RNA metabolism during cytodifferentiation in the cellular slime mold Polysphondelium pallidum

407

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95762

RNA METABOLISM D U R I N G C Y T O D I F F E R E N T I A T I O N IN T H E CELLULAR SLIME MOLD POLYSPHONDELIUM PALLIDUM

R A Q U E L R. S U S S M A N

Department o[ Biology, Brandeis University, Waltham, Mass. (U.S.A.) ( R e c e i v e d J u n e 26th, 1967)

SUMMARY

I. Studies of the cellular slime molds have demonstrated the appearance and disappearance of specific developmentally regulated macromolecules. In order to investigate whether the control mechanism(s) resides at the transcription level, a study of RNA metabolism was undertaken. 2. The data show that a considerable proportion of the RNA originally present in the vegetative amoebae is degraded during the developmental sequence and that resynthesis occurs at an appreciable rate throughout. A major portion of the nascent material appears to be ribosomal RNA (rRNA). No difference could be detected between the newly formed ribosomes in differentiating cells and the ribosomes of vegetative amoebae in respect to physicochemical properties, base composition, protein associations, or hybridization tests. The significance of ribosome biosynthesis is discussed.

INTRODUCTION

Cellular slime mold amoebae, after growth, come together into multicellular aggregates and construct fruiting bodies consisting of at least two differentiated cell types: thick walled, dormant spores and vacuolated stalk cells. These developmental events can occur in the absence of any nutrients and are accompanied b y significant decreases in major cell constituents including protein and RNA. Nevertheless, extensive protein synthesis does occur as shown b y the presence of functional polyribosomal complexes in significant amounts 1 and b y the accumulation of a number of specific enzymes *-4 and antigens 5. The synthesis of RNA has also been reported 6 and, on the basis of patterns of actinomycin D sensitivity, appears to be required for the subsequent synthesis of these enzymes 4,7,8 as well as for the normal course of morphogenesis and. of cytodifferentiation9. This paper is concerned with an initial examination of RNA metabolism in Polysphondylium pallidum. Although other slime mold species, notably Dictyostelium A b b r e v i a t i o n s : ¥ S , o.15 M NaC1, o.1 M v e r s e n e (pH 8.0); SSC, o.15 M NaC1, o,15 M sod i u m c i t r a t e (pH 7.o), d i l u t i o n g i v e n a as m u l t i p l e of this; r R N A , r i b o s o m a l R N A .

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R.R. SUSSMAN

discoideum, have been used more extensively in the past for developmental studies, P. pallidum was employed here because it can be grown axenically thereby eliminating ambiguities that might conceivably arise in the case of species still cultivated in the presence of a bacterial associate 6. Thus, the results obtained can serve as a useful standard of comparison. The data show that a considerable proportion of the RNA originally present in the vegetative amoebae is degraded during the developmental sequence and that resynthesis occurs at an appreciable rate throughout. A major portion of the nascent material appears to be ribosomal RNA (rRNA). No difference could be detected between the newly formed ribosomes in differentiating cells and the ribosomes of vegetative amoebae in respect to physicochemical properties, base composition, protein associations, or hybridization tests.

METHODS AND MATERIALS

Organism and experimental conditions P. pallidum strain P P - I was grown as previously described 1°. Cultures in 4oo ml of broth containing lecithin, milk powder, proteose peptone, in 0.05 M phosphate (pH 6.5) were shaken at 22 °. The amoebae grew with a doubling time of 5 h and were harvested in late log phase (at cell densities slightly below I . lO7 cells/ml). The cells were washed b y centrifugation 3 times in cold salt solution n, suspended at a density of 2. IoS/ml and o.I-ml aliquots were deposited on quartered Millipore tilters (0.8/~, black, 47 mm) resting on the same size absorbent pads with 0.5 ml o.oi M Mcllvain buffer (pH 4.8-5.o), plus streptomycin (5oo/~g/ml) and contained in 60 m m petri dishes. Incubation temperature was 16 °. Under these conditions, cyst formation b y individual amoebae is minimized and a high degree of morphogenetic synchrony is observed. The entire developmental sequence (see Fig. I) is completed in 55-60 h. Cells were harvested from the Millipore filters in cold water, centrifuged, and the pellets either used immediately or stored at --20 °. Sterility controls (aliquots spread on nutrient agar plates and incubated at 37 °) were routinely employed to demonstrate the absence of bacterial contamination.

