Developmental changes in RNA and protein synthesis during germination of Dictyostelium discoideum spores

DEVELOPMENTAL

BIOLOGY

67, 189-201 (1978)

Developmental Changes in RNA and Protein Synthesis during Germination of Dictyostelirrm discoideum Spores JUDITH

G. GIRI’

Roche Znstitute of Molecular

AND HERBERT

L. ENNIS

Biology, Nutley, New Jersey 07110

Received May 151978; accepted in revised form July 10, 1978 Spore germination in Dictyostelium discoideum is a synchronous developmental process involving three distinct stages: activation, swelling, and emergence of amoebae. The regulation of protein and RNA synthesis during normal germination in the wild-type D. discoideum has been compared with the events that occur during abortive germination of mutant HE-1 and cycloheximide-treated wild-type spores, both of which are blocked at the emergence stage. Some aspects of the regulation of the synthesis of different types of RNA were investigated. RNA was isolated from germinating spores labeled at hourly intervals after activation. The RNA was fractionated by oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. Analysis of the RNA shows that the synthesis of mRNA, tRNA, and rRNA is developmentally regulated during germination. During the initial stages, the majority of the RNA made is mRNA and tRNA, while during later stages of germination, more rRNA is synthesized. In order to detect the presence of functional mRNA at different stages, RNA was isolated from dormant and germinating spores. Translatable mRNA was assayed by the ability of the RNA to stimulate incorporation of radioactive amino acids into protein in a wheat germ cell-free extract. The results show that dormant spores contain translatable polyadenylated mRNA, and the fraction of total RNA with mRNA activity increases during germination. From an analysis of the proteins synthesized during germination, it is evident that the synthesis of proteins is also developmentally regulated. Four classes of proteins could be distinguished on gels, depending on the time of onset and duration of their synthesis. A comparison of proteins made in vivo with those synthesized in vitro indicates that there are qualitative changes in the population of translatable mRNAs during germination, presumably due to differential gene transcription. Some of these changes observed in vitro correlate well with the differences in proteins synthesized in intact germinating spores. RNA and protein synthesis were compared in mutant HE-1 and in wild-type germination. In the mutant, unlike the wild type, there is no change in the proportion of mRNA made during germination, and rRNA represents a smaller portion of the total RNA made at any interval than in the wild type. In addition, it is noteworthy that the pattern of protein synthesis during abortive germination in the mutant is very different from that observed in the wild type. The control over the synthesis of different types of RNA and proteins during germination appears abnormal in the mutant, suggesting that developmental regulation is defective in the mutant. INTRODUCTION

The slime mold Dictyostelium discoideum is a particularly well-suited organism for the study of the control of gene expression during development. The life cycle can be broken down into a few relatively simple steps amenable to biochemical and genetic analysis; the life cycle can be synchronized;

and the cell number and growth conditions can be easily controlled (Sussman and BrackenburY, 197@* Control of gene expression during spore germination has not yet been investigated in detail. Spore germination in D. discoideum involves three distinct stages: activation, swelling, and emergence of amoebae (Cotter and Raper, 1966; Cotter, 1975). Germination is a synchronous developmental process which is completed within 3.5 hr after activation of dormant spores. Spores

i Present address: College of Physicians and Surgeons, Columbia University, Department of Human Genetics and Development, 701 West 168th Street, New York, New York 10032. 189

