Synthesis of macromolecules during microcyst germination in the cellular slime mold Polysphondylium pallidum

,,EVELOPMENTAL

65, 251-259 (1978)

BIOLOGY

Synthesis

of Macromolecules during Microcyst Cellular Slime Mold Polysphondylium

HERBERT

L. ENNIS,

Roche Institute

DIANE

of Molecular

PENNICA, Biology,

AND

Germination pallidurn JANET

in the

M. HILL

Nutley, New Jersey 07110

Received July 29, 1977; accepted in revised form April

7, 1978

Microcyst germination in the cellular slime mold Polysphondylium pallidum is a useful model for studying macromolecular changes necessary for or coincident with the transition from one cell type (cyst) to another (amoebae). Protein synthesis starts soon after cysts are incubated under permissive conditions, as evidenced by the incorporation of precursors and the appearance of polysomes. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins made at intervals during germination shows that protein synthesis is developmentally regulated during this process. RNA synthesis also begins early during germination. Cysts contain polyadenylated RNA that can stimulate the incorporation of radioactive amino acids into protein in an in vitro wheat germ protein synthesizing system. The concentration of poly(A)-containing RNA increases during germination and during inhibition of protein synthesis by cycloheximide. INTRODUCTION

Microcyst formation is an alternate developmental pathway to fruiting body construction in the life cycle of the cellular slime mold Polysphondylium pallidum (Blaskovics and Raper, 1957). In liquid cuIture, under conditions of high osmotic pressure, single amoebae are able to develop synchronously into microcysts, which are surrounded by a bilayered cell wall (Toama and Raper, 1967a,b). Incubation of microcysts under conditions of low osmotic pressure results in synchronous germination (Hohl et al., 1970). Germination of microcysts can be used as a simple model to study developmentally regulated events. It is relatively easy to collect large numbers of cysts for biochemical studies; germination occurs in buffer and therefore no exogenous supplies of nutrients are required; and radioactive precursors to protein and nucleic acid are incorporated during germination. It has been recently demonstrated that protein and RNA synthesis occur during microcyst germination (O’Day, 1974, 1976; O’Day et al., 1976). Protein synthesis has

been shown to be required for germination (Cotter and Raper, 1968; O’Day, 1974; O’Day et al., 1976), whereas it is suggested that new RNA synthesis is not necessary (O’Day et al., 1976). An increase in the intracellular concentration of two enzymes, alkaline phosphatase (O’Day, 1974) and acid protease (O’Day, 1976), and an increase in the extracellular concentration of several lysosomal glycosidases (O’Day, 1974) have been reported. There is no evidence, however, that these enzymes are critical for germination. Consequently, we have investigated protein and RNA synthesis during microcyst germination. The present work describes the parameters that are important in using cyst germination as a paradigm for a developmental pathway. We have (a) established methods for studying the incorporation of precursors into macromolecules, (b) isolated and characterized RNA and protein species, and (c) demonstrated that protein synthesis is developmentally controlled. MATERIALS

AND

METHODS

Microcyst formation and isolation. Amoebae of strain WS-320 were grown in

251 0012.X06/78/0652-0251$02.00/O Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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DEVELOPMENTALBIOLOGY

a New Brunswick gyrotory water-bath shaker (at 100 rpm) in flasks containing sterile 10 mM K+-phosphate buffer, pH 6.7, 50 pg/ml streptomycin, and 2.5 X log/ml autoclaved Escherichia coli B. The doubling time at 23°C is approximately 8 hr. About 1 day after the cells had entered the lag phase of growth, 0.1 M KC1 (final concentration) was added (Toama and Raper, 1967a). The incubation was continued for an additional 3-4 days, at which time greater than 90% of the amoebae had encysted. The microcysts were collected by centrifugation at 5000g for 4 min and suspended in cold phosphate buffer containing 0.5% Nonidet P-40, which lyses all amoebae present but has no effect on the viability of the cysts. Using this procedure, suspensions of pure cysts are obtained. The cysts were then washed with cold phosphate buffer an additional three times. The cysts were used immediately or frozen at -20°C in phosphate buffer containing 20% dimethylsulfoxide (final concentration, v/v). Cysts frozen in this way are viable for at least 6 months, and routinely at least 90% germinate within 4-5 hr in subsequent experiments. Electron micrographs of detergenttreated-frozen cysts and each stage in their germination are morphologically identical to those of untreated cysts. Each batch of cysts was plated on nutrient agar to monitor bacterial or mold contamination. Conditions for germination. Frozen cysts were washed free of dimethylsulfoxide by centrifugation. Dimethylsulfoxide in a final concentration greater than 2% inhibits germination, so it is important to wash three times with cold phosphate buffer. The cysts were suspended at approximately 1 X 107/ml in 10 mM K+-phosphate buffer, pH 6.7, containing 0.75% ethanol. It was accidentally observed that ethanol, at this concentration, stimulated germination. Incubation was carried out in a New Brunswick gyrotory water bath shaker (at 100 rpm) at 23°C. Germination was monitored microscopically. Labeling with radioactive precursors.

