Printed in Sweden Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/113?-O?S9$0?.00/0
Experimental
Cell Research I13 (1978) 259-262
CYCLOHEXIMIDE-INDUCED PHYSARUM
of Biophysics
DELAY
IN
POLYCEPHALUM
CARL SCHEFFEY’ Department
MITOTIC
and J. J. WILLE*
and Theoretical Biology, The University Chicago, IL 60637, USA
qf Chicago,
SUMMARY Cycloheximide pulses applied to Physarum polycephalum surface plasmodia delay mitosis. Pulses applied in G2 cause a delay of mitosis which is linearly dependent on the phase in the cell cycle at which the pulse is applied. A 30 min pulse of 10 pg/ml cycloheximide starting in G2 at time t after mitosis induces an excess delay (delay in excess of pulse duration) of the next mitosis of (O.S)t- 1.3 h. The excess delays induced by 7 h pulses during G2 are at most 1 h larger. Pulses applied less than 30 min before mitosis induce only small delays.
The surface plasmodial form of the myxomycete, Physarum polycephalum, is a syncytium of synchronously dividing nuclei well suited to the study of mitotic timing. Its cell cycle has no Gl period, an S period lasting about 3.5 h, and a G2 period of 5 to 15 h[5, 10, 111. It has been proposed that the cell controls the length of its cycle by gradual accumulation of a “mitogen” or division protein which triggers mitosis upon reaching some critical level [2, 3, 10, 11, 131. If so, the delay of mitosis by a specific inhibitor of protein synthesis may shed some light on the nature of the mitogen and control of its synthesis. Cycloheximide inhibits eukaryotic protein synthesis by binding to at least two sites on the 80s ribosomal subunit [6]. At a concentration of 10 pg/ml, it inhibits over 90% of Physarum’s protein synthesis [9, 14, 15, 201. Previous investigators report its effects on Physarum’s mitotic timing [l, 4, 91. The present study extends these investigations, showing that a simple pattern of delays is induced by cycloheximide pulses in Physarum’s G2 period.
METHODS AND MATERIALS Mitotic
timing experiments
Microplasmodia were maintained as shake cultures and plated to form surface plasmodia, using the methods and culture medium previously reported [12]. Medium was buffered with saturated CaCO,. All experiments were performed on plasmodia between the second and fourth mitosis after plating. The original inoculum was cut out of the plasmodium before the experiment. Experiments were done at room temperature, which was held within a range of 2” in each experiment. Plasmodia were grown on millipore filter paper, which was supported-above medium level in the Petri plate bv a Schleicher and Schuel filter circle (No. 595;7 cm)-on top of glass beads or a steel grid. Plasmodia were exposed to different media by transferring them with the underlying millipore paper. Plasmodia were cut into replicate test and control pieces, all of approximately the same size. At the same time that test plasmodia were transferred to plates containing cycloheximide, control plasmodia were transferred to fresh medium and the time of the next mitosis observed. At the end of cycloheximide pulses, plasmodia were transferred successively to seven medium plates which served as washes, and finally transferred to a fresh medium plate, where the time of the ensuing mitosis was observed. Times of metaphase were observed with unfixed smears under phase contrast microscopy at 1000x. Only metaphase and stages of nuclear reconstruction * Present address: Department of Physiology-Anatomy, University of California, Berkeley, CA 94720, USA. 2 Present address: Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803. USA. E.rp Cd Rrs I13 ( 1978)
260
Scheffey and Wille
.4 .
8 .
. . /
.2 '$
1 04 0
.2
.4
.6
.8
1.0
Fig. 1. Abscissa: proportion of cell cycle completed when pulses begin. Mitosis at 0 and 1.0. S ends around 0.3; ordinate: excess delay (delay in excess of pulse duration) plotted as proportion of a control cell cycle. Excess mitotic delays induced by 30 min pulses of 10 pg/ml cycloheximide.
were taken as evidence that metaphase occurred. Nuclear reconstruction in plasmodia that had received a cycloheximide pulse had about the same schedule as in control plasmodia. If the difference between times of mitosis in the two test replicates plus the difference between times of mitosis in the two control replicates exceeded I h, an experiment was discarded and not considered for data analysis.
denly at 33 min (0.046 cycles) before mitosis and are less than 0.1 cycles for the remainder of the time until mitosis. A least squares fit to the delay-phase relation for points between phases 0.4 and 0.95 cycles after mitosis has slope 0.55 and a,=0.02 cycles. Each point in fig. 1 represents the difference between mean time of mitosis in two test replicates and mean time of mitosis in two control replicates; the average difference between replicates with a given treatment was 0.014 cycles. The length of the cell cycle following that in which cycloheximide was applied was observed in several cases and was about the length of a cell cycle in the untreated control.