0012-1606/78/0671-0189$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved

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do not contain polysomes, but polysomes and mRNA syntheses are developmentally appear during the swelling stage, coincident regulated during spore germination. Conwith the beginning of incorporation of la- sequently, spore germination in D. discoibeled amino acids into protein (Giri and deum appears to be a useful model for Ennis, 1977). RNA synthesis also begins studying regulatory mechanisms that occur during the swelling stage (Giri and Ennis, during this simple developmental event. 1977; Yagura and Iwabuchi, 1976). InhibiMATERIALS AND METHODS tors of protein or RNA synthesis interfere Spore germination. Spores of wild- type with spore germination, indicating that D. discoideum strain B (referred to as wt), both protein and RNA synthesis are reor in the germination-defective mutant HEquired for germination (Cotter et al., 1969; 1, were grown and stored as previously deGiri and Ennis, 1977). scribed (Ennis and Sussman, 1975). HE-l Several mutants defective in spore germination have been isolated. One of these, was called mutant B previously (Ennis and mutant HE-1 [previously called mutant B Sussman, 1975; Giri and Ennis, 1977). Isolation of RNA. To characterize the (Giri and Ennis, 1977)], swelled normally types of RNA synthesized during germinabut was shown to be blocked in emergence of amoebae. Electron micrographs showed tion (Figs. 2 and 3), 5-7 x 10’ spores were that the defect in germination is due to the activated and labeled with 50 pCi/ml of [63H]uracil (New England Nuclear Corp., inability of this mutant to digest the innermost spore coat layer; consequently, the 25.6 Ci/mmole). To label wt spores during the first hour of germination, as well as for amoeba is unable to emerge (unpublished the analyses of mutant HE-l, or wt spores data). Developmentally regulated changes in incubated in the presence of cycloheximide, the synthesis of macromolecules have been 100 &i/ml was added instead of 50 @.X/ml. described for other stages in the life cycle At the end of the labeling period, the cells and the of D. discoideum and in another slime were collected by centrifugation pellets were frozen. mold, Polysphondylium pallidurn (Alton The frozen samples were disrupted by and Lodish, 1977; Francis, 1976). In the grinding with dry ice in a cold mortar. The present study, we extend these observations samples were then suspended in cold 0.05 to include spore germination. The regulaM Tris-HCl, pH 7.5, containing 0.1% Mation of protein and RNA synthesis during caloid (Baroid Division, National Lead Co., spore germination in wild-type D. discoideum has been compared with those events Houston, Tex.) and 2% SDS. One and onethat occur during abortive germination of half volumes of a mixture of phenol: chloroform:isoamyl alcohol (66:33:1) was mutant HE-1 and cycloheximide-treated added and the phenol extraction was carwild-type spores. In the latter two situations, swelling occurs, but the emergence of ried out in the cold as previously described amoebae is blocked (Cotter et al., 1969; (Jacobson, 1976). The extraction was reEnnis and Sussman, 1975). The use of mu- peated three times. To the final aqueous layer, 0.2 vol of 2 M NaOAc, pH 5.1, and tants defective in germination and inhibitors that block germination can provide one 2.5 vol of 95% ethanol were added, and the way of identifying key events and control mechanisms in the developmental pathmRNA, messenger RNA; rRNA, ribosomal RNA; tRNA, transfer RNA; oligo(dT)-cellulose, oligothymiway. dylic acid-cellulose; poly(A), polyadenylic acid; We show that protein, rRNA,’ tRNA, poly(A)’ RNA, RNA containing polyadenylic acid ’ Abbreviations used: wt, wild-type D. discoideum; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis,

that binds to oligo(dT)-cellulose; poly(A)lacking polyadenylic acid that does oligo(dT)-cellulose; TCA, trichloroacetic

RNA, RNA not bind to acid.

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RNA was allowed to precipitate overnight at -20°C. The following day, the RNA was collected by centrifugation (lO,OOOg, 15 min), washed two to three times with 70% ethanol, and finally suspended in sterile distilled water. To prepare unlabeled RNA for in vitro translation, about 10” spores were used, yielding from 280 to 450 ODZSOunits, depending on the efficiency of disruption of the spores. Sedimentation of RNA in sucrose gradients. Labeled RNA samples were applied to Xi-30 or 5-20s sucrose gradients, prepared in 0.1 A4 Tris-HCl, pH 7.4, 0.1 M NaCl, and 0.5% SDS. Sedimentation was at 20,000 rpm for 18 hr at 22°C in a Spinco SW25-1 rotor. One-milliliter fractions were collected by the use of an Isco density gradient fractionator. To each fraction, 1.0 ml of water and 10 ml of Instabray (Yorktown Research) were added, and the total radioactivity in each fraction was determined by counting in a Beckman LS-100 liquid scintillation spectrometer. Oligo(dT) - cellulose chromatography. Oligo(dT)-cellulose (Type T3, Collaborative Research, Waltham, Mass.) fractionation of RNA was carried out at room temperature as previously described (Aviv and Leder, 1972). The “binding buffer” contained 0.01 M Tris-HCl, pH 7.5, and 0.5 M NaC1; the “elution buffer” was 0.01 M Tris-HCl, pH 7.5. The unbound fractions and bound fractions were pooled separately and, for in vitro translation experiments, concentrated by precipitation with ethanol. All unbound fractions used in in vitro translation were chromatographed twice. Translation of RNA fractions in the wheat germ cell-free system. The system described by Roberts and Paterson (1973) was used, with a few modifications. The wheat germ was from Niblack and it was not necessary to preincubate the extract as the endogenous activity was low (less than 5% of the activity). The final concentrations of constituents in the reaction mixture were as follows: 1.3 mJ4 ATP, 0.3 mM GTP, 10