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[4,5-3H]Leucine (New England Nuclear Corp.; specific activity, 5 Ci/mmole) at 20 &i/ml and [6-3H]uracil (New England Nuclear Corp.; specific activity, 21.9 Ci/ mmole) at 20 pCi/ml were routinely added to monitor incorporation into protein and RNA, respectively. Aliquots of 1.0 ml (containing 1 x lo7 cells/ml) were removed at intervals following the addition of labeled precursors and precipitated with an equal volume of cold 10% trichloroacetic acid. The trichloroacetic acid-insoluble material was collected on glass-fiber filters (Reeve Angel 934 AH or Whatman GF/C, 2.4 cm), dried, placed in vials containing Omnifluor (New England Nuclear Corp.), and counted in a Beck&an LS-100 scintillation counter. When incorporation into protein was measured, the labeled samples were boiled for 10 min prior to filtration. RNA extraction and analysis. Samples for RNA preparation were collected by centrifugation and frozen immediately. Frozen pellets representing about 5 x lOa cysts were disrupted by grinding with dry ice, then quickly suspended in cold 0.05 M Tris-HCl, pH 7.5, to which 0.1% Macaloid (Baroid Division, National Lead Co., Houston, Texas) and 2% sodium dodecyl sulfate (SDS) were added to inhibit nucleases. To this, 1.5-fold the volume of a mixture of phenol:chloroform:isoamylalcohol (66:33:1) was added (Jacobson, 1976). Phenol extraction was carried out in the cold for 5 min. The aqueous phase was collected and reextracted two more times with the phenol:chloroform:isoamylalcohol mixture. After the final extraction, 0.2 vol of 2 M sodium acetate, pH 5.1, and 2.5 vol of absolute ethanol were added to the aqueous phase. The RNA was allowed to precipitate overnight at -20°C. The following day, the RNA was collected by centrifugation (12,OOOg,20 min), washed two times with 70% ethanol, and finally suspended in sterile water. Sucrose density gradient analysis of RNA. RNA (1 ml) was applied to a 29-ml