Long pulses Fig. 2 shows the phase dependence of delay induced by pulses of 10 pg/ml cyclohexiRESULTS mide lasting 1 h or more. The experimental 30 min pulses protocol was the same as for 30 min pulses. Plasmodia at various phases in the cell Most of the points shown are for 7 h pulses, cycle were cut into duplicate test and dupli- which are divided into two experimental cate control pieces. In some cases, more series. These two series were done with replicates were used. Test plasmodia were cultures derived from different exspherulatransferred to medium containing 10 @g/ml tions of Physarum’s spherule (dormant) cycloheximide for 30 min, then to seven washes and finally transferred to fresh me.8 dium. Fig. 1 shows the excess delay (delay o" 00 minus pulse duration) of the next mitosis in 00 0 test plasmodia relative to their controls .6 I 0 plotted against proportion of the cell cycle completed when the pulses began. The “phase” was computed on the basis of cycle length in control plasmodia, which varied from 10 to 12 h. There are relatively high I L OJ delays (0.3-0.4 cycles) early in the cycle, 0 .2 .4 .6 .8 1.0 and a transition to smaller delays (0.1 2. Abscissa: time after mitosis when pulses begin, cycles) at about 0.3 cycles after mitosis. Fig. in cell cycles (hours); ordinate: excess delay in control cell cvcles. The phase of this transition approximately Excess-mitotic delays induced by long pulses of 10 coincides with the end of S. Delays increase pg/ml cycloheximide. 0, 7 h pulses, series A; 0. 7 h linearly through most of G2, but drop sud- pulses, series B; A, l-5 h pulses. E.rp Cd RPS 1I3 / 1978)
Cycloheximide-induced mitotic delay Table 1. Mitotic delays induced by 10 pgl ml cycloheximide pulses starting shortly before mitosis Time before control metaphase Pulse pulse started duration (min) (min) 16 18 21 :: 33 35 49
until until 30 until until 30 420 30
M M M M
Total delays of mitosis in test replicates (min) 32,45, 52, 56 15 39 40 25 360,60” 29, 32, 34,795 336
(1 Mitosis in this replicate was asynchronous.
form. Through the last half of the cell cycle, there is a linear relationship between phase and delay, which is indicated by the sightfitted line of slope 0.9. There is no systematic difference in G2 between the delays induced by 7 h pulses (dots and circles) and those induced by the shorter pulses of l-5 h duration (triangles). G2 excess delay is approximately constant with increasing pulse durations of over 1 h; this was verified by comparing pulses of 2.5 h with 7 h and 6 h with 12 h, using replicates cut from the same original plasmodium for both pulse durations. The delays induced by 30 min pulses were, however, smaller than those induced by 7 h pulses late in G2. A 7 h
261
pulse at 0.9 cycles after M induced an excess delay of about 0.48 cycles, while a 30 min pulse at the same phase induced a delay of about 0.38 cycles. Larger delays were observed in S. Seven hour pulses starting in S induced excess delays of 0.6 to 0.8 cycles, about double the excess delay induced by 30 min pulses in S. This comparison between 7 h and 30 min pulses in S was verified when both pulse durations were applied to replicates cut from the same original plasmodium. Increasing excess delay with increasing pulse duration was also seen in S for pulses of 5 to 1.50min duration (unpublished observations). When cycloheximide was applied less than 30 min before mitosis, plasmodia went through mitosis while still on the cycloheximide medium. Data for treatments shortly before mitosis are shown in table 1. In one case, four replicate test plasmodia cut from the same original plasmodium were transferred to 10 pg/ml cycloheximide at 3.5 min before control mitosis. Three of these replicates went through mitosis on the cycloheximide, all with 0.047 cycles total (rather than excess) delay. At that time, the remaining replicate had nuclear morphology similar to that of a normal plasmodium 30-90 min before mitosis. It was kept on cycloheximicle 7 h, during which time some of its nuclei showed early pro-
Table 2. Patterns of mitotic delays induced by various perturbations to Physarum
Perturbation 30 min pulse, 10 pg chex Long pulses, IO pg chex UV low dose refs [S, 211 UV high dose ref. [21] X-irradiation ref. [7]
Average delays early in cycle (cycles)” 0.3 0.7 0.15-0.3 Sensitive to much lower dose
n Phases refer to time after mitosis.