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mM creatine phosphate, 5 1.18of creatine phosphokinase, 20 mM Hepes, 80-100 mM KCl, 2.5 mil4 magnesium acetate, 2 mikf dithiothreitol, 20 fl complete amino acid mix lacking leucine, and 0.1 mM spermine. In addition, to a 50-d reaction mix, 20 ~1 of wheat germ extract and 0.3 pCi of [‘“Clleucine (314 mCi/mmole, New England Nuclear) were added, along with the appropriate amount of RNA. When [35S]methionine (10 @i, 600-1000 Ci/mmole) was used in the in vitro reaction, the complete amino acid mix lacked only methionine. The reaction mixture was incubated for 1 hr at 25°C. The label incorporated into TCA-insoluble protein was measured either by adding an equal volume of 10% TCA and boiling the sample for 10 min or by incubating with 0.2 ml of 0.5 N NaOH (containing 10 pg/ml of casamino acids) for 10 min at 37°C neutralizing with 0.2 ml of 0.5 N HCl, then precipitating with an equal volume of 10% TCA. The TCA-insoluble material was collected on GF/C filters (2.4 cm, Whatman), and the filters were dried and counted. For SDS-PAGE analysis of the translation products, an aliquot of the [35S]methionine-labeled reaction mixture, containing about 30,000 cpm, was applied to slab gels. Background due to unincorporated [35S]methionine was abolished by incubating the gels overnight in a solution of 10% TCA and 25% 2-propanol prior to preparation for autoradiography (Holland et al., 1977). RESULTS

Incorporation of [3H]UraciZ into RNA during Germination Incorporation of labeled uracil into RNA in the wild type (wt) has been previously shown to begin during the swelling stage, about 40-60 min after activation (Giri and Ennis, 1977; Yagura and Iwabuchi, 1976) (Fig. 1). In mutant HE-l and cycloheximide-treated wt spores, both of which have been shown to swell but are blocked in emergence (Cotter et al., 1969; Ennis and Sussman, 1975), incorporation into RNA

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HOURS FIG. 1. Incorporation of [3H]uracil into RNA during germination. Spores were activated and labeled with [3H]uracil during germination as described previously (Giri and Ennis, 1977). One-milliliter samples containing 2.5 x lo7 spores were removed at the indicated intervals, and incorporation into the TCA-insoluble fraction was determined. (0) Incorporation by wt spores; (0) incorporation by wt spores in the presence of 300 pg/rnl of cycloheximide added immediately after activation; (0) incorporation by mutant HE-1 spores.

proceeds at a slower rate than in the untreated wt (Fig. l), and less label is incorporated. The types of RNA synthesized were determined during each stage of germination of wt spores, cycloheximide-treated wt spores, and mutant HE-l spores. Total RNA labeled at hourly intervals during germination with [3H]uracil was fractionated by oligo(dT)-cellulose chromatography to obtain an estimate of the fraction of total RNA synthesized during each stage that contains poly(A) tails. It is evident from Table 1 that during normal germination ( wt spores), the portion of label in the poly(A)+ fraction decreases, while the portion of total labeled RNA in the unbound [poly(A)-] fraction increases during germination. The fraction of poly(A)+ RNA decreases from 48% of the 0- to 1-hr-labeled RNA to 25% of the total RNA labeled from 2 to 3 hr

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after activation. During the same intervals, the label in the poly(A)- fraction rises from 52 to 75% of the total labeled RNA. In contrast to the results obtained in wt, mutant HE-l, or cycloheximide-treated wt spores, little change in the poly(A)+ fraction is detectable during germination (Table 1). Each sample of RNA described in Table 1 was further analyzed by sucrose density sedimentation. The results obtained with the poly(A)- fraction are presented in Fig. 2. It can be seen that material cosedimenting with D. discoideum rRNA is detectable as early as the first hour of germination of wt spores (Fig. 2A). At this stage (O-l hr after activation), a large part of the poly(A)- RNA sediments coincident with 4 S RNA, and is presumably tRNA. At later stages of germination, l-2 hr (Fig. 2B) and 2-3 hr after activation (Fig. 2C), an increasing portion of the poly(A)- RNA sediments as rRNA. It is not possible to obtain a precise determination of the amount of 4 S and rRNA in the unbound fraction because of the TABLE OLmo(dT)-Cm.LuLosE LABELED Samples and labeling period (hr)

1

CHROMATOGRAPHY DURING GERMINATION” Peyapeagme Po$$-

of

OF RNA

Peyaretlagme

of

Po$i;)+

wt o-1 l-2 2-3 Mutant HE-1 o-1 l-2 2-3 wt + cycloheximide o-1 2-3

52.0 59.4 75.0

48.0 40.6 25.0

50.6 56.1 54.3

49.4 43.9 45.7

52.2 57.2

47.8 42.8

n Activated spores were labeled with r3H]uracil for 1-hr periods during germination. The RNA was extracted and applied to oligo(dT)-cellulose columns as described in 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. These fractions were then analyzed on sucrose gradients, as shown in Figs. 2 and

