Optile Protoplasts Appear to Be in Excellent Physiological

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

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

* A preliminary communication was presented at the Annual Meeting of the National Academy of Sciences on April 25, 1962. Investigations supported by grant no. CA-6375 from the National Cancer Institute (USPHS). t New address: Dean, Graduate School of Biomedical Sciences, University of Texas at Houston. 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 convection," 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 the Effect of Use and Disuse on Neuromuscular Functions. ed. E. Gutmann (Prague: Czechoslovak Academy 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. THIMANNt BIOLOGICAL LABORATORIES, HARVARD UNIVERSITY Communicated May 21, 1965 Living protoplasts produced by removal of cell walls from higher plant tissue offer good experimental material for attacking a number of long-standing problems such 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," 2 or by using large, degenerating cells from ripe fruit which are losing their walls naturally.3 4 Cocking (1960)5 succeeded in preparing protoplasts from tomato root tissue by digestion with cellulase from Myrothecium verrucaria. In 1962 he reported 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 response to IAA has been studied intensively and is much better understood. These coleoptile protoplasts appear to be in excellent physiological condition, and their behavior on exposure to detergents and to various hydrolytic enzymes has been observed with a view to determining what macromolecules are essential to protoplast Downloaded by guest on September 28, 2021 VOL. 54, 1965 BOTANY: RUESINK AND THIMANN 57

integrity. Some preliminary observations have also been made of their reaction to IAA. Preparation of Enzyme.-Since commercial sources of cellulase were found much too weak to dissolve 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., were grown 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 contents poured 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 reciprocal shaking at 220C for 14 days resulted in nearly maximal amounts of cellulase in the medium. After filtration, the solution was evaporated to 40 ml under reduced pressure at 430C in 2 hr. This evaporation 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 fraction coming out at 35-70% of saturation was redissolved in 1-2 ml of distilled water (the smallest amount needed to get complete solution), and desalted with a 1.3 X 11-cm column of Sephadex G-25 at 20C, using 0.1% NaCl to maintain ionic strength and prevent binding. The cellulase activity passed rapidly through the column in the front-running brown-pigmented band, ions and certain pigments being retained. Three 1-ml fractions were usually collected; these were frozen and retained activity for as long as 6 months through repeated freezing and thawing. Table 1 shows 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 with intermittent 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 in 4-6 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 cases no buffer was added, although 0.025 M NaH2PO4 at pH 6.5 was suitable when buffering was desired. 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 mannitol. After 10 min of allowing the protoplasts TABLE 1 to settle, the solution was pipetted off the top to SEPARATION OF CELLULASE ACTIVITY FROM about 0.1 ml, and the protoplasts were washed FUNGAL FILTRATE a second time in the same way. Specificactivity Total To 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 38,000 coleoptile sections were producing protoplasts, 40 ml concentrate 1,000 40,000 four peeled sections were split and half of each 0-35%S 2,000 was plasmolyzed in 0.5 M mannitol, the other half 35-70% 20,000 30,000 being used to prepare protoplasts as above. Di- Supernatant 3,000 ameters of 180 protoplasts were measured and the INlost active 1.0 ml of volumes computed by assuming the protoplasts 3 ml Sephadex eluate 13,000 13,000 to be perfect spheres. At the same time, the Units are arbitrary and based on a viscometric lengths and diametersof the plasmolyzed contents of 114 cells of the subepidermal layer from the other halves were measured and their volumes computed by assuming the cells to be cylinders with slightly rounded ends. The length of each cell wall was also measured so that the osmotic potential of the cells involved could be computed by the plasmometric method. lo For 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 onto warmed microscope slides. A thin ring of silicone grease was applied to the slide just inside the ring to prevent liquid from being drawn under the parafilm by capillarity. After adding cells and covering with a coverslip, the number of good protoplasts was determined by systematically scan- Downloaded by guest on September 28, 2021 58 BOTANY: RUESINK AND THIMANN PROC. N. A. S.

