Vol. 140, No. 2
JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 490-497 0021-9193/79/11-0490/08$02.00/0
Translational Regulation of Polysome Formation During Dormancy of Physarum polycephalum WILLIAM R. JEFFERY Department of Zoology, University of Texas, Austin, Texas 78712 Received for publication 14 August 1979
The translational activity of actively growing microplasmodia and dormant microsclerotia of Physarum polycephalum was investigated by analyzing the distribution of ribosomes in polysomes. Microplasmodial post-mitochondrial fractions contained substantial amounts of polysomes and ribosomal subunits but very few native monosomes. During the starvation period which preceded microsclerotium formation, polysome levels remained constant, whereas the subunit titer began to increase. During encystment ribosomal subunits continued to accumulate as the level of polysomes gradually decreased. Dormant microsclerotia contained a large surplus of stored ribosomal subunits but no detectable polysomes. However, incubation of microsclerotia with concentrations of cycloheximide sufficient to slow polypeptide elongation without affecting initiation caused the gradual reappearance of polysomes at the expense of the subunits. Under these conditions the percentage of subunits driven into polysomes reached values similar to those of actively growing microplasmodia. Microsclerotia returned to nutrient medium contained very low levels of polysomes during the lag period which preceded germination. These were formed with preexisting, stored messenger ribonucleic acid. During the germination period, polysome levels were markedly increased. This elevation was dependent on new ribonucleic acid transcription. It is concluded that dormant microsclerotia contain functional messenger ribonucleic acid and ribosomes which are subject to translational repression at the level of initiation. As a response to adverse environmental conditions such as starvation, dessication, or suboptimal temperature, the plasmodial slime mold Physarum polycephalum can undergo a form of dormnancy known as sclerotization (15). During the sclerotization process the plasmodial cytoplasm is partitioned into small multinucleate cysts by intemal membrane formation (31). Aggregations of these cysts, called sclerotia, are characterized by a low metabolic rate (10) and the ability to remain viable for up to several years (15). Return to favorable environmental conditions induces the dormant sclerotia to revert to actively growing plasmodia. Dramatic biochemical changes accompany the induction of sclerotization by nutrient depletion (25-27). During the starvation period preceding encystment, macromolecular synthesis continues with biosynthetic precursors derived from the partial catabolism of DNA, RNA, and protein (27) and the utilization of plasmodial glycogen reserves (11). Although the biochemical changes which occur during the starvation period have been extensively studied, very little is known about the metabolic activities of the dormant sclerotium. For example, the funda490
mental question ofwhether cysts actively engage in protein synthesis during dormancy or store mRNA and ribosomes for use during plasmodial regeneration is unresolved. In the present study translational activity during the transition from active growth to dormancy and during plasmodial regeneration from dormant sclerotia has been investigated by analyzing the distribution of ribosomes in polysomes. It is demonstrated that plasmodial polysomes are gradually dissociated during the encystment periods; that functional, but relatively inactive, mRNA and ribosomes are sequestered in the mature sclerotium; and that polysomes are reformed with stored sclerotial mRNA during plasmodial regeneration. It is concluded that protein synthesis is translationally repressed at the level of initiation during dormancy.
MATERIALS AND METHODS Culture methods. P. polycephalum (Carolina strain) was grown as microplasmodia at 220C in agitated suspension culture on a rotary shaker with the medium described previously (7). Dormant microsclerotia were fonned from mid-exponential-phase microplasmodial cultures by starvation in a citrate-
TRANSLATIONAL REGULATION OF DORMANT PHYSARUM
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buffered (pH 4.6), nonnutrient salts medium (12). Germination of dormant microsclerotia was induced by their transfer back to the routine culture medium. Polysome preparation. Polysomes were prepared from microplasmodia and microsclerotia by a modified version of the method of Brewer (4). Mid-exponentialgrowth-phase microplasmodial cultures and dormant or germinating microsclerotial cultures were harvested by centrifugation at room temperature at 100 x g for 1 min. The supernatant was decanted, and the pellet was resuspended in 5 volumes of ice-cold 50 mM Trishydrochloride (pH 7.2), 100 mM KCl, 100 mM MgCl2, 200 mM sucrose, and 75 mM EDTA and homogenized by 15 up and down strokes of a ground glass homogenizer. Increasing the EDTA concentration from 25 mM (4) to 75 mM was consistently found to yield larger polysomes. The post-mitochondrial fraction was prepared from the homogenate by centrifugation at 20,000 x g for 10 min at 4°C. The supernatant fractions were removed from the tubes and immediately layered on
density gradients
or
stored in liquid N2.
Zone sedimentation. Zone sedimentation of the post-mitochondrial supernatant fraction was carried out at 40C through 12 ml, 10 to 50% linear sucrose density gradients. To preserve the integrity of the polysomes, it was necessary to dissolve the sucrose in 50 mM Tris-hydrochloride (pH 7.2), 100 mM KCl, and 100 mM MgCl2. The gradients were centrifuged in the Beckman SW41 rotor for 90 min at 35,000 rpm. The absorbance at 254 nm was recorded by pumping the gradients through an LKB Uvicord I spectrophotometric cell. Determination of ribosome distribution. The absorbance profiles obtained were traced onto paper, and the area contributed by the absorbance of sucrose (derived from blank gradients). was subtracted. The regions corresponding to ribosomal subunits (RSU), native monosomes, and RNase-produced monosomes and disomes (and occasionally trisomes) (Fig. 1A and B) were cut out and weighed. The level of polysomes was obtained by subtracting the weight of the native monosomal area from that of the total monosome and disome (and occasionally trisome) areas of RNasetreated samples. The level of RSU was estimated directly from the weight of the area below the 60S and 40S peaks. The percentage of ribosomes in polysomes was obtained by dividing the weight of the polysomal area, obtained from RNase-treated profiles as described above, by the weight of the total ribosomal area.
Measurement of poly(A) content. The polyadenylic acid [poly(A)] content of RNA, extracted from the post-mitochondrial fraction of sclerotia by procedures described previously (1), was determined by the method of Jeffery (14). This involved bringing portions of the RNA preparation to 10 mM Tris-hydrochloride (pH 7.6), 200 mM NaCl, and 5 mM MgCl2 and mixing them with an excess of [3H]polyuridylic acid (5.14 Ci/ mmol; New England Nuclear Corp., Boston, Mass.). The mixture was incubated at 25°C for 15 min to promote annealing and then brought to 10 ,ug/ml with pancreatic RNase and incubated for 1 h at 37°C. Finally the digests were chilled, and the nucleic acids were precipitated with 10% cold trichloroacetic acid and collected for liquid scintillation counting on glass fiber filters.