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FRACTION NUMBER FIG. 2. Sucrose density gradient sedimentation of poly(A)RNA. Spores were labeled with [3H]uracil for Ihr periods. The RNA was extracted and applied to oligo(dT)-cellulose columns as described in Materials and Methods. The RNA fraction that did not bind to ohgo(cellulose was pooled for each sample, and a portion was applied to a sucrose gradient. These are the same samples described in Table 1. (A-C) RNA isolated from wt D. discoideum; (D-F) RNA isolated from HE-l mutant; (G, F) RNA from wt spores incubated in the presence of 300 pg/ml of cycloheximide. The samples in A, D, and G were prepared from spores labeled from 0 to 1 hr after activation; those in B and E from 1 to 2 hr after activation; and those in C, F, and H from 2 to 3 hr. The positions of sedimentation of D. discoideum 17 and 25 S rRNAs are shown by the arrows. Cycloheximide was added immediately after activation. Fifteen to thirty percent gradients were used,

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. Con-

sequently, from Fig. 2 it can be estimated that in germinating wt spores, the proportion of 4 S RNA in the poly(A)- RNA made at O-l hr is 37%, at l-2 hr 30%, and at 2-3 hr 21%. The fraction of rRNA (17 S plus 25 S) in the poly(A)- RNA at O-l hr is 47%, at

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l-2 hr 61%, and at 2-3 hr 73%. It is evident from these results that more rRNA accumulates later during germination than during the first hour after activation, while 4 S RNA accumulation decreases during germination. During germination of mutant HE-l, rRNA represents a smaller portion of the poly(A)- RNA at the corresponding times, as seen in Figs. 2D-F. It is also evident that rRNA is not synthesized in cycloheximidetreated cells, and the major portion of labeled poly(A)- RNA made during germination sediments as 4 S RNA (Figs. 2G and W. The sedimentation profiles of the poly(A)+ RNA are shown in Fig. 3. Since all of the samples showed similar profiles, only the 2- to 3-hr-labeled samples are shown. Most of the poly(A)-containing RNA sediments between 8 and 27 S, with the major fraction between 13 and 18 S. The poly(A)+ RNA fraction, as seen from these gradients, is not contaminated to a large extent with rRNA. This can be deduced also from a comparison of Figs. 3A and C. In Fig. 3C, the poly(A)’ fraction from cycloheximide-treated cells is shown, in which, as noted above (Figs. 2G and H), no rRNA is synthesized, yet the sedimentation pattern of the poly(A)+ RNA is similar to that of the wt poly(A)+ fraction seen in Fig. 3A. Translation of RNA Isolated Stages of Germination

at Different

The ability of RNA found in dormant spores and during various stages of germination to stimulate the in vitro incorporation of amino acids into protein was determined. Unlabeled RNA from unactivated spores and from spores at 1 hr (swollen spores) and at 2.5 hr after activation (emergence of amoebae) were fractionated on oligo(dT)cellulose and tested for mRNA activity in the wheat germ cell-free synthesizing system. From the data which are summarized in Table 2, four conclusions can be derived.

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FIG. 3. Sucrose density gradient sedimentation of poly(A)+ RNA. Labeled RNA samples were fractionated on oligo(dT)-cellulose columns, the bound fractions were eluted and pooled, and a portion was applied to sucrose gradienta. The RNA samples labeled from 2 to 3 hr are shown: These are the same samples as described in Table 1. (A) wt RNA, (B) RNA from mutant HE-l; (C) RNA from wt incubated in the presence of cycloheximide. Five to twenty percent gradients were used.

(a) Total unfractionated RNA isolated from dormant spores and during germination is able to stimulate the incorporation of amino acids into protein in the wheat germ extract. 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. Hybridization of total RNA with [3H]poly(U) (results not presented) also

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TABLE

2

In Vitro TRANSLATION

OF RNA FROM GERMINATING SPORES: STIMULATION OF [‘%]LEUCINE INCORPORATION INTO THE TCA-INSOLUBLE FRACTIONS

Source

of RNA

Spores wt at 1 hr wt at 2.5 hr Mutant HE-1 at 2.5 hr wt + cycloheximide at 2.5 hr

Specific (cpm

195 278 546 235 604

stimulatory incorporated/ag RNA) *, ’

20

39 24 n.d.d n.d.

activity of

5257 7786 8611 n.d. n.d.