ning the whole depression under phase contrast (150 X), scoring those showing spherical shape and a smooth surface. A green interference filter was used on the microscope lamp. After determining the initial number, 10 X of test solution was added carefully to the slide, and the protoplasts were 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 magnification. When it was desired to lower the osmotic potential of the solutions, 10 X of 0.2 M mannitol 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 lipase Worthington; pancreatic lipase-Worthington PLII; bacterial phospholipase C-Worthington PHL-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 exception to spherical nonrigid protoplasts (see Fig. 1). All contained either one large vacuole or a number of smaller ones, surrounded by cytoplasm which appeared quite viscous with little apparent Brownian motion. In the viable protoplasts, vigorous cyclosis was usually apparent either as sheets of cytoplasm flowing around a large central vacuole or as narrow streams of cytoplasm slipping through the partitions between smaller vacuoles. Large plastids were common and phase-dense granules the size of mitochondria were universally present. Some preparations included a few naked tonloplasts, easily distinguished by their lack of phase-dense cytoplasm. Figure 2 shows the relative distribution of the volumes of cytoplasm of plasmolyzed cells and of isolated protoplasts in 0.5 M mannitol. Note that protoplasts were produced from cells of a large range of sizes and that correlation in numbers was very good for the larger sizes. This correlation agrees well with the visual observation that the cytoplasm in each plasmolyzed coleoptile cell was almost always seen as 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 min exposure in 0.5 M mannitol. The plasmolytic treatment was followed by 1 min in toluidine-blue to enhance contrast. Right: Phase-contrast picture at the same magnification of some of the living protoplasts, prepared from the other half of the coleoptile shown at left. Both X 630. Downloaded by guest on September 28, 2021 VOL. 54, 1965 BOTANY: RUESINK AND THIMANN 59

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 3 spherical body (not included in usual 220 protoplast counts), sometimes complete -0 with vacuole and apparently normal X'' cyclosis. Unlike bacteria, coleoptiles a <- -2 2-3 3-4 6-7 -9 of the age used show virtually no cell SIZE CATEGORES (yvxw7xj0-4) divisions.1' FIG. 2.-Comparison of the number of indi- viduals having specified volumes of cell contents. In the cells plasmolyzed in 0.5 M Open circles, intact cells plasmolyzed in 0.5 M mannitol, the ratio of protoplast volume mannitol; filled circles, protoplasts from the to 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 was 0.46 M or about 11 atmospheres. Reactions of the Protoplasts to Treatment.-Exposure to 0.02 M NaOH or HC1 in the 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 bursting within 5 min. When 1 volume of a 0.5 M solution of urea or glycerol was mixed with protoplasts in an equal volume of 0.5 M mannitol on a slide, the percentage survival 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 protoplasts satisfactorily but was inconvenient because its greater density caused the protoplasts to float. Table 2 shows the survival of protoplasts and the cytoplasmic streaming observed TABLE 2 PROTOPLAST SURVIVAL AND CYCLOSIS AFTER EXPOSURE FOR 1 HR TO VARIOUS ENZYMES Per cent Per cent No. of expts. Treatment surviving streaming 1 0.05% Wheat germ lipase 96 92 1 0.05% Phospholipase D 86 94 1 Control pH 7.5 82 93 2 0.1% Pancreatic lipase 86 75 2 Control pH 7.5 97 74 3 0.05% Phospholipase C 90 58 2 Control pH 7.5 83 50 2* 0.5% Trypsin 72 50 2* Control 92 98 2 0.1% Pronase 74 62 2 Control pH 7.5 87 90 4 0.1% Trypsin and 0.1% phospholipase C 94 56 4 Control pH 7.5 85 72 2 0.07% Trypsin, 0.07% wheat germ lipase, and 67 43 0.07% phospholipase D 2 Control pH 7.5 67 72 1 0.03% RNase 60 44 1 Control (unbuffered at pH 7.0) 87 G4 3 0.03% RNase and 0.1% trypsin 78 40 2 Control pH 7.5 88 67 2 0.03% RNase, 0.1% pancreatic lipase, and 0.1% 71 40 phospholipase C 2 Control pH 7.5 87 58 * One with, one without buffer at pH 7.5. Where mixtures are indicated, trypsin was added last. Downloaded by guest on September 28, 2021 60 BOTANY: RUESINK AND THIMANN PROC. N. A. S.

following exposure to a series of hydrolytic enzymes. Survival was only slightly decreased by 1-hr digestion in the proteolytic enzymes; the decreased streaming may have been due to their penetration through the plasma membranes without deg- radation of these membranes. Surprisingly, no significant attack on the membranes was initiated by any of the lipases or phospholipases. In the thought that enzymatic activity might be decreased by the osmoticum, the activities of the trypsin and wheat germ lipase were assayed by the WillstAtter methods.'2 The presence of 0.5 M mannitol was found to decrease trypsin activity by only 17 per cent 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 membrane integrity is not dependent on exposed protein or lipid. In contrast to the enzymes, the anionic detergent, taurocholic acid, and the cationic detergent, hexadecyltrimethyl ammonium bromide, caused rapid disruption of the protoplasts. The cationic compound was over 30 times more effective than the anionic (Fig. 3). The basic proteins cytochrome c and protamine sulfate were also effective, 50 per cent bursting being produced by concentrations of 200 and 100 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 the protoplasts was 0.01 per cent with EDTA present. As shown in Table 2, RNase did not 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 various concentrations 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 the digestion and the auxin incubation. Here most of the protoplasts floated on the surface and were more difficult to handle and count; some were lost by both bursting and sinking. As seen by the lowest line of Figure 4, no change in survival occurred after 30 min in any IAA concentration. Survival was essentially the same after only 10 min incubation, indicating that the losses were due to handling problems. To determine whether IAA would influence survival under more drastic conditions, protoplasts were incubated in IAA in 0.5 M mannitol for 50 min and then