491
RESULTS Characteristics of microplasmodial polysomes. The sedimentation proffies of post-mitochondrial fractions prepared from exponentially growing, vegetative microplasmodia are shown in Fig. 1. Ribosome distribution in polysomes and lighter particles was somewhat different than that previously reported for Physarum microplasmodia (4, 24). The major differences observed were an enrichment of RSU and a deficiency in single ribosomes (Fig. 1A). RNase treatment of the post-mitochondrial fraction before centrifugation converted the polysomes to lighter particles, mainly monosomes and disomes, but did not affect the RSU levels (Fig. 1B; no effect of RNase on RSU levels was observed in 25 separate experiments). An RNaseinsensitive, UV-absorbing material previously identified as glycogen particles (28) remained in the high-molecular-weight region of the gradient after RNase treatment. Incomplete polysome digestion by RNase resulting in accumulation of disomes has also been reported in other eucaryotes (19, 21). Since it is unusual for actively growing cells to contain proportionately larger RSU than monosome pools (2), further experiments were designed to determine whether this result was caused by the extraction conditions. For example, it is possible that the high RSU levels were due to polysome runoff without reinitiation during the sample preparation. To test this possibility, cycloheximide which slows polypeptide elongation (29) was added to the extraction media and the gradients. As shown in Fig. 1C, cycloheximide addition did not reduce RSU levels, suggesting that they were not caused by runoff. It is also unlikely that the high RSU levels were due to the excessive amounts of EDTA and Mg2e required to inhibit endogeneous RNase activity in Physarum (4) since they were also present when the concentrations of these components in the extraction media were significantly reduced (Fig. 1D). Similar levels of RSU were also observed when polysomes were extracted with various EDTA-Mg2+ ratios including the 25 mM/100 mM ratio originally employed by Brewer (4). The apparent excess of RSU could also be due to selective binding of polysomes and monosomes to membranes or other insoluble microplasmodial structures. This possibility was checked by the addition of Triton X-100 and sodium deoxycholate to the extraction media. As shown in Fig. 1E, no alterations in the distribution of ribosomal material between polysomes, monosomes, and RSU were subsequently found. Thus, the results suggest that a high proportion of ribosomes exist in an RSU pool in Physarum microplasmodia. Large
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JEFFERY
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FIG. 1. Zone sedimentation of the post-mitochondral firacton from P. polycephalum microplasmodia. (A) Poatnitochondrial fractions under normal extractin conditn. The arrows represent the position of40, 608, and 808 ribosomal markers derived from Ehrlich ascites tumor ceUs centrifuged on parallel gradients. (B) Post-mitochondrial fractions treated with 10 pg of pancreatic RNase A (RNase) per ml for 30 mmn on ice before centrifugatin. (C) Post-mitochondrial fractions prepared under conditions in which the extraction media and gradient were suppemented with 20 pg of cycloheximide per ml (D) Post-mitochondrial firactions prepared with extraction media composed of 50 mM Tris-hydrochloride (pH 7.6, 50 mM KCl4 I mM MgCI2, and 20 mM sucrose. The gradients contained the same concentration of salts. (E) Post-mitochondrial fractions prepared in the presence of 0.5% Triton X-100) and 1% sodium deoxychokate. The sedimentation direction is from right to left. The vertical dashed lines represent the posion which the absorbance scale is halved. The hatched and shaded surfaces ui A and B represent the areas from which the levels ofpolysomes and RSU, respectively, were cakulated as described in the text. in
RSU pools are also found in yeast (20) and the fungus Mucor (22). Polysome dissociation and RSU accumulation during microwlerotium formation. Dormant microsclerotia are formed from
starved microplasmodial cultures asynchronously between 24 and 36 h after the shift to nonnutrient medium. The sedimentation profiles of post-mitochondrial fractions derived from cultures at selected intervals in the starvation-encystment period and from mature microsclerotia are shown in Fig. 2A through D. Since the high-molecular-weight regions of the gradients contain glycogen particles as well as polysomes, indirect methods are required for the measurement of polysome titers. This was facilitated by measuring the levels of monosomes and disomes which appeared in RNase-treated post-mitochondrial fractions (Fig. 2E through H). Although lower proportions of ribosomes distributed in polysomes were observed in the present study (Table 1) compared with previous measurements (24), it is believed that the current estimations are more accurate since they exclude the glycogen components. The changes detected in polysome and RSU levels during sclerotization are summarized in Fig. 3. It can be seen that polysome levels remained substantial during the starvation period, decreased markedly during and after encystment, and were undetectable in microsclerotia by 72 h after the medium shift. During a similar interval, the level of RSU was increased threefold (Fig. 3), suggesting that encysting microsclerotia accnuulate and store ribosomes. The initial decline in RSU and polysomes observed after the medium shift is due to "transfer shock" and not the starvation condition itself, since a similar effect occurred when microplasmodia were switched to fresh nutrient medium. The inability to detect polysomes in mature microsclerotia could be due to depressed translatl activity. This observation, however, could also be explained by polysome compartmentalization or the presence of an activated cyst-specific, polysome-uncoupling agent in the microsclerotia. The possibility of a polysomeuncoupling agent activated during the extraction procedure was tested by mixing microsclerotia and microplasmodia before homogenization. As shown in Fig. 4A the sedimentation profiles obtained from the post-mitochondrial fractions of this mixture exhibited polysomes excluding the possibility of an uncoupling agent. The absence of detectable polysomes in the microsclerotia was not due to compartmentalization either, since they were not released after detergent treatment (Fig. 4B) or by more extensive homogenizations. Thus, it appears that mature microsclerotia are translationally inactive. Induction of polysomes in dormuant microsclerotia. The lack of an essential component, such as mRNA, or the active repression of
TRANSLATIONAL REGULATION OF DORMANT PHYSARUM
VOL. 140, 1979
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FIG. 2. Zone sedimentation of the post-mitochondrial fractions from P. polycephalum during the transition from active growth to dormancy. (A) 1 h, (B) 24 h, (C) 36 h, and (D) 72 h after the switch to nonnutrient medium. (E to H) RNase-treated counterparts of A to D. Other details are similar to those in the legend to
Fig. 1. an otherwise completely functional protein synthetic machinery could cause the translational deficiency seen in dormant microsclerotia. It was therefore of interest to determine whether microsclerotial RSU could be driven into polysomes. Low levels of cycloheximide reduce polypeptide elongation rates without affecting initiation (30) and can be used to saturate free cytoplasmic mRNA with ribosomes (18). As shown in Fig. 5, incubation of mature microsclerotia 10 with ltg of cycloheximide per ml, a concentration less than that necessary for maximal inhibition of protein synthesis in Physarum (6), resulted in the gradual appearance of polysomes at the expense of RSU. After 24 h of incubation, about 40% of the ribosomal material was observed in polysomes, a value which approximated that found in exponentially growing microplasmodia (Table 1). The induction of polysomes by cycloheximide also shows that our procedures are capable of extraction of these particles from microplasmodia, a finding consistent with the idea that no polysome compartmentalization occurs during dormancy. The induction of polysome formation in dormant microsclerotia by cycloheximide cannot be accounted for by new transcription. Similar percentages of RSU were driven into polysomes in microsclerotia pretreated with cordycepin or actinomycin D before cycloheximide addition to inhibit RNA synthesis (Table 1). Moreover, the relatively small increase in poly(A) titer ob-
served in cycloheximide-treated microsclerotia (Table 2), which was probably due to drug-specific interference with the normal turnover of this sequence (1), is inconsistent with the possibility that net poly(A)-RNA synthesis is the cause of polysome formation. These results suggest that mRNA, ribosomes, and the other components necessary for protein synthesis are stored in the sclerotia and that translation is repressed at the level of initiation during dormancy.