’ RNAs isolated from the above sources were fractionated on oligo(dT)-cellulose and tested for mRNA activity in the wheat germ cell-free synthesizing system. For this experiment, the RNA samples were obtained from oligo(dT)-cellulose and concentrated and dialyzed as described in Materials and Methods. ’ Corrected for endogenous reaction. ’ Micrograms per 5Oql reaction volume. d Not determined.

showed that the fraction of total RNA containing poly(A) increased during germination. (b) Poly(A)+ RNA is found in dormant spores as well as in swollen spores and amoebae. The specific activity of this mRNA apparently is greater in swollen spores and emerging amoebae than in dormant spores. (c) Ninety to ninety-five percent of the mRNA activity of the total RNA is due to the poly(A)+ fraction. As can be seen, very little stimulation of protein synthesis is observed using the RNA that does not bind to oligo(dT)-cellulose. In addition, as will be shown later (Fig. 7, slot 5), analysis of the translation products by SDSPAGE indicates that the small amount of stimulation observed by the poly(A)- fraction does not represent a unique class of mRNA. (d) The HE-l mutant and cycloheximide-treated wt spores also have translatable mRNA. RNA prepared from cycloheximide-treated spores has a similar activity to RNA isolated from the wt 2.5 hr after activation, whereas RNA isolated from the mutant at 2.5 hr is comparable to RNA obtained from the wt at 1 hr after activa-

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tion. The dependence of the in vitro reaction on the concentration of poly(A)+ RNA is shown in Fig. 4A, and the kinetics of in vitro protein synthesis directed by these RNAs in Fig. 4B. As mentioned in the legend to Fig. 4, the RNA samples were all treated in the same way and were dialyzed prior to addition to the in vitro reaction. Under these conditions, the l- and 2.5~hr RNAs gave very similar results in both experiments, but RNA from spores was always less efficient in stimulating incorporation. These results give further evidence that mRNA from dormant spores is translated less efficiently than mRNA from germinating spores. Incorporation of [3H]Proline into Protein during Germination The incorporation of radioactive proline into protein shows that protein synthesis begins during or immediately prior to spore swelling (Fig. 5) (Giri and Ennis, 1977; Yagura and Iwabuchi, 1976), and is first observed at about 30 min after activation. As seen in Fig. 6B, and as described previously (Giri and Ennis, 1977), four classes of proteins are synthesized during wt germination: (a) proteins labeled from 0 to 1 hr, but not detected at later times, such as bands A and F; (b) proteins synthesized in larger amounts during the first hour of germination than at later times, such as bands B and G; (c) proteins not labeled from 0 to 1 hr, but which appear at later times, such as bands C, E, and H; and (d) proteins which are synthesized throughout germination, such as bands D and J. It is evident that in mutant HE-l, not only is the rate of incorporation into protein decreased (Fig. 5), but the pattern of proteins synthesized is also different (Fig. 6A), suggesting that the regulation of the synthesis of proteins in the mutant is different from that in the wt. For example, bands J and C (Fig. 6B) are not made or are made in low amounts in mutant HE-l. The synthesis of protein E, however, which we sug-

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FIG. 4. Translation of poly(A)+ RNA from germinating spores. RNA was obtained either from unactivated spores or from spores at 1 and 2.5 hr after activation, and fractionated on oligo(dT)-cellulose columns. The bound fractions were concentrated and translated in the wheat germ system as described in Materials and Methods. (0) RNA from spores (not activated); (0) RNA from spores 1 hr after activation; (A) 2.5~hr RNA; (Cl) no added RNA. (A) Dependence on RNA concentration. Incubations were carried out in 59 pl as described in Materials and Methods. (B) Kinetics of in vitro protein synthesis. Reaction mixtures contained 1.5 H of mRNA. Ten-microliter aliouots were removed at the indicated intervals and incorporation into the TCAinsoluble material was determined.

8

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gested earlier (Giri and Ennis, 1977) is D. discoideum actin, appears to be synthesized in mutant HE-1 as it is in the wt. Comparison of in Vitro Translation Products with the Proteins Synthesized in Intact Cells during Germination

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FIG. 5. Incorporation of [3H]prolme into protein during germination. The procedures for labeling with [3H]proline and measuring incorporation were described previously (Giri and Ennis, 1977). One-milliliter samples containing lo7 spores were removed at the indicated intervals. (0) Incorporation by wt spores; (Cl) incorporation by wt spores incubated in the presence of 309 gg/ml of cycloheximide added immediately after activation; and (0) incorporation by mutant HE1 spores.

Figure 7 shows SDS-PAGE of proteins synthesized in wheat germ extracts, directed by unfractionated or oligo(dT)-cellulose-fractionated RNA. RNA was obtamed from ungerminated wt spores (0 l-n) or from spores at 1 hr (swollen spores) or 2.5 hr after activation (emergence). Slots 1 and 2 represent proteins synthesized when 0-hr spore RNA was used, slots 3 and 4 when 1-hr RNA was used, and slots 5, 6, and 7 when 2.5hr RNA was used as mRNA. For comparison, slots 10, 11, and 12 show the pattern of proteins synthesized in uiuo during germination, with the same labeled extracts as used in Fig. 6B (run again on 12.5% gel for comparison). As seen, band E, which is a major translation product in uiuo after the first hour of germination (but not present from 0 to 1 hr), is also synthesized when l- or 2.5~hr RNA directs in vitro