CI0 I'0

F6 0.67 0 0.20 Mexo~decyI5trimhy Tossocholote Q ammorium \\ .4 .050 brode

< a RNase + P04 > .2 £ RNose Unbuc_\ed 0 RN e EDTA

10 100 1000 10 100 I000 OONCENTRATION (pg/ml) FIG. 3.-Left: Protoplast survival after 1 hr in RNase, expressed as the fraction of survival in the controls at 1 hr (which was 80-100%), alone and with 0.025 M phosphate at pH 6.2 or 10-4 M EDTA at pH 6.5. Fractions near the points indicate the ratio of stable protoplasts showing streaming in RNase to those streaming in the controls. Right: Similar representation of survival in a cationic and an anionic detergent, both in 0.025 M phosphate at pH 7.5. Downloaded by guest on September 28, 2021 VOL. 54, 1965 BOTANY: RUESINK AND THIMANAN 61

0.2 M mannitol was added to reduce 1.0 the 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 plast survival in each auxin concentra-

Discussion.-Cellulase preparations 0 | | | , -6 -D are noted for their low activity in rela- 2 0 -I -2 IAA-3 -4 -5 tion to the cellulose-digesting activity of LOG CONCENTRATION (mgr/Ute* the organism from which they are de- protoplastsFIG. 4.-Theremainingfraction30ofmintheafterinitialthenumberadditionof rived. This enzyme preparation was no of IAA. Average deviations were 6% with 4 trials at pH 6.5, with 2 trials in unbuffered exception, and unless kept at maximum mannitol, and 12%3%with 3 trials in sucrose. concentration it was not active enough to remove the walls within 2-3 hr. It was noted that although all of the tissue was softened during the digestion, the inner cell layers retained enough cell wall to restrain their cell contents; the protoplasts were derived primarily from the outer parenchyma layers. The method of fractionation probably removed much of the recently described hydrolyzing factors from Myrothecium verrucaria filtrate, which are very active on cotton but inactive on carboxymethyl- cellulose.'3 On the other hand, the preparation contained many of the proteins present in the fungal filtrate, some of which may have been polysaccharidases besides cellulase and hence may have participated in the hydrolysis. Electro- phoresis on acrylamide gel at pH 9.4 showed, however, that the predominant amount 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 had no 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 highlyr refractile plastids that the smaller granules which make streaming observable could not be detected. Cocking reported "cyto- TABLE 3 plasmic motion" which was probably cyc- RESISTANCE TO OSMOTIC STRESS AFTER A most 50-MIN TO IN 0.5 M losis in his protoplasts, but previous EXPOSUREMANNITOLIAA workers used high ionic concentrations in Survival after IAA Survival of their stabilizing solutions and failed to re- IAA concn. treatment controls the phenomenon, casting some doubt Gg/liter) M M portport ~~~~~~~~~~~500043 40 on the vitality of their material. 25,000 47 43 5,000 32 37 The plasmalemma of plant cells is usu- 500 51 51 ally visualized as a bimolecular layer con- 50 68 66 sisting of protein and lipid components. 255 5361 4552 Unless the vulnerable sites of such com- 0.5 46 52 ponents were geometrically isolated from 0.05 46 60 attack, one would have expected proteases Average of all 50.6 50.2 and lipases to hydrolyze the membrane and The data show the percentage of protoplasts cause cell lysis. Trypsin splits either intactmannitol.after 10 min subsequent treatment in 0.4 M Downloaded by guest on September 28, 2021 62 BOTANY: RIJESINK AND THIMANN PROC. N. A. S.