Polysome reformation during microsclerotial germination. Dormant microsclerotia can be induced to form microplasmodia by return to nutrient medium. Excystment occurs after a lag phase of 24 to 36 h. As shown in Fig. 6 and quantitated in Fig. 7, low levels of polysomes can be detected in microsclerotia as early 1 h after the addition of nutrient medium. Polysome formation at this time is resistant to actinomycin D (Fig. 7). The low levels of polysomes present throughout the lag phase, which include only about 10 to 20% of the available ribosomes, were not due to a deficiency in mRNA since, as was observed previously in dormant microsclerotia, 40 to 50% of the ribosomes could be driven into the polysomes by cycloheximide. However, in contrast to the situation observed earlier in microsclerotia, 50% of the ribosomes were accumulated in polysomes after only 1 h of cycloheximide treatment. Thus, the rate of polysome accumulation
as
494
J. BACTERIOL.
JEFFERY
in cycloheximide-treated microsclerotia appears to be significantly stimulated after nutrient addition. During the excystment period, polysome levels dramatically increased at the expense of RSU (Fig. 6 and 7). At this time the proportion of ribosomes in polysomes rose to the highest levels (53% at 48 h; 65% at 58 h) encountered in this investigation. Since this rise was considerably depressed by actinomycin D administered during the lag phase, it is probably dependent on new transcription in the excysted microplasmodia.
DISCUSSION The primary purpose of the present investigation was to determine whether translational activity persists during sclerotization, a form of dormancy experienced by plasmodia of the acellular slime mold P. polycephalum. To avoid the uncertainties related to variable precursor permeability and pool size during development, this question was approached by examining the levels of ribosomes incorporated into polysomes. The results demonstrate that, although significant levels of polysomes are present throughout
.:
FIG. 3. Relative levels of polysomes and RSU in the post-mitochondrial fractions of P. polycephalum during the transition from active growth to dormancy. Polysome and RSU levels were determined as described in the text. The shaded area represents the approximate duration of the encystment period.
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TABLE 1. Effect of drugs on polysome levels in exponentially growing microplasmodia and dormant microscierotia of P. polycephalum %
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Drug treatment
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44.2
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±
6.6 (6)
Microsclerotiab
Undetectable 44.2 ± 2.5 (12)
Undetectable 36.4 ± 3.1 (3) Undetectable 46.2 ± 4.5 (3)
aThe percentage of ribosomal material in polydescribed in the text. b Microsclerotia dormant for 12 weeks were utilized. c The data are expressed as mean percentage ± standard error. The number of experiments appears in somes was determined as
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FIG. 4. Zone sedimentation of post-mitochondrial fractions derived from (A) a mixture of microplasmodia and microsclerotia and (B) detergent-treated microsclerotia of P. polycephalum. In A, equal volumes of microplasmodia and microsclerotia were mixed before homogenization. In B, the extraction media contained 0.5% Triton X-100 and 1% sodium deoxycholate. Other details are similar to those in Fig. 1.
the starvation period which precede dormancy and the lag phase before germination, translational activity is severely repressed at the level was 10 of initiation in the dormant sclerotium. ig/nml. 'The cordycepin concentration utilized was 200 ug/ The persistence of substantial levels of polyml (9). somes during the starvation period and the enf Microsclerotia were preincubated in cordycepin or cystment process is consistent with earlier radioactinomycin D for 2 h before the addition of 10 ug of active precursor incorporation studies, which cycloheximide per mL Cordycepin and actinomycin D suggested a continuation of protein sythesis at were shown to inhibit the incorporation of [3Hjuridine into the add-insoluble material of 96-h-starved mi- moderate rates during the sclerotial induction crosclerotia by 92 and 83%, respectively (Jeffery, un- period (27). The contrast between Physarum plasmodia and starved mammalian cells is published data). 'The actinomycin D concentration used was 300 marked in this regard. Mammalian polysomes are rapidly dissociated after the beginning of plg/ml (26). parenthese. d The cycloheximide concentration utilized
TRANSLATIONAL REGULATION OF DORMANT PHYSARUM
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FIG. 5. Polysome formation in cyclohexumidetreated microsclerotia of P. polycephalum. Dorwa microsclerotia, cultured for 13 days after the siwitch to nonnutrient medium, were supplemented wiith 10 pg of cycloheximide per ml. At various interva~ Is the post-mitochondrial fractions were prepared anci subjected to zone sedimentation. (A) Post-mitochondrLal fraction of the 13-day microsclerotiun. Post- mitochondrial fractions derived from cultures (B) I1 (C) 6 h, and (D) 24 h after cycloheximide addition. I h details are similar to those in Fig. 1.