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PENNICA,

AND

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5-20% sucrose density gradient in 0.1 A4 ble material was collected on GF/C filters, Tris-HCl, pH 7.5, 0.1 M NaCl, and 0.5% and the filters were then dried and counted SDS. Sedimentation was at 20,OOOgfor 18 in Omnifluor (New England Nuclear) in a spectromehr at 22°C in a Spinco SW 25.1 rotor. One- Beckman LS-100 scintillation milliliter fractions were collected, the ab- ter. All values were corrected for the ensorbance (at 260 nm) was determined, and dogenous reaction in the absence of added the sample was then mixed with 1 ml of RNA. Hybridization of RNA with fH]poly(U). Hz0 and 10 ml of Instabray (Yorktown Research); total radioactivity in each frac- The reaction was carried out as previously described (Bishop et al., 1974). The total tion was counted. reaction of 50 ~1 contained 1 pg of [5-3H] chromatography. OLigo(dT)-cellulose poly(U) (50 pmoles, 0.033 @i, 19,800 cpm) Oligo(dT)-cellulose (Type T3, Collaboraand 5 and 10 pg of RNA, in 2~ SSC (done tive Research, Waltham, Massachusetts) fractionation of RNA was carried out at in duplicate). The reaction was incubated at 45°C for 15 min and diluted to 1 ml with room temperature as previously described cold 2~ SSC. Pancreatic RNase (25 pg/ml) (Giri and Ennis, 1977). The “binding buffer” contained 0.01 M Tris-HCl, pH 7.5, was added and the reaction mixture was and 0.5 N NaCl; the “elution buffer” was incubated for 60 min at 25°C. The RNaseresistant hybrids were precipitated with 1.5 0.01 M Tris-HCl, pH 7.5. The unbound ml of cold 10% trichloroacetic acid, in the fractions and bound fractions were pooled presence of 25 pg of bovine serum albumin separately. Translation of RNA fractions in the added to the reaction as a carrier. The wheat germ cell-free system. The system precipitates were collected by filtration as described by Roberts and Paterson (1973) described previously and counted. The was used, with a few modifications. The added RNA was in the linear range and did wheat germ was obtained from Niblack and not saturate the [3H]poly(U). it was not necessary to preincubate the RESULTS extract since the endogenous activity was low (less than 5% of the activity). The final Kinetics of Germination concentrations of constituents in the reacThe kinetics of appearance of amoebae is tion mixture were as follows: 1.3 mM ATP, given in Fig. 1. Emergence of amoebae 0.3 m&f GTP, 10 m&f creatine phosphate, starts at approximately 2 hr after incuba5 pg of creatine phosphokinase, 20 mM tion in phosphate buffer and proceeds rapHepes, 80-100 n-&f KCl, 2.5 n-&f magnesium acetate, 2 n-J4 dithiothreitol, 20 $kf complete amino acid mix lacking methionine, and 0.1 mM spermidine. In addition, to a 50+1 reaction mix, 20 ~1 of wheat germ extract, 10 pC!i of 35[S]methionine (600-1000 mCi/mmole; Amersham/Searle Co.), and 20 pg of RNA were added. Incorporation is proportional to the amount of RNA added and is therefore a reflection of the mRNA content of the sample. The reaction mixture 0 2 3 4 was incubated for 1 hr at 25°C. The label incorporated into trichloroacetic acid-insolFIG. 1. Kinetics of germination of P. pallidurn miuble protein was determined by adding 10% crocysts. Conditions for germination are described untrichloroacetic acid and boiling the sample der Materials and Methods. Germination is scored as for 10 min. The trichloroacetic acid-insoluthe percentage of amoebae present at any time.

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DEVELOPMENTAL BIOLOGY

idly thereafter. Germination is complete at 3-4 hr. Using the light microscope, we are not able to adequately score cyst swelling, which is known to occur before the emergence of amoebae (Hohl et al., 1970). Therefore, this parameter of germination is not presented in Fig. 1. Germination is temperature dependent (data not presented). It occurs rapidly at 27 and 32”C, but is prevented completely at

37°C. Protein and RNA Synthesis during Germination (a) Incorporation of radioactive precursors. Protein synthesis, as evidenced by the incorporation of labeled amino acid, begins immediately after cysts are incubated in phosphate buffer (Fig. 2). This occurs well before amoebae emerge. In contrast, RNA synthesis proceeds much more slowly than protein synthesis. The incorporation into RNA is low early during germination but proceeds more rapidly later on. It is not known whether this is due to poor penetration of the [3H]uracil into cysts or to a large pool early during germination. The kinetics of incorporation is the same when proline, methionine, or an amino acid mixture is used instead of leucine and when adenine or [32P]phosphate is used instead of uracil.

VOLUME 65,1978

was no loss of polysomes in the 3-hr sample (data not presented).

(c) Developmental regulation ofproteins during cyst germination. SDS-polyacrylamide gel electrophoresis of proteins isolated from growing amoebae (Fig. 4A, lane 2), cysts (lane 3), and cyst walls (lane 4) was performed. It is evident that there are major differences in the proteins found in growing amoebae compared to those found in the soluble portion of the cysts. In addition, there are at least eight proteins unique to the cyst wall. The walls also appear to contain some proteins found in the soluble portion of the cyst. However, this may be due to slight contamination by cytoplasm.

TIME (hod

2. Protein and RNA synthesis during cyst germination. Germinating cysts were labeled as described under Materials and Methods. Radioactive compounds were added at zero time. (0) Incorporation into protein; (A) incorporation into RNA. FIG.