peptide or ester linkages of the carboxyl group of basic amino acids such as lysine and arginine; the observed resistance to tryptic attack shows that such common linkages are not present at the surface of the protoplast envelope. The resistance of Avena protoplasts to both trypsin and lipase digestion was similar to that reported for yeast protoplasts.'4 In contrast is the finding that protoplasts of Bacillus megaterium, normally much more stable than higher plant protoplasts, were rapidly lysed by these enzymes."5 These findings indicate basic structural differences between the membranes 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 of the cellulase remained present throughout, yet the behavior of the protoplasts did not change markedly with time, (2) the protoplasts were spherical with no angular material of any sort, and were quite flexible when exposed to small currents on the slide, (3) no rigid shell was left on bursting, and (4) the protoplasts did disintegrate rapidly in detergents and in ribonuclease. Furthermore, exposure of the protoplasts to pectinase (Rohm and Haas Pectinol Concentrate 42E and/or 41P) or to a 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, that is, upon hydrogen bonding and van der Waals forces. The cationic detergent gives 50 per cent bursting in 1 hr at a concentration of 3.3 X 10-5 M while 1.1 X 10-3 M anionic detergent is required. This 33-fold difference suggests that an anionic moiety is being disorganized by the detergent, and is similar to results reported for bacterial protoplasts.'6 The disintegration in RNase reported here was completely unexpected. The enhanced sensitivity in EDTA probably reflects removal of heavy-metal interference 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 of the 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/ml RNase, and may therefore have been due to penetration.' RNase penetrated into barley and onion root cells in 40 min,9 and it induced mitotic abnormalities in onion and lily roots within 2 hr.20 In Spirodela and in corn leaves it powerfully inhibits anthocyanin formation, a reaction which is almost certainly not localized in the plasmalemma. 17 A change in calcium transport followed RNase treatment of Elodea leaves, but the site of this effect was unclear.2' However, Tanada showed that in Mung bean roots 100 ,g/ml RNase produced an effect on ion uptake within 10 min.22 Cells treated in RNase for 20 min were said to show decreased staining by 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 of RNA in cell membranes. Perhaps more significantly, Miasuda24 noted that RNase changed the plasmolysis pattern of Avena coleoptiles from concave to convex, indicating a decreased binding of cytoplasm to wall. By two methods of ghost formation and three types of RNA determination, membranes of B. rnegaterium, strain I, have been shown to contain 1-2 per cent RNA.2' Using a different method to Downloaded by guest on September 28, 2021 VOL. 54, 1965 BOTANY: RUESINK AND THIMANN 63

form ghosts, strain KM of the same bacterium yielded membranes containing 11 per cent RNA by orcinol determination.26 But even with their high RNA content, these latter membranes were stable in RNase although disintegrated by wheat germ lipase. The present data would indicate that in higher plants, RNA, even if present only in small amount, functions as a structural component of the membrane of greater importance for its integrity than protein or phospholipid. An alternative explanation of the effect of RNase might be that it is acting as a base, much in the same way as the cationic detergent. RNase is reported to have its isoelectric point at 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 explanation were to prove correct, it would point to the critical presence of numerous anionic structures in the membrane. It would also suggest that other reported effects of RNase, like those cited above, might have the same basis. On the other hand, apparent bursting by large molecular species might always be due to the presence 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 IAA enters these protoplasts, and the cells normally do respond to auxin by increased growth. Peeled tissues were long ago shown to take up auxin readily.Y2 In the present experiments peeled coleoptile sections were placed in 0.1 M glucose with or without 5 mg/liter IAA; the tissues in auxin increased 25 per cent in length in 9 hr, those without auxin only 13 per cent. The bursting in glycerol indicates that the uncharged glycerol molecule easily penetrates the membrane of the protoplast; inhibition of cyclosis in these protoplasts by 0.05 M calcium indicates that a charged ion can penetrate. Since Avena coleoptile protoplasts are derived from the tissue usually used to detect auxin, we conclude that the bursting response reported in root 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 streaming. Each cell normally yields one such protoplast. The protoplasts burst when the external osmotic pressure is lowered, or when exposed to detergents or to ribonuclease. They are stable to proteolytic and lipolytic enzymes, however, alone or in combination, and thus a critical structural component may be RNA. They show 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). Downloaded by guest on September 28, 2021 64 GENETICS: MASTERS AND PARDEE PROC. N. A. S.

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 REPLICATION DURING 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 synthesized only during parts of the cell cycle in Escherichia coli and Bacillus subtillis.1 Periods of little net enzyme synthesis alternated with periods of rapid synthetic activity. The synthesis of each of the enzymes studied was taking place in a manner which we have termed autogenous,2 that is, the enzyme synthesis occurred under the influence of the control mechanisms present in the normally growing cell (i.e., subject to the influence of feedback loops). In addition, we measured the potential for synthesis of certain other enzymes (i.e., the rate of synthesis under induced or derepressed conditions). This potential proved to vary discontinuously. It remained constant for the length of a cell cycle and then quite 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 synthetic activity 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;3 one can attempt to correlate the order of synthetic events with the order of replication 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 Downloaded by guest on September 28, 2021