Incorporation o! labeled precursors [14C]Uracil. To label vegetative cells, 20 #C of [14C]uracil (3o mC/mmole) was added to 400 ml of culture medium. After incubation the cells were harvested, washed and either used immediately or resuspended and reincubated in cold medium when a chase was required. Developing cells were labeled b y depositing them on the quartered Millipore filters resting on quartered support pads saturated with 0.4 ml o.oi M Mcllvain buffer containing I/~C uracil (30 mC/mmole). After the desired period of incubation the cells were either harvested immediately or chased b y transferring the Millipore filters to fresh support pads saturated with buffer containing 1.5 mM cold uracil and incubating further. [asP]Phosphate. To label vegetative cells, 2-1o 5 amoebae were suspended in io ml of the nutrient medium lacking cold phosphate but containing 1-3 mC of sterile, neutralized carrier free [32p]phosphate. The culture was shaken at 22 °. When Biochim. Biophys. Acta, 149 (1967) 4o7-421

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METABOLISM IN CELLULAR SLIME MOLD

409

the cell density reached 5" Ioe/m 1, the amoebae were harvested and used immediately or chased b y washing in IO mM cold phosphate and reincubating them in nutrient medium containing cold phosphate. Developing cells were labeled by saturating the filter support pads with 0.4 ml of IO mM citrate buffer (pH 4.8) containing I00 #C [32p~phosphate and. when necessary, they were chased b y transferring the filter to pads containing o.oi M Mcllvain buffer.

Cell extracts Washed cell suspensions (ioS/ml) were ruptured at 4 ° b y 60-80 manual strokes in a glass homogenizer. Alternately, the cells were lysed in o.I ~o sodium deoxycholate.Unbroken cells and large debris were removed b y centrifugation for IO min at 15oo ×g.

Ribosome isolation and puri/ication Extracts were prepared in 5 mM magnesium acetate, 5o mM NH4C1, 1.5 mM dithiothreitol, 5 mM Tris buffer (pH 7.4) and centrifuged 20 min at 20 ooo ×g. The supernatant was centrifuged 12o min at lO5 ooo ×g. This cycle of low and high spins were repeated 3-4 times and the final pellet was suspended in 20 mM magnesium acetate, ioo mM NaC1, IO mM phosphate buffer (pH 7.2). In a few instances the centrifugations were preceded b y differential precipitation of the ribosomes from the crude extracts by (NH,)2SO 4 following KURLAND'S method TM. Sedimentation analyses of the final product were carried out in 5-20 % linear sucrose gradients (prepared in the same phosphate buffer) centrifuged at 25 ooo rev./min for 2 h at 4-6 °. Crude extracts and purified ribosomal preparations (1-3.4 mg RNA/ml) were also centrifuged in a Spinco Model E ultracentrifuge at 5-8 °, equipped with schlieren optics. The observed sedimentation values were corrected for temperature and viscosity.

Sucrose density sedimentation o / R N A The method of GILBERT13 was employed because the nucleolytic activity of slime mold extracts is very high. Harvested cells were washed in cold water and the final pellets containing 2.1o7-1.io 8 cells were gently stirred with o.I ml of io % sodium dodecyl sulfate in a 3 °0 bath. Total cell breakage is almost immediate. After a few minutes, 0. 9 ml of water (at room temperature) was added and the now clear cell extract was layered over a 29-ml linear sucrose gradient (15-3 ° %, w/w in 0. 5 % sodium dodecyl sulfate, IOO mM NaC1, 5 mM Tris (pH 7.4). The tubes were centrifuged at 24000 rev./min for 18 h at 19 ° in the SW-25 rotor of a Spinco Model L centrifuge, punctured, and the fluid removed from below with a finger pump, passed through the o.5-ml continuous flow cell of a Gilford recording spectrophotometer and collected in aliquots of 30 drops. Radioactivity of the fractions was determined by precipitating with cold trichloroacetic acid (15 °/o final concentration) in the presence of 200 #g bovine serum albumin, collecting and washing the precipitates on Millipore filters, drying, and counting in vials with IO ml Liquifluor in a NuclearChicago Scintillating counter. Biochim. Biophys. Acta, 149 (t967) 4o7-421

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R.R. SUSSMAN

Base compositions o[ RNA species Cells, labeled over many hours with [zzP]phosphate and then chased (see below) were lysed in sodium dodecyl sulfate and centrifuged through a sodium dodecyl sulfate-sucrose gradient as described. The 23-S, 16-S, and 4-S peaks were separated and each extracted with an equal volume of cold, water saturated-phenol. Non-labeled purified P. pallidum RNA was added as carrier, and the phenol extraction repeated 2 or 3 times. The final aqueous phase was precipitated with 2 vol. cold 95 % ethanol, left at --20 ° for at least i h and centrifuged. The precipitate was dissolved in i ml IO mM acetate buffer (pH 5.2) and passed through a I cm x I 5 cm Sephadex G-25 (coarse) column in the same buffer. The excluded volume (4 ml) was precipitated with 0. 3 M sodium acetate and 2 vol. ethanol in the cold and redissolved in buffer. The purified [32P]RNA species were hydrolyzed with 0. 3 M KOH at 3 °o for 18 h. Tile hydrolysate was neutralized with formic acid and the 2',3'-mononucleotides separated in a Dowex-I column 14. The 0.8 cm ×22 cm column was washed with concentrated formic acid and then to neutrality with H20. 200-500/zg of the neutralized hydrolysate was applied, followed by extensive washing with distilled H20. Two continuous gradients of formic acid were used to elute the nucleotides, first 2 M formic acid fed into a 5o-ml reservoir of H~O, eluted CMP and AMP and then 8 M formic acid into the same reservoir eluted GMP and UMP. The eluent was collected in 2-ml fractions at a speed of i ml/min, under positive air pressure. The absorption at 260 mtz was recorded continuously and the radioactivity of the fractions measured, after drying on planchettes, in a gas-flow counter. The recovery of the mononucleotides was estimated by comparing the expected and observed adsorption of the hydrolysed carrier RNA. The molar extinction ratios obtained were typical of each nucleotide in formate with a single exception: The hydrolyzed 4-S RNA yielded a peak which was eluted after UMP, and represented 18 °/o of the total amount. This as yet unidentified material had a molar extinction ratio of 1.8 at 250 : 260 m/z and of I.O at 280 : 26o m#. I t was omitted in calculating the proportions of the four usual bases.