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FIG. 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins synthesized during germination. Germinating wt spores and spores of mutant HE-l were labeled for I-hr periods with [“S]methionine, and the extracts obtained after sonication were applied to 10% polyacrylamide slab gels. Electrophoresis was performed as previously described (Giri and Ennis, 1975). (1) Spores labeled O-l hr; (2) spores labeled l-2 l-q and (3) spores labeled 2-3 hr after activation. (A) HE-l mutant, (B) wt.

synthesis, but spore RNA does not direct its synthesis. Other proteins such as X, made throughout germination in Go, are also found among the in vitro translation products. By comparing the translation products obtained using total RNA (slots 2, 4, and 7) with those obtained using the poly(A)+ fraction as mRNA (slots 1, 3, and 6), it is evident that most, if not all, classes of mRNA are represented in the poly(A)+ fraction. In slot 5, the RNA that did not bind to oligo(dT)-cellulose after the second passage was used, and as seen by comparison with slot 6, no unique classes of mRNA are present in the unbound fraction of 2.5hr RNA. As was described previously (Table 2),

RNA isolated at 2.5 hr from wt spores treated with cycloheximide stimulated incorporation of radioactivity into TCA-insoluble protein in wheat germ extracts. However, it is noteworthy that when RNA from cycloheximide-treated spores was translated in cell-free extracts, the pattern of proteins made in vitro (Fig. 7, slot 9) is different from that of proteins made using 2.5-hr wt RNA as mRNA (slot 7). For example, less protein E is made (and other differences can be easily seen). There are only a few differences, however, between the proteins synthesized in vitro using 2.5hr HE-l mutant RNA (slot 8) and wt RNA (slot 7), in contrast to the many differences seen in uiuo (Fig. 6).

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FIG. 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro synthesized proteins directed by RNA isolated from it spores, cycloheximide-treated wt spores, and mutant HE-1 spores. In uitro translation in wheat germ extracts and electrophoresis of [%]methionine-labeled samples were carried out as described in Materials and Methods and previously (Giri and Ennis, 1977). Each sample applied to a 12.5% gel contained 30,000 cpm. Protein synthesis directed by: (1) poly(A)+ wt spore RNA (0 hr); (2) total wt spore RNA (0 hr); (3) poly(A)+ wt spore RNA (1 hr); (4) total wt spore RNA (1 hr); (5) poly(A)2.5-hr wt RNA [after second passage through oligo(dT)-cellulose]; (G)poly(A)+ wt spore RNA (2.5 hr); (7) total wt spore RNA (2.5 hr); (8) total mutant HE-1 spore RNA (2.5 hr); (9) total wt cycloheximide-treated spore RNA (2.5 hr). Slots 10, 11, and 12 are the same in viuo labeled samples as in Fig. 6B. DISCUSSION

Spore germination in D. discoideum proceeds in three stages: activation, swelling, and emergence of amoebae. Synchronous germination can be induced by a variety of agents (Cotter, 1975), and after activation with 20% dimethyl sulfoxide, as in the experiments described, the entire sequence is completed within 3.5 hr after activation. RNA and protein syntheses begin during the swelling stage (Figs. 1 and 5) (Giri and Ennis, 1977; Yagura and Iwabuchi, 1976). Both are required for completion of germination, as inhibitors of protein or RNA synthesis were shown to interfere with germination (Cotter et al., 1969; Giri and Ennis, 1977). In an attempt to investigate in more detail the type of regulatory events occurring

during germination, the synthesis of different types of RNA was first examined. RNA was isolated from germinating spores labeled at hourly intervals after activation. The newly synthesized RNA samples were fractionated by oligo(dT)-cellulose chromatography into a fraction not retained by the column, poly(A)- RNA-representing 4 S RNA, rRNA, and a small amount of mRNA; and into a fraction bound to the column, poly(A)+ RNA-consisting mainly of polyadenylated mRNA. The results indicated (Table 1) that: (a) The amount of label in the poly(A)- fraction increased during wt germination; and (b) the portion of label in the poly(A)+ fraction decreased from 48% of the total label in the 0- to l-hrlabeled RNA to 25% in the 2- to 3-hr-labeled sample. Further analysis of the