starvation, releasing the mRNA as a free icytoplasmic particle (17, 32). Presumably, the mobilization of plasnodial autocatabolic proc esses by starvation somehow prevents polysome dlissociation in Physarum until the time of enIcystment. Although protein synthesis appears to b e arrested in dormant sclerotia, functional mlINA, ribosomes, and the other factors and precursors necessary to support tranalation must be present. This is evidenced by the accumulati4 on of polysomes in cycloheximide-treated scle trotia under conditions in which new transcriptiion is blocked. The appearance of polysomes in c :ycloheximide-treated sclerotia suggests that t ranslation is repressed at the level of initiatii on in dormant Physarum. Potentially translaLtable mRNA, stored in the small-particle fracti on of mammalian celLs, can also be driven into polysomes by cycloheximide treatment (8, 16). It is impressive that similar levels of ribosome are eventually accumulated in the polysomes (Df cycloheximide-treated sclerotia as are observred in
495
exponentially growing cells. Since this situation occurred in the presence of an excess of RSU, it argues for the existence of similar proportions of potentially active mRNA in the cytoplasm of both developmental stages. The possibility that low levels of protein synthesis occur in dormant sclerotia is not excluded by the absence of detectable polysomes in gradients prepared from their lysates. Indeed, that the gradual accumulation of polysomes after polypeptide translocation is slowed by cycloheximide suggests that initiation is occurring at low rates in the sclerotia. This is also supported by the detection of low levels of radioactive amino acid incorporation into the acid-insoluble fraction during dormancy (Jeffery, unpublished data). The existence of depressed, but not entirely eliminated, translational activities in dormant Physarum is consistent with what has been observed during dormancy in other eucaryotic organisms such as fungal spores (3), plant seeds (23), and unfertilized sea urchin eggs (13, 19). Nutrient addition to dormant sclerotia results in the appearance of polysomes within 1 h. Polysome formation at this time appears to be based on the utilization of preformed mRNA conserved in the sclerotium, rather than newly synthesized transcripts, since it is completely resistant to actinomycin D. This finding is consistent with the results of an earlier report which showed that actinomycin D did not affect protein synthesis after sclerotia were returned to
nutrient medium (5). Although only low levels of polysomes were observed during the long lag period which preceded sclerotial excystment, the proportion of TABLE 2. Poly(A) content of normal and
cycloheximide-treated microscierotia of P. polycephaluma [3H]poly(U) hyPoyA bridizedP)ly RNA MiCrosclerotiumb (A2/ cpm/ pg4tg % MYcpm/ml Am1, of of RNA
Control Cyclohexiniide-
5.4 6.2
26,840 4,970 40,110 6,469
RNAd
147 191
0.015 0.019
treatede a Poly(A) content was measured by [3H]polyuridylic acid ([3H]poly(U)) association as described previously (14). Microsclerotia dormant for 12 weeks were used. c A26, Absorbance at 260 nm. The poly(A) content was calculated from the specific activity of [3H]poly(U). ' Microsclerotia were incubated in 10 jig of cycloheximide per ml for 24 h. b
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FIG. 7. Relative levels of polysomes and RSU in the post-mitochondrial fractions of P. polycephalum during the transition from dormancy to active growth. Polysome and RSU levels were determined as described in the text. In some experiments, 300X g of actinomycin D per ml was added to the cultures with the nutrient medium. The shaded area represents the approximate duration of the excystment period.
potentially translatable mRNA in the cytoplasm, as measured by the ability of cycloheximide to drive ribosomes into the polysomes, remained high. It is significant that the rate of polysome accumulation in cycloheximidetreated sclerotia was much greater after nutrient addition. These observations suggest that the rate of translational initiation is markedly en-
hanced, although not to its potential maximum, after nutrient addition. The biochemical nature of the processes responsible for translational repression in dornant sclerotia and its activation after nutrient addition are currently unknown. Since all the necessary requirements for protein synthesis are available in sclerotia, it is conceivable that their translational activities may be limited by relative quantitative deficiencies of key substances or by active inhibitory mechanisms. The possibilities include a limited titer of one or more of the initiation factors or a protein-mediated repression of stored mRNA, ribosomes, or other translational elements. These possibilities are presently being investigated in this laboratory. ACKNOWLEDGMENTS The expert technical assistance of Mary Robinson is grate-
fully acknowledged. Financial support was provided by Public Health Service grant GM-25119 from the National Institute of General Medical Sciences and National Science Foundation grant PCM 7724767. LITERATURE CITED 1. Adams, D. S., and W. R. Jeffery. 1978. Polyadenylic acid degradation by two distinct processes in the cyto-
plasmic RNA of Physarum polycephalum. Biochemistry 17:4519-4524. 2. Baglioni, C., C. Vesco, and M. Jacobs-Lorena. 1969. The role of ribosomal subunits in mammalian cells. Cold Spring Harbor Symp. Quant. Biol. 34:555-565. 3. Brambl, R., L. D. Dunkle, and J. L. Van Etten. 1978. Nucleic acid and protein synthesis during fungal spore germination, p. 94-118. In J. E. Smigh and D. R. Berry (ed.), The filamentous fungi: developmental mycology.
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Halsted Press, New York. 4. Brewer, E. N. 1972. Polysome profiles, amino acid incorporation in vitro, and polysome reaggregation following disaggregation by heat shock through the mitotic cycle in Physarum polycephalum. Biochim. Biophys. Acta 277:639-645. 5. Chet, L, and H. P. Rusch. 1970. RNA and protein synthesis during germination of apherules in Physarum polycephalum. Biochim. Biophys. Acta 224:620-622. 6. Cummins, J. E., E. N. Brewer, and H. P. Rusch. 1965. The effect of actidione on mitosis in the slime mold Physarum polycephalum. J. Cell Biol. 27:337-341. 7. Daniel, J. W., and H. Baldwin. 1964. Methods for the culture of plasmodial Myxomycetes. Methods Cell Physiol. 1:9-41. 8. Fan, H., and S. Penman. 1970. Regulation of protein synthesis in mammalian cells. I. Inhibition of protein synthesis at the level of initiation during mitosis. J. Mol. Biol. 50:655-670. 9. Fouquet, H., R. Wick, R. Bohme, and H. W. Sauer. 1975. Effects of cordycepin in Physarum polycephalum. Arch. Biochem. Biophys. 168:273-280. 10. Goodman, E. M., and T. Beck. 1974. Metabolisn during differentiation in the slime mold Physarum polycephalum. Can. J. Microbiol. 20:107-111. 11. Goodman, E. M., and H. P. Ruwch. 1970. Ultrastructural changes during spherule formation in Physarurn polycephalum. J. Ultrastruct. Res. 30:172-183. 12. Guttes, E., and S. Guttes. 1963. Starvation and cell wall formation in the myxomycete Physarumpolycephalum. Ann. Bot. N. S. 27:49-53. 13. Humphreys, T. 1969. Efficiency of translation of messenger RNA before and after fertilization in sea urchins. Dev. Biol. 20:435-456. 14. Jeffery, W. R. 1977. Polyadenylation of maternal and newly-synthesized RNA during starfish oocyte maturation. Dev. Biol. 57:98-108. 15. Jump, J. A. 1954. Studies on sclerotization in Physarum polycephalum. Am. J. Bot. 41:561-567. 16. Lee, G. T., and D. L Englehardt. 1979. Peptide coding capacity of polysomal and non-polysomal messenger RNA during growth of animal cells. J. Mol. Biol. 129: 221-233. 17. Lee, S. Y., V. Krismanovic, and G. Brawerman. 1971. Initiation of polysome formation in mouse sarcoma 180 ascites cells. Utilization of cytoplasmic messenger RNA. Biochemistry 10:895-900. 18. Lodish, H. F. 1971. Alpha and beta globin messenger ribonucleic acid: different amounts and rates of initia-