Isolation o/[aH]DNA Vegetative cells, at a density of I. ioe/ml growing slowly in axenic medium prepared without milk powder were incubated with I #C/ml of [ZH]thymidine (15.9 C/ mmole) for 24 h, at 22 ° with shaking, until they reached 6-Ioe/ml. The harvested cells were washed 3 times with cold VS (o.15 M NaC1, o.I M versene (pH 8.0)) and the sediment extracted for 5 min in ice, with 5 ml VS and 5 ml cold phenol (saturated with VS). After centrifugation, the aqueous and phenol layers were discarded and the interphase extracted twice again in the same manner to eliminate most of the RNA. The final interphase was suspended in 2 ml VS, 2 ml phenol and 0.2 mf IO% sodium dodecyl sulfate and heated at 60 ° for 3-5 min. After centrifugation at room temperature, the aqueous layer contained the DNA. The interphase phenol was re-extracted at room temperature with more VS and the aqueous phase combined with the previous one. The DNA was precipitated twice with o.r vol. of 2 M potassium acetate and I vol. absolute ethanol, spooling it on a glass rod and using I • IOO SSC (SCC : o.15 M NaC1, o.15 M sodium citrate (pH 7.0)) to dissolve it. To purify it further, the soluBiochim. Biophys. Acta, 149 (1967) 4o7-42I

RNA METABOLISMIN CELLULARSLIME MOLD

411

tion of DNA was treated for 2 h at 37 ° with 2o #g/ml of ribonuclease that had been heated for lO min in a boiling bath, and later, with 50 #g/ml of self-digested pronase, for another 2 h. These treatments were followed by deproteinization with I M sodium perchlorate and I vol. chloroform containing o.I vol. isoamyl alcohol, shaking at room temperature for I h. The last treatment was repeated until no solid interphase appeared. The purified DNA was precipitated with I vol. absolute ethanol and stored at 4 ° as a solution in I : IOO SSC with a few drops of chloroform. The specific activity obtained varied between ioo and 200 counts/rain per #g DNA, and the yield was 60-80 ~o of the initial DNA, which in the strain used is 0.3 ~o of the dry weight.

Puri/ication o! rRNA [or hybridization The cell extract made by lysing with o. I °/o sodium dodecyl sulfate was treated with VS-saturated phenol, at room temperature. The aqueous phase was extracted twice more with phenol and then precipitated with 2 vol. of ethanol. After I h in the freezer, the RNA was separated b y centrifugation at 500o rev./min for IO rain and dissolved in I ml sodium dodecyl sulfate buffer (o.oi M Tris, o.I M NaC1, o.ool M EDTA, o.5 O//osodium dodecyl sulfate (pH 7.4)). This solution was passed through a 20 cm × I cm column of Sephadex G-25 coarse, equilibrated with sodium dodecyl sulfate buffer at room temperature, and the excluded fraction of about 3-4 ml, precipitated again with o.I M NaC1 and 2 vol. ethanol. The sediment was dissolved in I ml sodium dodecyl sulfate buffer and fractionated twice in a 15-3o % sodium dodecyl sulfate-sucrose gradient, isolating the ribosomal peaks and recovering the RNA by precipitation with ethanol. The last sediment was dissolved directly in 2 ×SSC and stored in the freezer.

Hybridization technique The procedure used was that described b y GILLESPIE* AND SPIEGELMAN 15. The DNA was denatured with 0.3 M KOH for 15-2o min at room temperature, and neutralized with HC1. The solution was then diluted with 2 × SSC to a concentration of 2 ~ ug/ml and 5 ml passed through each nitrocellulose membrane filter (B-6, coarse of Schleicher and Schuell) with vacuum, washed with IOO ml of 2 ×SSC and dried, first at room temperature for 6 h and later at 80 ° for 2 h. Incubation of the filters with RNA was performed in 5 ml of 2 x SSC in screwcap vials, at 65 ° for 12 h. After cooling the vials in ice, the filters were washed with 50 ml of 2 x SSC on each side, placed in 5 ml of 2 × SSC containing 20 #g/ml boiled ribonuclease and incubated at room temperature for I h. The washing procedure was repeated and the filters dried before adding liquiflor and counting. The noise level was determined with each concentration of [32P]RNA by placing an empty filter in the same vial with the DNA filter and processing it in the same way. The values obtained were always corrected for noise.