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RNA and Protein Synthesis in D. discoideun

poly(A)- RNA fraction by sedimentation in sucrose density gradients (Fig. 2) showed that, during the initial stages of germination, a larger portion of the total RNA corresponds to 4 S RNA, and during later stages, to rRNA. It is possible that the proportion of newly made RNA which is tRNA could be incorrect if -C-C-A turnover rather than synthesis was occurring. It is known that terminal incorporation into -C-C-A groups of tRNA occurs in the presence of inhibitors of RNA which completely inhibit net tRNA synthesis (Deutscher, 1973). It has been previously shown (Giri and Ennis, 1977; and unpublished data) that no incorporation of [3H]uracil into RNA occurs during inhibition of RNA synthesis by a variety of inhibitors. Consequently, it seems unlikely that the incorporation into 4 S RNA observed is due to -C-C-A turnover on tRNA. Preliminary experiments (not presented) also indicated that newly synthesized rRNA is incorporated into ribosomes during germination. It is not immediately clear why the germinating spores need to synthesize new ribosomes, as a pool of excess ribosomes not participating in polysomes is present (Giri and Ennis, 1977). From these experiments, it is evident that 4 S RNA (presumably tRNA), rRNA, and mRNA are synthesized de nouo during germination, and their synthesis is developmentally regulated. It appears that a greater fraction of the total RNA synthesized at the initial stages of germination is polyadenylated than during later stages, and that the fraction comprising rRNA increases as germination proceeds. In order to detect the presence of functional mRNA at different stages of germination, RNA was isolated from unactivated dormant spores and from spores 1 hr (swelling stage) and 2.5 hr (emergence stage) after activation. Translatable mRNA was assayed by the ability of the RNA to stimulate incorporation of amino acids into protein in wheat germ extracts. Unfractionated RNA from dormant spores as well as RNA

Spores

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from spores after 1 and 2.5 hr of germination were active in directing in vitro protein synthesis (Table 2). It is evident that the fraction of total RNA with mRNA activity increases during germination. The results also indicate that most of the mRNA activity in the RNA samples was due to the poly(A)+ fraction. The residual activity in the unbound fraction probably represents partly the inefficiency of the column in removing all of the poly(A)+ RNA (after two passages) and might also be due to mRNAs with shortened poly(A) sequences that failed to bind. Gel analysis of the translation products of fractionated poly(A)RNA (Fig. 7, slot 5) and poly(A)+ RNA (slot 6) suggests that the unbound RNA does not represent a unique class of mRNA, as no major unique translation products are detected. The presence of mRNA stored in spores has been reported in other systems, for example, in the aquatic fungus Blastocludiella emersonii (Johnson et al., 1977). It will be of interest to study the cellular location of mRNA in dormant spores since it is already known that no polysomes are present (Giri and Ennis, 1977). It also appears from our experiments that the poly(A)+ RNA from dormant spores has a lower specific activity (Table 2) and is translated less efficiently in wheat germ extracts than poly(A)+ RNA from spores at 1 and 2.5 hr after activation (Fig. 4). Similar results were reported for mRNA from unincubated Artemia salina cysts (Grossfeld and Littauer, 1976). By comparing the products obtained when RNAs from different stages of germination are translated in cell-free extracts with the proteins synthesized during germination (in Go), we hoped to gain some information about the control of gene expression during germination. From an analysis of the proteins synthesized in viva during germination (Fig. 6B), it is evident that the synthesis of various proteins is developmentally regulated, as numerous changes in the pattern of proteins synthe-

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sized occurs during germination. Four classes of proteins could be distinguished on these gels: (a) proteins labeled early (O-l hr) but not labeled at later times during germination; (b) proteins which are synthesized in larger quantities at early stages than at later times; (c) proteins which appear only after the first hour of labeling; and (d) proteins labeled throughout germination. A comparison of proteins made in uiuo with those synthesized in vitro (Fig. 7) presents evidence that there are qualitative changes in the population of translatable mRNAs during germination, presumably due to differential gene transcription. It can be seen that band E, which is a major translation product during germination (presumably actin) and appears only after the first hour of germination (Fig. 6B and Fig. 7, slots lo-12), is also seen as a major product in vitro when l- and 2.5~hr RNAs are used to direct protein synthesis (Fig. 7, slots 3 and 6), but is not present when RNA isolated from dormant spores is used as mRNA (Fig. 7, slots 1 and 2). In contrast, band X, which is synthesized throughout germination, is also synthesized in cell-free extracts using RNA from any stage in germination (Fig. 7, slots 1-7). The time of appearance of band E, for example, indicates that its synthesis is probably regulated at the level of transcription, as spores do not contain translatable mRNA for E protein and in uiuo E appears after the first hour of germination. More detailed experiments are necessary, however, to prove that transcriptional control is responsible for the observed changes in E protein synthesis during germination. A different approach used in attempting to elucidate some of the regulatory mechanisms in germination was to observe the events that occur under abnormal conditions, resulting either from mutation or from the use of inhibitors. RNA synthesis during germination was examined in the absence of protein synthesis in cycloheximide-treated spores. It has