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tion of translation. J. Biol. Chem. 246:7131-7138. 19. MacKintosh, F. R., and E. Bell. 1969. Regulation of protein synthesis in sea urchin eggs. J. Mol. Biol. 41: 365-380. 20. Mills, D. 1974. Isolation of polyribosomes from yeast during sporulation and vegetative growth. Appl. Microbiol. 27:944-949. 21. Mirkes, P. E. 1972. Polysomes and protein synthesis during development of Ilyanassa obsoleta. Exp. Cell Res. 74:503-508. 22. Orlowski, M., and P. S. Sypherd. 1978. Regulation of translation rate during morphogenesis in the fungus Mucor. Biochemistry 17:569-575. 23. Payne, P. L. 1976. The long-lived messenger ribonucleic acid of flowering plant seeds. Biol. Rev. 51:329-363. 24. Plaut, B. S., and G. Turnock. 1975. Coordination of macromolecular synthesis in the slime mold Physarum polycephalum. Mol. Gen. Genet. 137:211-225. 25. Sauer, H. W. 1973. Differentiation in Physarum polycephalum, p. 375-405. In J. M. Ashworth and J. E. Smith (ed.), Microbial differentiation. Cambridge University Press, New York. 26. Sauer, H. W., K. Babcock, and H. P. Rusch. 1969. Changes in RNA synthesis associated with differentiation (spherulation) in Physarum polycephalum. Biochim. Biophys. Acta 195:410-421. 27. Sauer, H. W., K. L. Babcock, and H. P. Ruwch. 1970. Changes in nucleic acid and protein synthesis during starvation and spherule formation in Physarum polycephalum. W. Roux. Arch. 165:110-124. 28. Schwarzer, M., and R. Braun. 1977. Preparation of polysomes from synchronous macroplasmodia of Physarumpolycephalum. Biochim. Biophys. Acta 479:501505. 29. Siegel, M., and H. Sisler. 1964. Site of action of cycloheximide in celLs of Saccharomyces pastorianus. I. Effect of the antibiotic on cellular metabolism. Biochim. Biophys. Acta 87:70-82. 30. Stanners, C. P. 1966. The effect of cyclohexinide on polyribosomes from hamster cells. Biochem. Biophys. Res. Commun. 24:758-764. 31. Stewart, P. A., and B. T. Stewart. 1961. Membrane formation during sclerotization of Physarum polycephalum. Exp. Cell Res. 23:471-478. 32. Yap, S. H., R. K. Strait, and D. A. Shafritz. 1978. Effect of a short term fast on the distribution of cytoplasmic albumin messenger ribonucleic acid in rat liver. Evidence for formation of free albumin messenger ribonucleoprotein particles. J. Biol. Chem. 253:4944-4950.
JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 490-497 0021-9193/79/11-0490/08$02.00/0
Translational Regulation of Polysome Formation During Dormancy of Physarum polycephalum WILLIAM R. JEFFERY Department of Zoology, University of Texas, Austin, Texas 78712 Received for publication 14 August 1979
The translational activity of actively growing microplasmodia and dormant microsclerotia of Physarum polycephalum was investigated by analyzing the distribution of ribosomes in polysomes. Microplasmodial post-mitochondrial fractions contained substantial amounts of polysomes and ribosomal subunits but very few native monosomes. During the starvation period which preceded microsclerotium formation, polysome levels remained constant, whereas the subunit titer began to increase. During encystment ribosomal subunits continued to accumulate as the level of polysomes gradually decreased. Dormant microsclerotia contained a large surplus of stored ribosomal subunits but no detectable polysomes. However, incubation of microsclerotia with concentrations of cycloheximide sufficient to slow polypeptide elongation without affecting initiation caused the gradual reappearance of polysomes at the expense of the subunits. Under these conditions the percentage of subunits driven into polysomes reached values similar to those of actively growing microplasmodia. Microsclerotia returned to nutrient medium contained very low levels of polysomes during the lag period which preceded germination. These were formed with preexisting, stored messenger ribonucleic acid. During the germination period, polysome levels were markedly increased. This elevation was dependent on new ribonucleic acid transcription. It is concluded that dormant microsclerotia contain functional messenger ribonucleic acid and ribosomes which are subject to translational repression at the level of initiation. As a response to adverse environmental conditions such as starvation, dessication, or suboptimal temperature, the plasmodial slime mold Physarum polycephalum can undergo a form of dormnancy known as sclerotization (15). During the sclerotization process the plasmodial cytoplasm is partitioned into small multinucleate cysts by intemal membrane formation (31). Aggregations of these cysts, called sclerotia, are characterized by a low metabolic rate (10) and the ability to remain viable for up to several years (15). Return to favorable environmental conditions induces the dormant sclerotia to revert to actively growing plasmodia. Dramatic biochemical changes accompany the induction of sclerotization by nutrient depletion (25-27). During the starvation period preceding encystment, macromolecular synthesis continues with biosynthetic precursors derived from the partial catabolism of DNA, RNA, and protein (27) and the utilization of plasmodial glycogen reserves (11). Although the biochemical changes which occur during the starvation period have been extensively studied, very little is known about the metabolic activities of the dormant sclerotium. For example, the funda490
mental question ofwhether cysts actively engage in protein synthesis during dormancy or store mRNA and ribosomes for use during plasmodial regeneration is unresolved. In the present study translational activity during the transition from active growth to dormancy and during plasmodial regeneration from dormant sclerotia has been investigated by analyzing the distribution of ribosomes in polysomes. It is demonstrated that plasmodial polysomes are gradually dissociated during the encystment periods; that functional, but relatively inactive, mRNA and ribosomes are sequestered in the mature sclerotium; and that polysomes are reformed with stored sclerotial mRNA during plasmodial regeneration. It is concluded that protein synthesis is translationally repressed at the level of initiation during dormancy.