Delays depend linearly on phase starting at this phase (cycles)”
Transition point to small delays just before mitosis (min before mitosis)
0.35 0.6 0.6
33-35 33-35 None 14 2C.60
Before 0.5
Slope of linear delay-phase relation 0.55 0.9-I .o -0.5 0.4
262
Scheffey and Wille
phase type morphologies. It was then carried through washes, and eventually went through synchronous mitosis with the excess delay predicted by the linear delayphase relation for long pulses in G2.
ing the course of these investigations. Dr Paulo Dice read the manuscript and made many helpful suggestions. Part of this work was completed at the Department of Biochemistry and Biophysics at the University of Pennsylvania School of Medicine. This work was supported in part from grants to Dr S. A. Kauffman from the National Science Foundation (Grant GB-36067) and from the University of Chicago Cancer Research Center. Project I 11-B.
DISCUSSION cell cycle may be divided into three sections, each with a distinct pattern of mitotic delays induced by cycloheximide. Periods approximately coinciding with S, most of G2, and the last half hour of the cell cycle each have delays with a different dependence on pulse phase and duration, summarized in table 2. The table also shows that similar patterns of delay have been reported for other perturbations to Physarum [7, 8, 211. The linear dependence of delay upon phase for these perturbations can be interpreted along the lines of the discussion of phase4elay curves by Mitchison [33. It would be desirable to predict how cycloheximide would affect the cytoplasmic concentration of proteins involved in mitotic timing. Unfortunately, cycloheximide has complex consequences to protein metabolism. It is known, for example, to depress degradation of certain proteins in other organisms [17]. Perhaps the most relevant data available are studies of cycloheximide’s effect on the level of specific proteins in Physarum [16, 18, 191. It appears reasonable, but not certain, that the level of proteins important in mitotic timing would decrease during a cycloheximide pulse. The extent of the decrease could be dependent on phase in the cell cycle; no evidence is available that bears on this possibility.
Physarum’s
We wish to thank Dr Stuart A. Kauffman for his encouragement and many stimulating comments dur-
REFERENCES 1. Cummins, J E, Blomquist, J C & Rusch, H P. Science 154 (1966) 1343. 2. Kauffman, S A & Wille, J J, J theor biol 55 (1975) 47. 3. Mitchison, J M, The biology of the cell cycle. Cambridge University Press, Cambridge (1971). 4. Murakami, K & Ohta, J, Plant cell physiol 12 (1971) 797. 5. Nvgaard, 0 F, Guttes, S & Rusch, H P, Biochim biophys acta 38 (l%O) 298. 6. Obrig, T G, Gulp, W J, McKeehan, W L & Hardesty, B, J biol them 246 (1971) 174. ’ 7. Sachsenmaier, W, Bohnert, E, Clausnizer, B & Nygaard, 0 F, FEBS lett 10 (1970) 185. 8. Sachsenmaier, W, Probleme der biologischen Reduplication (ed P Sitte) p. 139. Springer-Verlag, Berlin (1%6). 9. Sachsenmaier, W, von Foumier, D & Gurtler, K F. Biochem bioohvs res commun 27 (1967) 655. 10. Sachsenmaier, W’& Rusch, H P, Exp cell res 36 (1964) 124. 11. Sudbery, P E & Grant, W D, Exp cell res 95 (1975) 405. 12. Wille, J J & Kauffman, S A, Biochim biophys acta 407 (1975) 158. 13. Zeuthen, E & Williams. N, Proc set int symp for cellular chemistry: Nucleic acid metabolism; cell differentiation, and cancer growth (eds. E Cowdry & S Seno) p. 203. Oxford, New York, Pergamon Press (1969). 14. Cummins, J E & Rusch, H P, J cell biol 31 (1966) 577. 15. Haugli, F, Ph.D. Thesis, University of Wisconsin (1971). 16. Mitchell, J L & Rusch, H P, Biochim biophys acta 297 (1973) 503. 17. Woodside, K H, Biochim biophys acta 421 (1976) 70. 18. Hiittermann, A, Porter, M T & Rusch, H P, Arch Mikrobio174 (1970) 90. 19. -Ibid (1970) 283. 20. Cummins. J E. Brewer. E N & Rusch, H P, J cell biol 27 (1965) 337. 21. Devi, V R, Guttes, E & Guttes, S, Exp cell res 50 (1968) 589. Received August 3, 1977 Revised version received December 15, 1977 Accepted December 20, 1977
Experimental
Cell Research I13 (1978) 259-262
CYCLOHEXIMIDE-INDUCED PHYSARUM
of Biophysics
DELAY
IN
POLYCEPHALUM
CARL SCHEFFEY’ Department
MITOTIC
and J. J. WILLE*
and Theoretical Biology, The University Chicago, IL 60637, USA
qf Chicago,
SUMMARY Cycloheximide pulses applied to Physarum polycephalum surface plasmodia delay mitosis. Pulses applied in G2 cause a delay of mitosis which is linearly dependent on the phase in the cell cycle at which the pulse is applied. A 30 min pulse of 10 pg/ml cycloheximide starting in G2 at time t after mitosis induces an excess delay (delay in excess of pulse duration) of the next mitosis of (O.S)t- 1.3 h. The excess delays induced by 7 h pulses during G2 are at most 1 h larger. Pulses applied less than 30 min before mitosis induce only small delays.