(b) Appearance ofpolysomes during germination. The kinetics of formation of polysomes during germination can also be used as an index of protein synthesis. As shown in Fig. 3, the majority of ribosomes in cysts is present as free monosomes. Polysomes appear rapidly after incubation of cysts under conditions which promote germination. A significant level of polysomes is present as early as 0.5 hr, and they continue to accumulate to higher levels later on. The absence of polysomes at zero time is not due to preferential degradation during preparation of early samples. When equal aliquots of cells from 0 and 3 hr were mixed and analyzed for polysomes, there

FIG. 3. Polysome formation during cyst germination. Samples were prepared as previously described (Giri and Ennis, 1977) by grinding with solid CO?. Approximately 6 Azm units were applied to sucrose density gradients (15-30%). Analysis of the gradients was performed as previously described (Giri and Ennis, 1977).

ENNIS, PENNICA, AND HILL

Cyst Germination

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in P. pallidum

I23

94 68 42 1.7

25.7

17.2

A

2

FIG. 4. SDS-polyacrylamide gel electrophoresis of proteins made during cyst germination. (A) Stained gels. Gels were stained with Coomassie blue. (1) Standards: phosphorylase B, 94,ooO, bovine serum albumin, 68,ooO; Escherichia coli EF-Tu, 42,ooO; a-chymotrypsinogen, 25,700; and myoglobin, 17,200. The numbers beside the gels are the estimated molecular weights x 1O-3. (2) Extract of amoebae. (3) Soluble extract of cysts. (4) Cyst walls. Amoebae were grown in association with autoclaved bacteria as described under Materials and Methods. Cysts or amoebae were disrupted by sonication and centrifuged at 10,OOOgfor 10 min. The soluble portion (supernatant) was collected. The cyst walls (pellet) were washed by cycles of suspension and centrifugation, two times in gel buffer containing 0.5% Nonidet P-40 and two times in gel buffer. Fifty micrograms of protein was applied to each lane in the gel. (B) Autoradiograms. Samples of 2 X 10’ cysts were labeled for 1.25~hr periods with [?S]methionine (50-100 aCi/ml). The cysts were collected and disrupted by sonication. The extract was centrifuged at 10,OOOg for 10 min and the supernatant was collected. Polyacrylamide gel electrophoresis (12.5%) and autoradiography were performed as previously described (Giri and Ennia, 1977). Approximately 50,000 cpm was applied to each lane. (1) Cysts labeled O-l.25 hr; (2) cysts labeled 1.25-2.5 hr; (3) cysts labeled 2.5-3.75 hr.

Gel analysis of proteins labeled with [35S]methionine at 1.25hr intervals during germination (Fig. 4B) shows that protein synthesis is developmentally regulated during this process. At 2.5 hr only 48% amoebae have emerged from the cysts, and at 3.75 hr over 95% are present as amoebae. A detailed account of the regulation of the synthesis of these proteins will be presented in another communication. For the present,

it is suffkient only to note that a large number of changes in the pattern of proteins synthesized at the different times occur. Stained gels of these samples show that there are no significant changes in the concentration of the major proteins during germination (data not shown). (d) RNA synthesis during germination. The types of RNA synthesized during each stage of germination were determined. To-

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DEVELOPMENTAL BIOLOGY

tal RNA labeled at hourly intervals during germination was fractionated by oligo(dT)cellulose chromatography. Fractionation of the total RNA on oligo(dT)-cellulose can provide an estimate of the amount of poly(A)-containing RNA labeled during each stage of germination (Table 1). It is TABLE

1

OLIco(dT)-CELLULOSE CHROMATOGRAPHY OF RNA LABELED DURING MICROCYST GERMINATION” Labeling period 0-4 o-1 1-2 2-3

Percentage of la- Percentage of lfbe1 inRpot(A) be1 inRp$(A)) 77.7 80.3 74.3

22.3 19.7 25.7

n Cysts were labeled with [YH]uracil for 1-hr periods during germination. The RNA was extracted and applied to oligo(dT)-cellulose columns as described under Materials and Methods. The total radioactivity recovered in the pooled fractions that did not bind to oligo(dT)-cellulose and in the pooled bound fractions was estimated.