Chemicals [14C~Uracil (30 mC/mmole) and ~3H]uracil (5.61 C/mmole) were purchased from New England Nuclear Corp.; sterile, neutralized carrier-free sodium E3~Plphosphate * The kind advice and instruction of Dr. D. GILLESPIE,of Brandeis University, in the performance of hybridization assays is gratefully acknowledged.

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R. R. SUSSMAN

from Cambridge Nuclear Corp.; ribonuclease, crystallized from ethanol, from Worthington Biochem. Corp.; sodium dodecyl sulfate and sodium deoxycholate from Fisher Chemical Co.

Analytical procedures Protein was determined b y the LOWRY assay 16 with crystalline bovine serum albumin as a standard; RNA b y the orcinol reaction 17, with yeast RNA as a standard.

RESULTS

Net changes in dry weight, protein and RNA content Washed amoebae were deposited on filters and harvested after varying periods of incubation. Dry weight, protein, and RNA determinations were carried out on replicate cell samples. Table I summarizes the data. During morphogenesis, the dry weight diminished to 59-73 % of its initial value. Roughly proportionate net losses were observed in protein content (58-62 %) and RNA (57-7) %). The results are similar to those reported previously for D. discoideum is. Substitution of citrate for phosphate buffer or addition of uracil (IOO mM) to the support pad fluid did not affect the values shown in Table I. TABLE I NET CHANGES IN MAIN COMPONENTS DURING MORPHOGENESlS A m o e b a e were g r o w n in liquid m e d i u m to 6. ioS]ml, sedimented and w a s h e d w i t h salt solution 3 times, r e s u s p e n d e d in salt solution, at 2. I o s / m l a n d o. 5 ml spread on each filter at zero time. Filters were i n c u b a t e d a t I6 ° and at the indicated t i m e s two of t h e m h a r v e s t e d together. Cells were w a s h e d w i t h IO ml of cold distilled H,O. The s e d i m e n t w a s r e s u s p e n d e d in 2 ml H,O, t h e cells c o u n t e d a n d o. i-ml aliquots used for d r y w e i g h t s in triplicate. The r e s t of the suspension w a s divided for duplicate d e t e r m i n a t i o n s of R N A and protein. N u m b e r s in first line r e p r e s e n t m e a n s of four s u c h e x p e r i m e n t s i s t a n d a r d deviations. The second line is the p e r c e n t r e m a i n i n g at each stage, w h e n Stage i is considered ioo %. Stage i, t h; Stage 2, 2I h; Stage 3, 44 h; Stage 4, 69 h.

Component

Stage z (no aggregation)

Stage 2 (pseudoplasmodium )

Stage 3 (culmination)

Stage 4 (mature ]ruits)

8.6io.9 ioo

7 . 7 i i.o6 90

6.olo.6 7°

5.7~o.62 66

P r o t e i n ( m g / i o s cells) 5 . 5 t o . 9 % of initial ioo

5.2~o.12 94

3.9~o.87 71

3.3io.II 6o

R N A ( m g / i o 8 cells) ~/o of initial

1.44-4-o.22 93

1.25to.16 81

1.o6io.17 68

Dry weight (mg/io 8 cells) ~o of initial

1.55~o.II ioo

Incorporation o[ E14C]uracil into the acid insoluble [faction during morphogenesis Washed cells were deposited on the filters and exposed to El*C]uracil commencing at zero time as described in the METHODS section. At intervals, replicate cell samples were harvested, precipitated in IO % cold trichloroacetic acid, deposited on Biochim. Biophys. Acta, 149 (1967) 4o7-42I

RNA

413

METABOLISM IN CELLULAR SLIME MOLD

Millipore filters, washed with trichloroacetic acid, dried and counted at 35 % efficiency in a gas flow counter. Fig. I shows that the uracil was incorporated at a constant rate (67 ° #ffmoles/h) for about 15 h and then the apparent rate declined to zero at about 22 h. The latter was shown to be due to equilibration rather than cessation of RNA synthesis since replicate cell samples, incubated for 22 h in the absence of [14Cluracil and exposed to it thereafter, incorporated the isotope at a significant rate (13o iffmoles/h) though smaller than the initial level. The reduction in the rate of incorporation coincides with the end of the cell aggregation period and the beginning of the fruiting body construction. It cannot be ascribed to decreased availability of the assimilated uridine since the cold trichloroacetic acid-soluble fraction possessed relatively constant radioactivity (75-1o5 counts/min per ro T cells) throughout the entire developmental period. Ultimately the rate of incorporation declined to zero at about 50 h, coinciding with the termination of fruiting body construction. Cells, pulse labeled at this time, failed to incorporate uracil. In the samples collected up to the terminal stage of fruit construction, 95-97 % of the radioactivity in the trichloroacetic acid precipitates was solubilized by treatment with crystalline ribonuclease. In samples collected at the very end, 85-9 ° % of the radioactivity was ribonuclease sensitive. In cells that had incorporated [14C]uracil between 25-46 h of incubation on Millipore filters, all of the assimilated radiooSpecific activity of RNA o

