of numerousanionic structures in the membrane. It would also suggest that other reportedeffects of RNase, like those cited above, might have the same basis. On the otherhand, apparent bursting by large molecular species might always be due to thepresence of small molecules as contaminants, particularly if these are actively ac-cumulated. Careful further work will be needed to settle this problem.Cocking reported responses of root protoplasts to IAA,6 but no such responses

could be found in the present work. Several considerations indicate that IAAenters these protoplasts, and the cells normally do respond to auxin by increasedgrowth. Peeled tissues were long ago shown to take up auxin readily.Y2 In thepresent experiments peeled coleoptile sections were placed in 0.1 M glucose with orwithout 5 mg/liter IAA; the tissues in auxin increased 25 per cent in length in9 hr, those without auxin only 13 per cent. The bursting in glycerol indicates thatthe uncharged glycerol molecule easily penetrates the membrane of the protoplast;inhibition of cyclosis in these protoplasts by 0.05M calcium indicates that a chargedion can penetrate. Since Avena coleoptile protoplasts are derived from the tissueusually used to detect auxin, we conclude that the bursting response reported inroot cells6 is not directly related to the effect of auxin on growth.Summary.-Peeled Avena coleoptiles, on exposure to a concentrated cellulase

from Myrothecium, yield spherical protoplasts, showing vigorous cytoplasmic stream-ing. Each cell normally yields one such protoplast. The protoplasts burst whenthe external osmotic pressure is lowered, or when exposed to detergents or to ribo-nuclease. They are stable to proteolytic and lipolytic enzymes, however, alone orin combination, and thus a critical structural component may be RNA. Theyshow no visible response to IAA.

* Supported by a fellowship from the National Science Foundation.t Supported in part by a grant from the National Science Foundation, no. G21799.1 Levitt, J., G. W. Scarth, and R. D. Gibbs, Protoplasma, 26, 237 (1936).2 Vreugdenhil, D., Acta Bot. Neerl., 6, 472 (1957).3 Kuster, E., Protoplasma, 3, 223 (1927).4Tornava, S. R., Protoplasma, 32, 329 (1939).5 Cocking, E. C., Nature, 187, 962 (1960).6Ibid., 193, 998 (1962).7Whitaker, D. R., K. R. Hanson, and P. K. Datta, Can. J. Biochem. Physiol., 41, 671 (1963).8 Gruen, H. E., Plant Physiol., 34, 158 (1959).9 Whitaker, D. R., Arch. Biochem. Biophys., 43, 253 (1953).10 Ray, P. M., and A. W. Ruesink, J. Gen. Physiol., 47, 83 (1963).

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11 Avery, G. S., Jr., and P. R. Burkholder, Bull. Torrey Bot. Club, 63, 1 (1936).12 Sumner, J. B., and G. F. Somers, in Laboratory Experiments in Biological Chemistry (New

York: Academic Press, 1944), pp. 134, 147.13 Selby, K., and C. C. Maitland, Biochem. J., 94, 578 (1965).14 de Kloet, S. R., G. J. W. van Dam, and V. V. Koningsberger, Biochim. Biophys. Acta, 55,

683 (1962).15 Landman, 0. E., and S. Spiegelman, these PROCEEDINGS, 41, 698 (1955).16 Gilby, A. R., and A. V. Few, Nature, 179, 422 (1957); J. Gen. Microbiol., 23, 19 (1960).17 Radner, B. S., and K. V. Thimann, Arch. Biochem. Biophys., 102, 92 (1963).18 Hanson, J. B., Plant Physiol., 35, 372 (1960).9 Jensen, W. A., and A. D. McLaren, Exptl. Cell Res., 19, 414 (1960).20 Kaufmann, B. P., and N. K. Das, these PROCEEDINGS, 40, 1052 (1954).21 Lansing, A. I., and T. B. Rosenthal, J. Cell. Comp. Physiol., 40, 337 (1952).22 Tanada, T., Plant Physiol., 31, 251 (1956).23 O'Brien, T. P., N. Feder, and M. E. McCully, Protoplasma, 59, 368 (1964).24 Masuda, Y., Physiol. Plantarum, 12, 324 (1959).25 Weibull, C., and L. Bergstrom, Biochim. Biophys. Acta, 30, 340 (1958).26 Vennes, J. W., and P. Gerhardt, Science, 124, 535 (1956).27 Thimann, K. V., and C. L. Schneider, Am. J. Bot., 25, 627 (1938).

SEQUENCE OF ENZYME SYNTHESIS AND GENE REPLICATIONDURING THE CELL CYCLE OF BACILLUS SUBTILIS*

BY MILLICENT M\ASTERSt AND ARTHUR B. PARDEE

BIOLOGY DEPARTMENT, PRINCETON UNIVERSITY

Communicated by Colin S. Pittendrigh, May 26, 1965

In a previous communication we reported that certain enzymes were synthesizedonly during parts of the cell cycle in Escherichia coli and Bacillus subtillis.1Periods of little net enzyme synthesis alternated with periods of rapid syntheticactivity. The synthesis of each of the enzymes studied was taking place in amanner which we have termed autogenous,2 that is, the enzyme synthesis oc-curred under the influence of the control mechanisms present in the normallygrowing cell (i.e., subject to the influence of feedback loops). In addition, wemeasured the potential for synthesis of certain other enzymes (i.e., the rate ofsynthesis under induced or derepressed conditions). This potential proved tovary discontinuously. It remained constant for the length of a cell cycle and thenquite abruptly doubled.

Thus, in both situations there occurred a cyclic event which was clearly dis-cernible. In the case of autogenous synthesis, this event was a period of syntheticactivity during the normal growth cycle. When synthetic potential was measured,it was the periodic doubling of the cell's ability to make the enzyme.

Since it has been shown that the B. subtilis genome is replicated sequentially;3one can attempt to correlate the order of synthetic events with the order of replica-tion of genes. For this purpose the autogenous synthesis of four enzymes of B.subtilis has been studied [histidase, aspartate transcarbamylase (ATCase), orni-thine transcarbamylase (OTCase), and dehydroquinase (DHQase) ]. In addition,the potential for sucrase synthesis was measured. All the information

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