The surface plasmodial form of the myxomycete, Physarum polycephalum, is a syncytium of synchronously dividing nuclei well suited to the study of mitotic timing. Its cell cycle has no Gl period, an S period lasting about 3.5 h, and a G2 period of 5 to 15 h[5, 10, 111. It has been proposed that the cell controls the length of its cycle by gradual accumulation of a “mitogen” or division protein which triggers mitosis upon reaching some critical level [2, 3, 10, 11, 131. If so, the delay of mitosis by a specific inhibitor of protein synthesis may shed some light on the nature of the mitogen and control of its synthesis. Cycloheximide inhibits eukaryotic protein synthesis by binding to at least two sites on the 80s ribosomal subunit [6]. At a concentration of 10 pg/ml, it inhibits over 90% of Physarum’s protein synthesis [9, 14, 15, 201. Previous investigators report its effects on Physarum’s mitotic timing [l, 4, 91. The present study extends these investigations, showing that a simple pattern of delays is induced by cycloheximide pulses in Physarum’s G2 period.
METHODS AND MATERIALS Mitotic
timing experiments
Microplasmodia were maintained as shake cultures and plated to form surface plasmodia, using the methods and culture medium previously reported [12]. Medium was buffered with saturated CaCO,. All experiments were performed on plasmodia between the second and fourth mitosis after plating. The original inoculum was cut out of the plasmodium before the experiment. Experiments were done at room temperature, which was held within a range of 2” in each experiment. Plasmodia were grown on millipore filter paper, which was supported-above medium level in the Petri plate bv a Schleicher and Schuel filter circle (No. 595;7 cm)-on top of glass beads or a steel grid. Plasmodia were exposed to different media by transferring them with the underlying millipore paper. Plasmodia were cut into replicate test and control pieces, all of approximately the same size. At the same time that test plasmodia were transferred to plates containing cycloheximide, control plasmodia were transferred to fresh medium and the time of the next mitosis observed. At the end of cycloheximide pulses, plasmodia were transferred successively to seven medium plates which served as washes, and finally transferred to a fresh medium plate, where the time of the ensuing mitosis was observed. Times of metaphase were observed with unfixed smears under phase contrast microscopy at 1000x. Only metaphase and stages of nuclear reconstruction * Present address: Department of Physiology-Anatomy, University of California, Berkeley, CA 94720, USA. 2 Present address: Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803. USA. E.rp Cd Rrs I13 ( 1978)
260
Scheffey and Wille
.4 .
8 .
. . /
.2 '$
1 04 0
.2
.4
.6
.8
1.0
Fig. 1. Abscissa: proportion of cell cycle completed when pulses begin. Mitosis at 0 and 1.0. S ends around 0.3; ordinate: excess delay (delay in excess of pulse duration) plotted as proportion of a control cell cycle. Excess mitotic delays induced by 30 min pulses of 10 pg/ml cycloheximide.
were taken as evidence that metaphase occurred. Nuclear reconstruction in plasmodia that had received a cycloheximide pulse had about the same schedule as in control plasmodia. If the difference between times of mitosis in the two test replicates plus the difference between times of mitosis in the two control replicates exceeded I h, an experiment was discarded and not considered for data analysis.
denly at 33 min (0.046 cycles) before mitosis and are less than 0.1 cycles for the remainder of the time until mitosis. A least squares fit to the delay-phase relation for points between phases 0.4 and 0.95 cycles after mitosis has slope 0.55 and a,=0.02 cycles. Each point in fig. 1 represents the difference between mean time of mitosis in two test replicates and mean time of mitosis in two control replicates; the average difference between replicates with a given treatment was 0.014 cycles. The length of the cell cycle following that in which cycloheximide was applied was observed in several cases and was about the length of a cell cycle in the untreated control.