2xlO 4

100

o

• Ratio RNAlprotein x1OO

5C

lx10 a

E

"F_

c

o

o ~~ ° o o D i

I.~a

20

Time (h)

/

I

40

i

6~1

~;

3'o'

Time (h)

~'o--

Fig. I. [14CJUracil u p t a k e d u r i n g differentiation. I . lO 7 a m o e b a e were deposited on each filter. The s u p p o r t i n g p a d contained i/2C of [14C]uracil (47 m C / m m o l e ) in 0. 5 ml of io mM McIlvain b u f f e r (pH 5), therefore the a m o u n t of uracil per filter w a s 21 m/zmoles. One set w a s labeled f r o m zero time on, a n d the second set w a s s t a r t e d on filters w i t h o u t isotope a n d t r a n s f e r r e d to p a d s w i t h the label a t 22 h. Filters were i n c u b a t e d at 16 ° and a t intervals, three of t h e m were h a r v e s t e d separately. The cells were w a s h e d twice w i t h io ml cold H~O. T h e r e s u s p e n d e d s e d i m e n t s were precipitated w i t h trichloroacetic acid and c o u n t e d as described in METHODS:Each p o i n t r e p r e s e n t s t h e m e a n of the triplicate samples. Fig. 2. D e c a y of stable [14C]RNA d u r i n g fruiting. Vegetative a m o e b a e were g r o w n in 400 ml axenic m e d i u m c o n t a i n i n g 2o~uC of [llCJuracil until t h e y reached 2 - 4. Io6]ml. The cells were sedimented, w a s h e d and r e s u s p e n d e d in fresh axenic m e d i u m , and i n c u b a t e d for an additional 2 - 3 h. The a m o e b a e were centrifuged down, w a s h e d a n d r e s u s p e n d e d in s a l t solution at a d e n s i t y of 2. Ios/ml. o.I ml w a s deposited on filters and tiffs w a s considered zero time. Filters were inc u b a t e d at 16 ° a n d at the indicated times, 5 of t h e m were h a r v e s t e d together, w a s h e d w i t h io ml cold H~O and the s e d i m e n t r e s u s p e n d e d in I ml cold H~O. This s u s p e n s i o n w a s divided as follows: o.i ml for cell c o u n t s ; o. i ml for R N A d e t e r m i n a t i o n s in duplicate; 0.04 ml for p r o t e i n determin a t i o n s in triplicate and 0. 5 ml for trichloroacetic acid p r e c i p i t a t i o n a n d radioactive counts. Two s u c h s u s p e n s i o n s were used for each stage in a given e x p e r i m e n t a n d four e x p e r i m e n t s performed. O - Q , specific a c t i v i t y of R N A , d e t e r m i n e d b y trichloroacetic-insoluble, ribonucleasesensitive c o u n t s / m g of R N A ; 0 - 0 , p r o p o r t i o n of R N A to p r o t e i n c o n t e n t in t h e s a m e sample.

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activity could be accounted for as uridine and cytidine ribotides. The harvested, washed cells were hydrolyzed with 0.5 M LiOH 19, incubated at 37 ° for 2 h, adjusted to pH 3.o with 4.5 % perchloric acid. and centrifuged. The supernatant was neutralized with LiOH. Aliquots were dried, extracted with ether-isopropanol (2:I, v/v) to remove the salt and the solids dissolved in water and analyzed b y paper electrophoresis ~°. 84 % of the original radioactivity was recovered in two spots, identified as UMP (61%) and CMP (23 %). No counts were recovered elsewhere.

Decay of stable E14ClRNA during [ruiting Amoebae were grown in liquid medium, in the presence of [14C]uracil. Before reaching the stationary phase the cells were sedimented, washed and resuspended in fresh medium, shaken at 22 ° for 2 more h, harvested and put on filters in buffer with I mM cold uracil. At different times and stages of differentiation, the cells were collected, washed, resuspended in H~O, and aliquots taken for determination of cell counts, protein, RNA, and trichloroacetic acid-precipitable counts. Fig. 2 is a summary of several experiments. During the entire developmental sequence, the net RNA content fell to 60 % of its initial value but in proportion to the loss of protein as demonstrated b y the constancy of the RNA:protein ratio. In this same period the specific radioactivity of the RNA fell to 57 % of its original value. Had no resynthesis of RNA occurred, the specific radioactivity would have remained at IOO %. The observed decrease means that, by the end of the fruit construction no more than 34 % of the original RNA could in fact have remained and this was diluted by RNA synthesized during development equivalent to 26 ~o of the original amount. This estimate of the isotope dilution is minimal since it is not corrected for recycling of the isotope during the turnover. However, the correction would probably be slight in any case, since for tile first 5 h of development the radioactivity of the trichloroacetic acid-soluble fraction was only about 2 % of the total radioactivity and, by the end of aggregation, had dropped to an undetectible level. In a companion experiment performed without the presence of cold uracil in the Millipore pad fluid, 49 % of the radioactivity originally present in the vegetative cell RNA was found to have been conserved in the RNA of the mature fruit.