MATERIALS AND METHODS Culture methods. P. polycephalum (Carolina strain) was grown as microplasmodia at 220C in agitated suspension culture on a rotary shaker with the medium described previously (7). Dormant microsclerotia were fonned from mid-exponential-phase microplasmodial cultures by starvation in a citrate-
TRANSLATIONAL REGULATION OF DORMANT PHYSARUM
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buffered (pH 4.6), nonnutrient salts medium (12). Germination of dormant microsclerotia was induced by their transfer back to the routine culture medium. Polysome preparation. Polysomes were prepared from microplasmodia and microsclerotia by a modified version of the method of Brewer (4). Mid-exponentialgrowth-phase microplasmodial cultures and dormant or germinating microsclerotial cultures were harvested by centrifugation at room temperature at 100 x g for 1 min. The supernatant was decanted, and the pellet was resuspended in 5 volumes of ice-cold 50 mM Trishydrochloride (pH 7.2), 100 mM KCl, 100 mM MgCl2, 200 mM sucrose, and 75 mM EDTA and homogenized by 15 up and down strokes of a ground glass homogenizer. Increasing the EDTA concentration from 25 mM (4) to 75 mM was consistently found to yield larger polysomes. The post-mitochondrial fraction was prepared from the homogenate by centrifugation at 20,000 x g for 10 min at 4°C. The supernatant fractions were removed from the tubes and immediately layered on
density gradients
or
stored in liquid N2.
Zone sedimentation. Zone sedimentation of the post-mitochondrial supernatant fraction was carried out at 40C through 12 ml, 10 to 50% linear sucrose density gradients. To preserve the integrity of the polysomes, it was necessary to dissolve the sucrose in 50 mM Tris-hydrochloride (pH 7.2), 100 mM KCl, and 100 mM MgCl2. The gradients were centrifuged in the Beckman SW41 rotor for 90 min at 35,000 rpm. The absorbance at 254 nm was recorded by pumping the gradients through an LKB Uvicord I spectrophotometric cell. Determination of ribosome distribution. The absorbance profiles obtained were traced onto paper, and the area contributed by the absorbance of sucrose (derived from blank gradients). was subtracted. The regions corresponding to ribosomal subunits (RSU), native monosomes, and RNase-produced monosomes and disomes (and occasionally trisomes) (Fig. 1A and B) were cut out and weighed. The level of polysomes was obtained by subtracting the weight of the native monosomal area from that of the total monosome and disome (and occasionally trisome) areas of RNasetreated samples. The level of RSU was estimated directly from the weight of the area below the 60S and 40S peaks. The percentage of ribosomes in polysomes was obtained by dividing the weight of the polysomal area, obtained from RNase-treated profiles as described above, by the weight of the total ribosomal area.
Measurement of poly(A) content. The polyadenylic acid [poly(A)] content of RNA, extracted from the post-mitochondrial fraction of sclerotia by procedures described previously (1), was determined by the method of Jeffery (14). This involved bringing portions of the RNA preparation to 10 mM Tris-hydrochloride (pH 7.6), 200 mM NaCl, and 5 mM MgCl2 and mixing them with an excess of [3H]polyuridylic acid (5.14 Ci/ mmol; New England Nuclear Corp., Boston, Mass.). The mixture was incubated at 25°C for 15 min to promote annealing and then brought to 10 ,ug/ml with pancreatic RNase and incubated for 1 h at 37°C. Finally the digests were chilled, and the nucleic acids were precipitated with 10% cold trichloroacetic acid and collected for liquid scintillation counting on glass fiber filters.
491
RESULTS Characteristics of microplasmodial polysomes. The sedimentation proffies of post-mitochondrial fractions prepared from exponentially growing, vegetative microplasmodia are shown in Fig. 1. Ribosome distribution in polysomes and lighter particles was somewhat different than that previously reported for Physarum microplasmodia (4, 24). The major differences observed were an enrichment of RSU and a deficiency in single ribosomes (Fig. 1A). RNase treatment of the post-mitochondrial fraction before centrifugation converted the polysomes to lighter particles, mainly monosomes and disomes, but did not affect the RSU levels (Fig. 1B; no effect of RNase on RSU levels was observed in 25 separate experiments). An RNaseinsensitive, UV-absorbing material previously identified as glycogen particles (28) remained in the high-molecular-weight region of the gradient after RNase treatment. Incomplete polysome digestion by RNase resulting in accumulation of disomes has also been reported in other eucaryotes (19, 21). Since it is unusual for actively growing cells to contain proportionately larger RSU than monosome pools (2), further experiments were designed to determine whether this result was caused by the extraction conditions. For example, it is possible that the high RSU levels were due to polysome runoff without reinitiation during the sample preparation. To test this possibility, cycloheximide which slows polypeptide elongation (29) was added to the extraction media and the gradients. As shown in Fig. 1C, cycloheximide addition did not reduce RSU levels, suggesting that they were not caused by runoff. It is also unlikely that the high RSU levels were due to the excessive amounts of EDTA and Mg2e required to inhibit endogeneous RNase activity in Physarum (4) since they were also present when the concentrations of these components in the extraction media were significantly reduced (Fig. 1D). Similar levels of RSU were also observed when polysomes were extracted with various EDTA-Mg2+ ratios including the 25 mM/100 mM ratio originally employed by Brewer (4). The apparent excess of RSU could also be due to selective binding of polysomes and monosomes to membranes or other insoluble microplasmodial structures. This possibility was checked by the addition of Triton X-100 and sodium deoxycholate to the extraction media. As shown in Fig. 1E, no alterations in the distribution of ribosomal material between polysomes, monosomes, and RSU were subsequently found. Thus, the results suggest that a high proportion of ribosomes exist in an RSU pool in Physarum microplasmodia. Large