been previously reported that cycloheximide allows normal swelling of spores, but inhibits the emergence of amoebae (Cotter and Raper, 1966; Cotter et al., 1969; Cotter, 1975). As shown in Figs. 2G and H in Fig. 3C, 4 S and poly(A)+ RNA are synthesized in the presence of cycloheximide, but the synthesis of rRNA is inhibited (Figs. 2G and H). The mRNA synthesized in the presence of cycloheximide was translatable in wheat germ extracts (Table 2), but the translation products showed many differences from the normal pattern (Fig. 7, slots 7 and 9). More direct information about regulatory processes can be obtained by using mutants blocked at specific steps in germination. A mutant, HE-l, was isolated (Ennis and Sussman, 1975) and was shown to be blocked at the emergence step. Syntheses of RNA and protein were compared during germination of this mutant and wt spores. In the mutant, unlike the Lot, no change was detected in the proportion of mRNA and tRNA made early and at later stages of germination (Table 1). In addition, rRNA represents a smaller fraction of the total RNA made at any interval than in the wild type (Fig. 2). Thus, the control over the synthesis of different types of RNA during germination is abnormal in the mutant. The most interesting result obtained with mutant HE-l is the difference in the pattern of protein synthesis observed during germination (Fig. 6A). This result suggests that the difference between mutant HE-l and the wt is not only quantitative or resulting from a slower rate of synthesis of macromolecules, but is likely to be due to a defect in regulating development. Even a cursory examination of the gel patterns of the wt (Fig. 6B) and HE-1 mutant proteins (Fig. 6A) shows many differences. Some proteins synthesized by wt spores are not made in the mutant (bands J and C), while new bands not found in the wt appear. A comparison of in vitro synthesized proteins directed by wt and HE-l mutant RNA (Fig. 7, slots 7 and 8) showed fewer differences

GIRI AND ENNIS

RNA and Protc sin Synthesis in D. discoideum Spores

than in uiuo (Fig. 6), suggesting a translational control (or posttranslational modif% cation) in viva. It is necessary, however, to compare more RNA samples obtained at different stages and to use better resolving gels to confirm this observation. The use of additional mutants blocked at different stages of germination will help dramatize changes occurring during normal germination and identify the gene products important for germination. Germination of D. discoideum spores is morphologically a relatively simple event. It is evident, however, from the results summarized above that at the molecular level, complex changes accompany this sequence of morphological events. Further detailed studies of germination at the molecular level should lead to an understanding of the regulation of this stage of the slime mold life cycle. REFERENCES T. H., and LODISH, H. F. (1977). Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum. Develop. Biol. 60, 180-296. AVIV, H., and LEDER, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Nat. Acad. Sci. USA 69,1408-1412. COTTER, D. A., and RAPER, K. B. (1966). Spore germination in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA S&880-887. COTTER, D. A. (1975). Spores of the cellular slime mold Dictyostelium discoideum. In “Spores VI” (P. Gerhardt, H. Sadoff, and R. Costilow, eds.), pp. 61-72. American Society of Microbiology, Washington, DC.

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riol. 100, 1020-1026. M. P. (1973). Synthesis and functions of the -C-C-A terminus of transfer RNA. In “Progress in Nucleic Acid Research and Molecular Biology” (J. N. Davidson and W. E. Cohn, eds.), pp. 51-92. Academic Press, New York. ENNIS, H. L., and SUSSMAN, M. (1975). Mutants of Dictyostelium discoideum defective in spore germination. J. Bacterial. 124, 62-64. FRANCIS, D. (1976). Changes in protein synthesis during alternate pathways of differentiation in the cellular slime mold Polysphondylium pallidum. Develop. Biol. 53,62-72. 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. 17,282-289. GROSSFELD, H., and LITTAUER, U. L. (1976). The translation in vitro of mRNA from developing cysts of Artemia salina. Eur. J. Biochem. 70, 589-599. HOLLAND, M. J., HAGER, G. L., and RIJTTER, W. J. (1977). Characterization of purified poly(adenylic acid)-containing messenger ribonucleic acid from Saccharomyces cerevisiae. Biochemistry 16, 8-16. JACOBSON, A. (1976). Analysis of mRNA transcription in Dictyostelium discoideum or slime mold messenger RNA: How to find it and what to do with it once you’ve got it. In “Methods in Molecular Biology: Eukaryotes at the Subcellular Level,” pp. 161-209. Marcel Dekker, New York. JOHNSON, S. A., LOVETT, J. S., and WILT, F. H. (1977). The polyadenylated RNA of zoospores and growth phase cells of the aquatic fungus, Blastocladiella. Develop. Biol. 56,329~342. 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, DEUTSCHER,

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and IWABUCHI, M. (1976). DNA, RNA and protein synthesis during germination of spores in the cellular slime mold Dictyostelium discoideum. Exp. Cell Res. 100, 79-87.