Sedimentation characteristics o/RNA synthesized during development Cells at different stages of development on filters were exposed to [3H]uracil for brief periods and then either harvested immediately or transferred to fresh support pads saturated with Mcllvain buffer containing a large excess of cold uracil and incubated for an additional 6 h to permit the decay of unstable RNA before harvesting. The cells were then lysed with sodium dodecyl sulfate and the extracts centrifuged in a sucrose-sodium dodecyl sulfate gradient (see METHODS). Fig. 3 shows some typical profiles of absorbance and radioactivity. The 2o-min pulses yielded very heterogeneous distributions of radioactivity, regardless of developmental stage. In contrast, pulses followed by chases with cold uracil yielded distributions in which the majority of counts were confined to the 23-S, I6-S and 4-S peaks. Longer periods of labeling (2 h or more) also yielded the latter pattern even when not followed by a chase. Biochim. Biophys. Acta, 149 (1967) 4o7-421

RNA

METABOLISM IN CELLULAR SLIME MOLD A not aggregated- pulse

A' not aggregated chase

0.4

4OO

02.

300

0.7:

200

i:

i v

Uw

O.1

i

415

.4- B pseudoplasmodium-pulse

100

?

-B' pseudoplas modium-c ha se

oo

"2

~" 0.:

8

oo~

0.: i i

o'~ 2 o

0.4 -C culmination-pulse

C' culmination-chase

400 .-c 3OO

0,3

200

0.2 !

0.1 0

-q

100 I

I

I

10

20

30

Fraction No.

~

I

20

3b

Fig. 3. Sucrose g r a d i e n t analysis of pulse-labeled and stable R N A at different stages of m o r phogenesis. 2. lO7 cells on a filter a t the specified stage (A, A' before aggregation; B, B ' pseudop l a s m o d i u m ; C, C' culmination) were exposed to 0.05 ml of p H ] u r a c i l (o. 5 mC/ml; 5.61 C/mmole) on t o p of the filter. After i n c u b a t i o n for 2o min, some filters were h a r v e s t e d (pulse) a n d o t h e r s were t r a n s f e r r e d to p a d s containing b u f f e r a n d i o / , m o l e s cold uracil a n d i n c u b a t e d f u r t h e r for several h o u r s (chase). The washed, s e d i m e n t e d cells were lysed w i t h o. i ml io % s o d i u m dodecyl sulfate and m a d e u p to I ml w i t h distilled H,O. The whole s a m p l e w a s layered o v e r 29 ml of s o d i u m dodecyl s u l f a t e - s u c r o s e g r a d i e n t (I 5-3 o %) and centrifuged at 24 ooo r e v . / m i n for 17-18 h a t 19 °. 3o-35 fractions were collected a n d t h e acid precipitable c o u n t s d e t e r m i n e d in a liquid scintillation c o u n t e r on Millipore filters. , A260 my; O - - - O , radioactivity.

Some properties o/ the ribosomes In extracts obtained b y either grinding or treatment with deoxycholate, 74 % of the R N A was sedimented by centrifugation at IOO ooo × g for 2 h. Examination in the analytical ultracentrifuge revealed one main peak with an approximate sedimentation constant at 80 S, two smaller peaks (60 S and 4 ° S) and a varying amount of polysomal material which is virtually absent in the mature fruits. The trichloroacetic acid-insoluble radioactivity from 14C-labeled amino acids is primarily concentrated in the polysomal region 1. Apparently, the majority of the ribosomes of this species are Biochim. Biophys. Acta, 149 (1967) 4o7-421

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R . R . SUSSMAN

T A B L E II COMPOSITION OF RIBOSOMES R i b o s o m e s f r o m v e g e t a t i v e a m o e b a e a n d f r o m cells in e a r l y c u l m i n a t i o n were isolated a n d purified as d e s c r i b e d in METHODS. A n a l i q u o t w a s u s e d for d r y w e i g h t s a n d t h e d e t e r m i n a t i o n s of R N A a n d p r o t e i n p e r f o r m e d o n w h o l e r i b o s o m e s . T h e a m o u n t s f o u n d b y t h e oricinol a n d LowRY m e t h o d s agree w i t h t h o s e g i v e n b y t h e d r y w e i g h t minus R N A .

Ribosomes

% o[ dry weight

Ratio 260 mjz:235 mlz

Extinction at 260 ml*

RNA

Protein

Vegetative

51 ± 4 "

49

1.7-1.8

98-1o9

Fruits

5 3 ± 8.