Long pulses Fig. 2 shows the phase dependence of delay induced by pulses of 10 pg/ml cyclohexiRESULTS mide lasting 1 h or more. The experimental 30 min pulses protocol was the same as for 30 min pulses. Plasmodia at various phases in the cell Most of the points shown are for 7 h pulses, cycle were cut into duplicate test and dupli- which are divided into two experimental cate control pieces. In some cases, more series. These two series were done with replicates were used. Test plasmodia were cultures derived from different exspherulatransferred to medium containing 10 @g/ml tions of Physarum’s spherule (dormant) cycloheximide for 30 min, then to seven washes and finally transferred to fresh me.8 dium. Fig. 1 shows the excess delay (delay o" 00 minus pulse duration) of the next mitosis in 00 0 test plasmodia relative to their controls .6 I 0 plotted against proportion of the cell cycle completed when the pulses began. The “phase” was computed on the basis of cycle length in control plasmodia, which varied from 10 to 12 h. There are relatively high I L OJ delays (0.3-0.4 cycles) early in the cycle, 0 .2 .4 .6 .8 1.0 and a transition to smaller delays (0.1 2. Abscissa: time after mitosis when pulses begin, cycles) at about 0.3 cycles after mitosis. Fig. in cell cycles (hours); ordinate: excess delay in control cell cvcles. The phase of this transition approximately Excess-mitotic delays induced by long pulses of 10 coincides with the end of S. Delays increase pg/ml cycloheximide. 0, 7 h pulses, series A; 0. 7 h linearly through most of G2, but drop sud- pulses, series B; A, l-5 h pulses. E.rp Cd RPS 1I3 / 1978)
Cycloheximide-induced mitotic delay Table 1. Mitotic delays induced by 10 pgl ml cycloheximide pulses starting shortly before mitosis Time before control metaphase Pulse pulse started duration (min) (min) 16 18 21 :: 33 35 49
until until 30 until until 30 420 30
M M M M
Total delays of mitosis in test replicates (min) 32,45, 52, 56 15 39 40 25 360,60” 29, 32, 34,795 336
(1 Mitosis in this replicate was asynchronous.
form. Through the last half of the cell cycle, there is a linear relationship between phase and delay, which is indicated by the sightfitted line of slope 0.9. There is no systematic difference in G2 between the delays induced by 7 h pulses (dots and circles) and those induced by the shorter pulses of l-5 h duration (triangles). G2 excess delay is approximately constant with increasing pulse durations of over 1 h; this was verified by comparing pulses of 2.5 h with 7 h and 6 h with 12 h, using replicates cut from the same original plasmodium for both pulse durations. The delays induced by 30 min pulses were, however, smaller than those induced by 7 h pulses late in G2. A 7 h
261
pulse at 0.9 cycles after M induced an excess delay of about 0.48 cycles, while a 30 min pulse at the same phase induced a delay of about 0.38 cycles. Larger delays were observed in S. Seven hour pulses starting in S induced excess delays of 0.6 to 0.8 cycles, about double the excess delay induced by 30 min pulses in S. This comparison between 7 h and 30 min pulses in S was verified when both pulse durations were applied to replicates cut from the same original plasmodium. Increasing excess delay with increasing pulse duration was also seen in S for pulses of 5 to 1.50min duration (unpublished observations). When cycloheximide was applied less than 30 min before mitosis, plasmodia went through mitosis while still on the cycloheximide medium. Data for treatments shortly before mitosis are shown in table 1. In one case, four replicate test plasmodia cut from the same original plasmodium were transferred to 10 pg/ml cycloheximide at 3.5 min before control mitosis. Three of these replicates went through mitosis on the cycloheximide, all with 0.047 cycles total (rather than excess) delay. At that time, the remaining replicate had nuclear morphology similar to that of a normal plasmodium 30-90 min before mitosis. It was kept on cycloheximicle 7 h, during which time some of its nuclei showed early pro-
Table 2. Patterns of mitotic delays induced by various perturbations to Physarum
Perturbation 30 min pulse, 10 pg chex Long pulses, IO pg chex UV low dose refs [S, 211 UV high dose ref. [21] X-irradiation ref. [7]
Average delays early in cycle (cycles)” 0.3 0.7 0.15-0.3 Sensitive to much lower dose
n Phases refer to time after mitosis.