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evident that during normal germination of cysts, the portion of label in the bound [poly(A)-containing or poly(A)+] fraction is constant throughout the process. Each sample of RNA described in Table 1 was further analyzed by sucrose density gradient sedimentation. Figure 5 (A-C) summarizes the data obtained with the RNA that did not bind to oligo(dT)-cellulose [poly(A)- RNA]. It can be seen that material cosedimenting with P. pallidum rRNA is present in germinating cysts as early as the first hour after activation (panel A). A large amount of the “unbound” RNA labeled O-l hr after activation sediments coincident with tRNA. At later times during germination, l-2 hr (panel B) and 2-3 hr (panel C), a greater portion of the unbound RNA sediments as rRNA. The mRNA species in P. pallidum are 17 and 26.5 S as determined by analytical sedimentation in a Spinco Model E centrifuge (data

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TOP

FRACTION

20

30 BOTTOM

NUMBER

FIG. 5. Sucrose density gradient sedimentation of RNA made during cyst germination. Cysts (5 x 10”) were labeled with [3H]uracil at hourly intervals during germination. The RNA was extracted and applied to oligo(dT)cellulose columns. The RNA fractions containing poly(A)+ and poly(A)- RNA were pooled for each sample, and a portion of each was applied to a sucrose gradient. These are the same samples as described in Table 1. (A) Poly(A)- RNA labeled O-l hr; (B) poly(A)- RNA, l-2 hr; (C) poly(A)) RNA, 2-3 hr; (D) poly(A)’ RNA, O-l hr; (E) poly(A)+ RNA, l-2 hr; (F) poly(A)’ RNA, 2-3 hr. The arrows indicate the sedimentation of P. pallidurn 4, 17, and 26.5 S RNAs. [3H]Uracil, 40 @i/ml, was used to label cysts O-1 hr during germination; 20 @i/ml was used for the other samples.

ENNIS, PENNICA, AND HILL

not shown). The large species of rRNA is larger than the corresponding molecule in D. discoideum but smaller than HeLa cell rRNA. It can be seen that the newly synthesized rRNA (closed circles) at each interval is actually a little larger than 26.5 S. Perhaps it has not yet been processed to the mature form. It is not possible to obtain a precise determination of the amount of tRNA and rRNA in the unbound fraction because of the overlap of the various peaks in the sucrose density gradients. However, a rough estimate of the relative amounts can be obtained by determining the fraction of the total RNA applied to the gradient which is present in the corresponding species. Consequently, from Fig. 5 it can be estimated that in germinating cysts, the proportion of tRNA in the unbound RNA fraction made from O-l hr is 40.5%, that from l-2 hr is 20.3%, and that from 2-3 hr is 17.6%. It is evident from these results that more rRNA is made during the later stages of germination than at early times. Conversely, the fraction of RNA that is tRNA decreases during germination. The sedimentation profiles of the poly(A)+ RNA are also shown in Fig. 5 (D-F). All of the samples showed similar profiles. Most of the poly(A)-containing RNA sedimented between 6 and 28 S, with the peak at 12-13 S. The poly(A)-containing fraction as seen from these gradients is not contaminated to a large extent with rRNA, although, as shown in panel E, some slight contamination is seen. (e) mRNA content at different stages of germination. The ability of cyst RNA and RNA isolated during various stages of germination to stimulate the in vitro incorporation of amino acids into protein was determined. Total RNAs from 0-hr cysts, from cysts at 1.5 hr after incubation (swollen), and from cysts at 3 hr (emergence of amoebae) were isolated and tested for mRNA activity in the wheat germ cell-free protein synthesizing system. RNA from cysts incubated

Cyst Germination

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in P. pallidum

for 3 hr in the presence of cycloheximide was also tested. From the data summarized in Table 2, three conclusions can be derived. (1) Total unfractionated RNA isolated from cysts and during germination is able to stimulate the incorporation of amino acids into protein in wheat germ extracts. Furthermore, the specific stimulatory activity of this RNA (measured as counts per minute per microgram of total RNA) increases during germination. This stimulatory activity is presumably a function of the fraction of the total RNA that is mRNA. Poly(A)-containing RNA binding to oligo(dT)-cellulose is found in cysts as well as in swollen cysts and amoebae (data not presented). (2) Hybridization of total RNA with [3H]poly(U) also showed that the fraction of total RNA containing poly(A) increased during germination. It has been consistently observed that the increase in hybridization of the RNA with poly(U) during germination was greater than the increase in the specific stimulatory activity. This may be due to the later lengthening of the poly(A) tracts on mRNA present early during germination. (3) Cysts treated with cycloheximide apparently accumulate mRNA to the same level as that TABLE