47

1.7-1.8

97-IO7

* S t a n d a r d d e v i a t i o n of t h e m e a n f r o m s e v e r a l e x p e r i m e n t s .

not bound to membranes because the same results were obtained with or without deoxycholate. The 8o-S ribosomes, purified as described in METHODS,were stable in 5 mM Mg ~+ with Tris buffer (pH 7.4) and in Io mM Mg ~+ with phosphate buffer (pH 7.4). Initial extractions could not be made in the latter because the ribosomes tended to aggregate and did not readily disperse. In the presence of 5 mM Mg 2+ with phosphate, the ribosomes dissociated into the 6o-S and 4o-S subunits. The sedimentation patterns of ribosomes purified from vegetative cells and mature fruits were identical. Table I I compares other physicochemical properties of the ribosomes from these two developmental stages. The ultraviolet absorption gave a 260 m#:235 m# ratio of about 1.7-1.8 , which agrees with a 5 ° % protein content, as determined b y chemical analysis. The only difference found between ribosomes isolated from vegetative cells and those from fruits was that more protein was associated with the latter after three sedimentations, but with one or two more washings the amount fell to the same level as that of the former.

A comparison between the rRNA [rom vegetative cells and ]rom [ruiting bodies Table I I I shows the base compositions of bulk RNA and of purified 23-S, I6-S and 4-S RNA. The G + C content of 43 %, found in both peaks of rRNA, similar to t h a t of D. discoideum ~1 is one of the lowest thus far reported for any organism excepting Tetrahymena pyri[ormis 22. The G + C content of the 4-S RNA is also lower than 4-S RNA from other organisms ~2. (The G + C content of P. pallidum DNA is 33 %, determined b y equilibrium density centrifugation and thermal denaturation.) No significant differences in base composition were detected between the rRNA from vegetative cells and from fruits. Fig. 4 shows that r R N A from these two developmental stages were also identical in their capacity to hybridize with P. pallidum DNA. Varying amounts of unlabeled rRNA, purified either from vegetative cells or from fruiting bodies, were hybridized with aliquots of D N A b y the method of GILLESPIE AND SPIEGELMAN15. After washing, the filters were reincubated with an amount of ~zP-labeled rRNA from fruiting cells shown in the control curves to be above the saturating level. No difference was observed in the abilities of either of the unlabeled RNA preparations to Biochim. Biophys. Acta, 149 (1967) 4o7-421

M

O

v

2

t~

t~

Vegetative Fruits Vegetative Fruits

16 S 16 S 23 S 23 S 4 S Bulk R~TA*

5 3 5 5 4 2

N u m b e r of experiment~ 17.8 q-o.86 18.34-o.47 18.o4-o.6 17.84-o.45 24.54-2. 3 17.54-o.5

CMP

29.04-0.82 3o.3!o.47 29.54-0. 7 29.o4-1.12 22.34-2. 3 28.94-2.9

AMP

25.7±0.4 24.04-o.83 25.44-0. 4 25.54-o.45 29.24-1. 3 25.94-2.9

GMP

27.74-0.38 27.o4-o.o 27.14-1.1 27.7-t-o.89 24.04-0. 7 27.74-0.3

UMP

1.2o 1.2o 1.21 1.2o 1.o6 1.2

A +G C+ U

1.14 1.o 5 i.io 1.13 1.13 1.16

G+ U -A + C

43.3 42.3 43.4 43.3 53.7 43.4

°/oGC

" The bulk R N A w a s non-radioactive, purified R N A f r o m wkole cells a n d t h e d e t e r m i n a t i o n s were m a d e b y ultraviolet a b s o r p t i o n of each nucleotide peak. The values r e p r e s e n t t h e m e a n moles p e r c e n t of t h e respective m o n o n u c l e o t i d e s ± the s t a n d a r d deviations f o u n d in the indicated n u m b e r of experiments.

Vegetative

Stage

RNA

Cells at the indicated stage, had been previously labeled w i t h 32P t for m a n y h o u r s a n d t h e n c h a s e d w i t h cold p h o s p h a t e . The 4 S, 16 S a n d 23 S were isolated and purified as described in METHODS. After h y d r o l y s i s w i t h 0. 3 M K O H at 3 °0 for 18 h, the 2',3'-mononucleotides were separated in a D o w e x I c o l u m n and t h e i r r a d i o a c t i v i t y m e a s u r e d (see E x p e r i m e n t a l details in M E T H O D S ) .