J. BACTERIOL.
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FIG. 1. Zone sedimentation of the post-mitochondral firacton from P. polycephalum microplasmodia. (A) Poatnitochondrial fractions under normal extractin conditn. The arrows represent the position of40, 608, and 808 ribosomal markers derived from Ehrlich ascites tumor ceUs centrifuged on parallel gradients. (B) Post-mitochondrial fractions treated with 10 pg of pancreatic RNase A (RNase) per ml for 30 mmn on ice before centrifugatin. (C) Post-mitochondrial fractions prepared under conditions in which the extraction media and gradient were suppemented with 20 pg of cycloheximide per ml (D) Post-mitochondrial firactions prepared with extraction media composed of 50 mM Tris-hydrochloride (pH 7.6, 50 mM KCl4 I mM MgCI2, and 20 mM sucrose. The gradients contained the same concentration of salts. (E) Post-mitochondrial fractions prepared in the presence of 0.5% Triton X-100) and 1% sodium deoxychokate. The sedimentation direction is from right to left. The vertical dashed lines represent the posion which the absorbance scale is halved. The hatched and shaded surfaces ui A and B represent the areas from which the levels ofpolysomes and RSU, respectively, were cakulated as described in the text. in
RSU pools are also found in yeast (20) and the fungus Mucor (22). Polysome dissociation and RSU accumulation during microwlerotium formation. Dormant microsclerotia are formed from
starved microplasmodial cultures asynchronously between 24 and 36 h after the shift to nonnutrient medium. The sedimentation profiles of post-mitochondrial fractions derived from cultures at selected intervals in the starvation-encystment period and from mature microsclerotia are shown in Fig. 2A through D. Since the high-molecular-weight regions of the gradients contain glycogen particles as well as polysomes, indirect methods are required for the measurement of polysome titers. This was facilitated by measuring the levels of monosomes and disomes which appeared in RNase-treated post-mitochondrial fractions (Fig. 2E through H). Although lower proportions of ribosomes distributed in polysomes were observed in the present study (Table 1) compared with previous measurements (24), it is believed that the current estimations are more accurate since they exclude the glycogen components. The changes detected in polysome and RSU levels during sclerotization are summarized in Fig. 3. It can be seen that polysome levels remained substantial during the starvation period, decreased markedly during and after encystment, and were undetectable in microsclerotia by 72 h after the medium shift. During a similar interval, the level of RSU was increased threefold (Fig. 3), suggesting that encysting microsclerotia accnuulate and store ribosomes. The initial decline in RSU and polysomes observed after the medium shift is due to "transfer shock" and not the starvation condition itself, since a similar effect occurred when microplasmodia were switched to fresh nutrient medium. The inability to detect polysomes in mature microsclerotia could be due to depressed translatl activity. This observation, however, could also be explained by polysome compartmentalization or the presence of an activated cyst-specific, polysome-uncoupling agent in the microsclerotia. The possibility of a polysomeuncoupling agent activated during the extraction procedure was tested by mixing microsclerotia and microplasmodia before homogenization. As shown in Fig. 4A the sedimentation profiles obtained from the post-mitochondrial fractions of this mixture exhibited polysomes excluding the possibility of an uncoupling agent. The absence of detectable polysomes in the microsclerotia was not due to compartmentalization either, since they were not released after detergent treatment (Fig. 4B) or by more extensive homogenizations. Thus, it appears that mature microsclerotia are translationally inactive. Induction of polysomes in dormuant microsclerotia. The lack of an essential component, such as mRNA, or the active repression of
TRANSLATIONAL REGULATION OF DORMANT PHYSARUM
VOL. 140, 1979
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FIG. 2. Zone sedimentation of the post-mitochondrial fractions from P. polycephalum during the transition from active growth to dormancy. (A) 1 h, (B) 24 h, (C) 36 h, and (D) 72 h after the switch to nonnutrient medium. (E to H) RNase-treated counterparts of A to D. Other details are similar to those in the legend to
Fig. 1. an otherwise completely functional protein synthetic machinery could cause the translational deficiency seen in dormant microsclerotia. It was therefore of interest to determine whether microsclerotial RSU could be driven into polysomes. Low levels of cycloheximide reduce polypeptide elongation rates without affecting initiation (30) and can be used to saturate free cytoplasmic mRNA with ribosomes (18). As shown in Fig. 5, incubation of mature microsclerotia 10 with ltg of cycloheximide per ml, a concentration less than that necessary for maximal inhibition of protein synthesis in Physarum (6), resulted in the gradual appearance of polysomes at the expense of RSU. After 24 h of incubation, about 40% of the ribosomal material was observed in polysomes, a value which approximated that found in exponentially growing microplasmodia (Table 1). The induction of polysomes by cycloheximide also shows that our procedures are capable of extraction of these particles from microplasmodia, a finding consistent with the idea that no polysome compartmentalization occurs during dormancy. The induction of polysome formation in dormant microsclerotia by cycloheximide cannot be accounted for by new transcription. Similar percentages of RSU were driven into polysomes in microsclerotia pretreated with cordycepin or actinomycin D before cycloheximide addition to inhibit RNA synthesis (Table 1). Moreover, the relatively small increase in poly(A) titer ob-
served in cycloheximide-treated microsclerotia (Table 2), which was probably due to drug-specific interference with the normal turnover of this sequence (1), is inconsistent with the possibility that net poly(A)-RNA synthesis is the cause of polysome formation. These results suggest that mRNA, ribosomes, and the other components necessary for protein synthesis are stored in the sclerotia and that translation is repressed at the level of initiation during dormancy.
Polysome reformation during microsclerotial germination. Dormant microsclerotia can be induced to form microplasmodia by return to nutrient medium. Excystment occurs after a lag phase of 24 to 36 h. As shown in Fig. 6 and quantitated in Fig. 7, low levels of polysomes can be detected in microsclerotia as early 1 h after the addition of nutrient medium. Polysome formation at this time is resistant to actinomycin D (Fig. 7). The low levels of polysomes present throughout the lag phase, which include only about 10 to 20% of the available ribosomes, were not due to a deficiency in mRNA since, as was observed previously in dormant microsclerotia, 40 to 50% of the ribosomes could be driven into the polysomes by cycloheximide. However, in contrast to the situation observed earlier in microsclerotia, 50% of the ribosomes were accumulated in polysomes after only 1 h of cycloheximide treatment. Thus, the rate of polysome accumulation
as
494
J. BACTERIOL.
JEFFERY
in cycloheximide-treated microsclerotia appears to be significantly stimulated after nutrient addition. During the excystment period, polysome levels dramatically increased at the expense of RSU (Fig. 6 and 7). At this time the proportion of ribosomes in polysomes rose to the highest levels (53% at 48 h; 65% at 58 h) encountered in this investigation. Since this rise was considerably depressed by actinomycin D administered during the lag phase, it is probably dependent on new transcription in the excysted microplasmodia.
DISCUSSION The primary purpose of the present investigation was to determine whether translational activity persists during sclerotization, a form of dormancy experienced by plasmodia of the acellular slime mold P. polycephalum. To avoid the uncertainties related to variable precursor permeability and pool size during development, this question was approached by examining the levels of ribosomes incorporated into polysomes. The results demonstrate that, although significant levels of polysomes are present throughout
.:
FIG. 3. Relative levels of polysomes and RSU in the post-mitochondrial fractions of P. polycephalum during the transition from active growth to dormancy. Polysome and RSU levels were determined as described in the text. The shaded area represents the approximate duration of the encystment period.