Delays depend linearly on phase starting at this phase (cycles)”
Transition point to small delays just before mitosis (min before mitosis)
0.35 0.6 0.6
33-35 33-35 None 14 2C.60
Before 0.5
Slope of linear delay-phase relation 0.55 0.9-I .o -0.5 0.4
262
Scheffey and Wille
phase type morphologies. It was then carried through washes, and eventually went through synchronous mitosis with the excess delay predicted by the linear delayphase relation for long pulses in G2.
ing the course of these investigations. Dr Paulo Dice read the manuscript and made many helpful suggestions. Part of this work was completed at the Department of Biochemistry and Biophysics at the University of Pennsylvania School of Medicine. This work was supported in part from grants to Dr S. A. Kauffman from the National Science Foundation (Grant GB-36067) and from the University of Chicago Cancer Research Center. Project I 11-B.
DISCUSSION cell cycle may be divided into three sections, each with a distinct pattern of mitotic delays induced by cycloheximide. Periods approximately coinciding with S, most of G2, and the last half hour of the cell cycle each have delays with a different dependence on pulse phase and duration, summarized in table 2. The table also shows that similar patterns of delay have been reported for other perturbations to Physarum [7, 8, 211. The linear dependence of delay upon phase for these perturbations can be interpreted along the lines of the discussion of phase4elay curves by Mitchison [33. It would be desirable to predict how cycloheximide would affect the cytoplasmic concentration of proteins involved in mitotic timing. Unfortunately, cycloheximide has complex consequences to protein metabolism. It is known, for example, to depress degradation of certain proteins in other organisms [17]. Perhaps the most relevant data available are studies of cycloheximide’s effect on the level of specific proteins in Physarum [16, 18, 191. It appears reasonable, but not certain, that the level of proteins important in mitotic timing would decrease during a cycloheximide pulse. The extent of the decrease could be dependent on phase in the cell cycle; no evidence is available that bears on this possibility.
Physarum’s
We wish to thank Dr Stuart A. Kauffman for his encouragement and many stimulating comments dur-
REFERENCES 1. Cummins, J E, Blomquist, J C & Rusch, H P. Science 154 (1966) 1343. 2. Kauffman, S A & Wille, J J, J theor biol 55 (1975) 47. 3. Mitchison, J M, The biology of the cell cycle. Cambridge University Press, Cambridge (1971). 4. Murakami, K & Ohta, J, Plant cell physiol 12 (1971) 797. 5. Nvgaard, 0 F, Guttes, S & Rusch, H P, Biochim biophys acta 38 (l%O) 298. 6. Obrig, T G, Gulp, W J, McKeehan, W L & Hardesty, B, J biol them 246 (1971) 174. ’ 7. Sachsenmaier, W, Bohnert, E, Clausnizer, B & Nygaard, 0 F, FEBS lett 10 (1970) 185. 8. Sachsenmaier, W, Probleme der biologischen Reduplication (ed P Sitte) p. 139. Springer-Verlag, Berlin (1%6). 9. Sachsenmaier, W, von Foumier, D & Gurtler, K F. Biochem bioohvs res commun 27 (1967) 655. 10. Sachsenmaier, W’& Rusch, H P, Exp cell res 36 (1964) 124. 11. Sudbery, P E & Grant, W D, Exp cell res 95 (1975) 405. 12. Wille, J J & Kauffman, S A, Biochim biophys acta 407 (1975) 158. 13. Zeuthen, E & Williams. N, Proc set int symp for cellular chemistry: Nucleic acid metabolism; cell differentiation, and cancer growth (eds. E Cowdry & S Seno) p. 203. Oxford, New York, Pergamon Press (1969). 14. Cummins, J E & Rusch, H P, J cell biol 31 (1966) 577. 15. Haugli, F, Ph.D. Thesis, University of Wisconsin (1971). 16. Mitchell, J L & Rusch, H P, Biochim biophys acta 297 (1973) 503. 17. Woodside, K H, Biochim biophys acta 421 (1976) 70. 18. Hiittermann, A, Porter, M T & Rusch, H P, Arch Mikrobio174 (1970) 90. 19. -Ibid (1970) 283. 20. Cummins. J E. Brewer. E N & Rusch, H P, J cell biol 27 (1965) 337. 21. Devi, V R, Guttes, E & Guttes, S, Exp cell res 50 (1968) 589. Received August 3, 1977 Revised version received December 15, 1977 Accepted December 20, 1977