2

In Vitro TRANSLATION OF RNA FROM GERMINATING CYSTS AND HYBRIDIZATION OF THE RNA TO r3H]POLY (U)” SogFAof

Cysts (0 hr) 1.5 hr 3 hr 3 hr + cycloheximide

Specific stimulatory activity (cpm incorporated/pg of RNA) 1264 1892 2258 2700

(cpm in RNaseresistant hybrid) 270 716 1052 808

n Total RNAs from 0-hr cysts, from 1.5-hr (swollen) and 3-hr (emergence of amoebae) germinating cysts, and from cysts incubated for 3 hr in the presence of cycloheximide (300 pg/ml) were isolated and tested for mRNA activity in the wheat germ cell-free synthesizing system as described under Materials and Methods. Hybridization of these RNAs to [“H]poly(U) was carried out as described under Materials and Methods.

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DEVELOPMENTALBIOLOGY

found in 3-hr untreated cysts. (fl Ribosome synthesis during germination. The data in section d show that ribosomal RNA is made during germination. It was next of interest to determine whether this ribosomal RNA was incorporated into ribosomes. The experiment summarized in Fig. 6 shows that, indeed, ribosomes are made during germination. However, more of the labeled RNA made from 2-3 hr after the initiation of germination is subsequently found in ribosomes than of that made during the early stages. The fraction of RNA labeled O-l hr eventually appearing in ribosomes is 36%; that of RNA labeled l-2 hr is 54%; and that of RNA labeled 2-3 hr is 74%. DISCUSSION

The results obtained in this investigation show that microcyst germination in P. pallidum can be used as a paradigm for studying macromolecular changes that occur during transition from one cell type (cyst) to another (amoeba). The organism is easily manipulated and amenable to biochemical analysis; germination is synchronous and is complete in a few hours; clearly identifiable morphological changes accompany this transition; the environment can be controlled; a large number of cells can be collected; and mutants should be readily obtainable. In our initial investigation on this aspect of slime mold development, we focus on (a) establishing methods for studying germination and (b) isolating and identifying proteins and RNA that are synthesized during germination. Protein synthesis as shown by incorporation of precursors and by appearance of polysomes starts soon after cysts are incubated under permissive conditions (Figs. 2 and 3). SDS-polyacrylamide gel analysis of proteins labeled with [35S]methionine at intervals during germination shows that protein synthesis is developmentally regulated during this process (Fig. 4B). A large number of changes occur in the quantity and types of proteins synthesized during these

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FIG. 6. Ribosome synthesis during cyst germination. Cysts (2 x 10”) were labeled with [3H]uracil (20 ,&i/pl) at hourly intervals during germination. After the hour of labeling, nonradioactive uracil (100 pg/pl) was added and incubation was continued for another hour. The celIs were then collected, suspended in approximately 1.5 ml of buffer containing 10 mM Tris-Cl (pH 7.8), 0.2 m&f magnesium acetate, and 10 m&f NH&l. The suspension was sonicated, the extract was centrifuged at 10,OOOgfor 5 min, and the supernatant was collected and dialyzed for 4 hr in the cold against the same buffer. One milliliter was layered on 29 ml of a 15-30% sucrose density gradient made up in the same buffer and then centrifuged in a Spinco SW 25.1 rotor at 21,090 rpm for 17 hr. One-milliliter samples were collected and analyzed for optical density and radioactivity. The fraction of total radioactivity in the gradient in ribosome subunits was calculated and is given in the text.