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Drug treatment
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44.2
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±
6.6 (6)
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Undetectable 44.2 ± 2.5 (12)
Undetectable 36.4 ± 3.1 (3) Undetectable 46.2 ± 4.5 (3)
aThe percentage of ribosomal material in polydescribed in the text. b Microsclerotia dormant for 12 weeks were utilized. c The data are expressed as mean percentage ± standard error. The number of experiments appears in somes was determined as
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FIG. 4. Zone sedimentation of post-mitochondrial fractions derived from (A) a mixture of microplasmodia and microsclerotia and (B) detergent-treated microsclerotia of P. polycephalum. In A, equal volumes of microplasmodia and microsclerotia were mixed before homogenization. In B, the extraction media contained 0.5% Triton X-100 and 1% sodium deoxycholate. Other details are similar to those in Fig. 1.
the starvation period which precede dormancy and the lag phase before germination, translational activity is severely repressed at the level was 10 of initiation in the dormant sclerotium. ig/nml. 'The cordycepin concentration utilized was 200 ug/ The persistence of substantial levels of polyml (9). somes during the starvation period and the enf Microsclerotia were preincubated in cordycepin or cystment process is consistent with earlier radioactinomycin D for 2 h before the addition of 10 ug of active precursor incorporation studies, which cycloheximide per mL Cordycepin and actinomycin D suggested a continuation of protein sythesis at were shown to inhibit the incorporation of [3Hjuridine into the add-insoluble material of 96-h-starved mi- moderate rates during the sclerotial induction crosclerotia by 92 and 83%, respectively (Jeffery, un- period (27). The contrast between Physarum plasmodia and starved mammalian cells is published data). 'The actinomycin D concentration used was 300 marked in this regard. Mammalian polysomes are rapidly dissociated after the beginning of plg/ml (26). parenthese. d The cycloheximide concentration utilized
TRANSLATIONAL REGULATION OF DORMANT PHYSARUM
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FIG. 5. Polysome formation in cyclohexumidetreated microsclerotia of P. polycephalum. Dorwa microsclerotia, cultured for 13 days after the siwitch to nonnutrient medium, were supplemented wiith 10 pg of cycloheximide per ml. At various interva~ Is the post-mitochondrial fractions were prepared anci subjected to zone sedimentation. (A) Post-mitochondrLal fraction of the 13-day microsclerotiun. Post- mitochondrial fractions derived from cultures (B) I1 (C) 6 h, and (D) 24 h after cycloheximide addition. I h details are similar to those in Fig. 1.
starvation, releasing the mRNA as a free icytoplasmic particle (17, 32). Presumably, the mobilization of plasnodial autocatabolic proc esses by starvation somehow prevents polysome dlissociation in Physarum until the time of enIcystment. Although protein synthesis appears to b e arrested in dormant sclerotia, functional mlINA, ribosomes, and the other factors and precursors necessary to support tranalation must be present. This is evidenced by the accumulati4 on of polysomes in cycloheximide-treated scle trotia under conditions in which new transcriptiion is blocked. The appearance of polysomes in c :ycloheximide-treated sclerotia suggests that t ranslation is repressed at the level of initiatii on in dormant Physarum. Potentially translaLtable mRNA, stored in the small-particle fracti on of mammalian celLs, can also be driven into polysomes by cycloheximide treatment (8, 16). It is impressive that similar levels of ribosome are eventually accumulated in the polysomes (Df cycloheximide-treated sclerotia as are observred in
495
exponentially growing cells. Since this situation occurred in the presence of an excess of RSU, it argues for the existence of similar proportions of potentially active mRNA in the cytoplasm of both developmental stages. The possibility that low levels of protein synthesis occur in dormant sclerotia is not excluded by the absence of detectable polysomes in gradients prepared from their lysates. Indeed, that the gradual accumulation of polysomes after polypeptide translocation is slowed by cycloheximide suggests that initiation is occurring at low rates in the sclerotia. This is also supported by the detection of low levels of radioactive amino acid incorporation into the acid-insoluble fraction during dormancy (Jeffery, unpublished data). The existence of depressed, but not entirely eliminated, translational activities in dormant Physarum is consistent with what has been observed during dormancy in other eucaryotic organisms such as fungal spores (3), plant seeds (23), and unfertilized sea urchin eggs (13, 19). Nutrient addition to dormant sclerotia results in the appearance of polysomes within 1 h. Polysome formation at this time appears to be based on the utilization of preformed mRNA conserved in the sclerotium, rather than newly synthesized transcripts, since it is completely resistant to actinomycin D. This finding is consistent with the results of an earlier report which showed that actinomycin D did not affect protein synthesis after sclerotia were returned to
nutrient medium (5). Although only low levels of polysomes were observed during the long lag period which preceded sclerotial excystment, the proportion of TABLE 2. Poly(A) content of normal and
cycloheximide-treated microscierotia of P. polycephaluma [3H]poly(U) hyPoyA bridizedP)ly RNA MiCrosclerotiumb (A2/ cpm/ pg4tg % MYcpm/ml Am1, of of RNA
Control Cyclohexiniide-
5.4 6.2
26,840 4,970 40,110 6,469
RNAd
147 191
0.015 0.019
treatede a Poly(A) content was measured by [3H]polyuridylic acid ([3H]poly(U)) association as described previously (14). Microsclerotia dormant for 12 weeks were used. c A26, Absorbance at 260 nm. The poly(A) content was calculated from the specific activity of [3H]poly(U). ' Microsclerotia were incubated in 10 jig of cycloheximide per ml for 24 h. b
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FIG. 7. Relative levels of polysomes and RSU in the post-mitochondrial fractions of P. polycephalum during the transition from dormancy to active growth. Polysome and RSU levels were determined as described in the text. In some experiments, 300X g of actinomycin D per ml was added to the cultures with the nutrient medium. The shaded area represents the approximate duration of the excystment period.
potentially translatable mRNA in the cytoplasm, as measured by the ability of cycloheximide to drive ribosomes into the polysomes, remained high. It is significant that the rate of polysome accumulation in cycloheximidetreated sclerotia was much greater after nutrient addition. These observations suggest that the rate of translational initiation is markedly en-
hanced, although not to its potential maximum, after nutrient addition. The biochemical nature of the processes responsible for translational repression in dornant sclerotia and its activation after nutrient addition are currently unknown. Since all the necessary requirements for protein synthesis are available in sclerotia, it is conceivable that their translational activities may be limited by relative quantitative deficiencies of key substances or by active inhibitory mechanisms. The possibilities include a limited titer of one or more of the initiation factors or a protein-mediated repression of stored mRNA, ribosomes, or other translational elements. These possibilities are presently being investigated in this laboratory. ACKNOWLEDGMENTS The expert technical assistance of Mary Robinson is grate-
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