intervals. Four classes of proteins can be distinguished on these gels (a) proteins labeled early (O-l.25 hr) but not labeled at later times during germination (an example of which is band A in Fig. 4B); (b) proteins that are synthesized in larger quantities at early stages than at later times (band B); (c) proteins that appear only after the first 1.25 hr of labeling (band C); and (d) proteins labeled throughout germination (band D). Similarly, it has also been shown that protein synthesis is developmentally regulated during microcyst formation (Francis, 1976). RNA synthesis also begins early during germination (Fig. 2). Analysis of the RNA made during germination indicates that the quality of RNA made early vs that made late during germination changes (Fig. 5). It is clear that a large fraction of the RNA

ENNIS, PENNICA, AND HILL

made O-l hr postgermination is smaller than ribosomal RNA and may be tRNA. The RNA made at l-2 or 2-3 hr is predominantly ribosomal. Ribosomes are also made during germination (Fig. 6). More of the RNA made late during germination is eventually found in ribosomes than of that made during the first ,hour, again showing that the synthesis of specific types of RNA is developmentally regulated. In order to detect the presence of mRNA at different stages of germination, RNA was isolated from cysts and from later stages. mRNA was assayed by the ability of the RNA to stimulate incorporation of amino acids into protein in wheat germ extracts and by the increase in poly(A) content of the RNA by hybridization with [3H]poly(U). Using these two parameters, it is quite clear that the fraction of total RNA with mRNA activity increases during germination (Table 2). We have shown that developmentally regulated changes in protein and RNA synthesis occur during cyst germination of P. pallidurn. We feel, consequently, that cyst germination could be a model system for studying the molecular events that regulate the synthesis of specific mRNAs and their subsequent expression. I thank Dr. D. H. O’Day, who provided a culture of

P. pallidum strain WS-320. REFERENCES BISHOP, J. O., ROSBASH, M., and EVANS, D. (1974). Polynucleotide sequences in eukaryotic DNA and RNA that form ribonuclease-resistant complexes with polyuridylic acid. J. Mol. Biol. 85, 75-86. BLASKOVICS, J. C., and RAPER, K. B. (1957). Encyst-

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ment stages of Dictyostelium. Biol. Bull. 113,58-88. COTTER, D. A., and RAPER, K. B. (1968). Spore germination in strains of Dictyostelium discoideum and other members of the Dictyosteliaceae. J. Bacterial. 96,1690-1695. FRANCIS, D. (1976). Changes in protein synthesis during alternate pathways of differentiation in the cellular slime mold Polysphondylium pallidum. De-

velop. Biol. 53,63-72. GIRI, J. G., and ENNIS, H. L. (1977). Protein and RNA synthesis during spore germination in the cellular slime mold Dictyostelium discoideum. Biochem.

Biophys. Res. Commun. 77,282-289. HOHL, H. R., MIURA-SANTO, L. Y., and COTTER, D. A. (1970). Ultrastructural changes during formation and germination of microcysts in Polysphondylium pallidum, a cellular slime mold. J. Cell. Sci. 7, 285-305. JACOBSON, A. (1976). “Methods in Molecular Biology. Eukaryotes at the Subcellular Level,” pp. 161-209. Marcel Dekker, New York. O’DAY, D. H. (1974). Intracellular and extracellular enzyme patterns during microcyst germination in the cellular slime mold Polysphondyliumpallidum. Develop. Biol. 36, 401-410. O’DAY, D. H. (1976). Acid protease activity during germination of microcysts of the cellular slime mold Polysphondyliumpallidum. J. Bacterial. 125,8-13. O’DAY, D. H., GWYNNE, D. I., and BLAKEY, D. H. (1976). Microcyst germination in the cellular slime mold Polysphondylium pallidum. Effects of actinomycin D and cycloheximide on macromolecular synthesis and enzyme accumulation. Exp. Cell Res.

97,359-365. ROBERTS, B. E., and PATERSON, B. M. (1973). Efficient translation of tobacco mosaic virus RNA and rabbit globin 9s RNA in a cell-free system from commercial wheat germ. Proc. Nat. Acad. Sci. USA 70, 2330-2334. TOAMA, M. A., and RAPER, K. B. (1967a). Microcysts of the cellular slime mold Polysphondylium pallidum. I. Factors influencing microcyst formation. J.

Bacterial. 94,1143-1149. TOAMA, M. A., and RAPER, K. B. (1967b). Microcysts of the cellular slime mold Polysphondylium pallidum. II. Chemistry of the microcyst walls. J. Bacteriol. 94, 1150-1153.