BASE COMPOSITION OF R N A FRACTIONS

TABLE III

H

©

N

©

t~ > 7J

Z

418

R. R. SUSSMAN

lool

100 8~C

BO"I~~ ~DRdi~coideurn

-~ 60-

8 6O "6 4O

'~ 40" ov

20

20" 2

_9

unlabeled

8

q

o

~ Input RNA(IJg)

T L2~

"~oRpaflidum

unlabeled

RNA

q~2

a

Unlabeled rRNA input (pg)

Fig. 4- Competition between P. pallidum rRNA from vegetative amoebae and that from fruiting cells for cistrons of P. pallidum DNA. lO-3O/~g of denatured P. pallidum [SH]DNA fixed to nitrocellulose filters were incubated at 65 ° for 12 h in 5 ml 2 X SSC containing the indicated amount of cold rRNA (purified as described in METHODS). The membranes were cooled, washed and re-incubated at 65 ° for I2 h with 5 ml 2 × SSC containing 5 fig of s2p-labeled fruit rRNA. After washing, treating with ribonuclease, and washing again, they were dried and counted. The counts were corrected for background and noise, and the/zg of labeled RNA annealed to I #g of DNA were calculated. The control, considered ioo %, was the amount of [s2p] RNA hybridized to DNA which had not been previously subjected to cold RNA. A, unlabeled vegetative rRNA; ©, unlabeled fruit rRNA; I , saturation curve of s2P-labeled vegetative rRNA; E], saturation curve of s'p-labeled fruit rRNA. Fig. 5. Competition between D. discoideum rRNA and P. pallidum rRI~A for sites on P. pallidum DNA. Conditions for hybridization were the same as in Fig. 4. First incubation with varying amounts of unlabeled rRNA. (~x-A, D. discoideum; 0 - 0 , P. pallidum). Second incubation with 5 fig of P. pallidum [s*p]rRNA.

e x c l u d e t h e l a b e l e d R N A . T h e s a m e results w e r e o b t a i n e d w h e n 32P-labeled r R N A f r o m v e g e t a t i v e cells was u s e d in t h e s e c o n d i n c u b a t i o n . Fig. 5 i n d i c a t e s t h a t a l t h o u g h P. pallidum a n d Dictyostelium discoideum h a v e r R N A of t h e s a m e G + C c o n t e n t , t h e r e is o n l y l i m i t e d h o m o l o g y b e t w e e n t h e m . T h u s u n l a b e l e d D. discoideum r R N A c o u l d e x c l u d e n o m o r e t h a n 45 % of l a b e l e d P. pallidum r R N A f r o m h y b r i d i z a t i o n w i t h P. pallidum D N A as c o m p a r e d w i t h 9 0 - 9 4 % exclusion by the homologous combination. T h e s a t u r a t i o n c u r v e s o b t a i n e d in s e v e r a l e x p e r i m e n t s s h o w e d t h a t a t p l a t e a u levels, b e t w e e n o . I a n d 0.2 % of t h e P. pallidum D N A w a s h y b r i d i z e d w i t h r R N A . T h i s is s i m i l a r to v a l u e s f o u n d in o t h e r o r g a n i s m s 2s,~4.

DISCUSSION

Uracil incorporation into R N A P. pallidum a m o e b a e i n c u b a t e d on M i l l i p o r e filters i n c o r p o r a t e [14C]uracil at a c o n s t a n t r a t e u n t i l t h e e n d of a g g r e g a t i o n a f t e r w h i c h t h e r a t e falls to a b o u t 20 % of t h e i n i t i a l l e v e l a n d t h e n s t a y s c o n s t a n t u n t i l t h e e n d of m o r p h o g e n e s i s . I n c o n t r a s t , t h e r a d i o a c t i v i t y ( a n d a b s o r b a n c e ) of t h e t r i c h l o r o a c e t i c a c i d - s o l u b l e p o o l rem a i n r e l a t i v e l y c o n s t a n t t h r o u g h o u t . U n f o r t u n a t e l y t h e s e d a t a a l o n e do n o t p r o v i d e an a c c u r a t e m e a s u r e of t h e r a t e s a t w h i c h t h e v a r i o u s classes of R N A are s y n t h e s i z e d . R e c e n t s t u d i e s in b a c t e r i a ~5 h a v e s h o w n t h a t t h e i n t r a c e l l u l a r p r e c u r s o r p o o l does

Biochim. Biophys. Acta, 149 (1967) 4o7-421

RNA

METABOLISM IN CELLULAR SLIME MOLD

419

not exchange freely with exogenously supplied material, and the entrance of the latter into the pool is drastically affected b y both RNA synthesis and breakdown, i.e. depends on the net rather than the absolute rate of RNA synthesis. In the slime molds this is further complicated b y the fact that the products of RNA breakdown do not reenter the precursor pool quantitatively but about 40 % disappears, presumably via catabolic activity (Table I). Consequently, a detailed estimate of the rate(s) of RNA synthesis must await information on the relative stabilities of the different RNA classes. A similar study in D. discoideum ~ has shown that E~H]uridine is incorporated at a constant rate throughout the developmental sequence falling to a negligible value only at the end of fruiting body construction. The radioactivity and absorbance of the trichloroacetic acid-soluble pool stayed constant throughout. With the same organism but under different experimental conditions, PANNBACKER21 reported that the rate of [14C]uracil incorporation rose approx. 4-fold at culmination compared to the initial stage. However, this coincided with an 8-fold increase in the specific activity of intracellular UTP and it was concluded that the real rate of RNA synthesis had therefore decreased by 50 %. Unfortunately, replicate determinations of the rate of RNA synthesis at four different stages of development varied by as much as 4-fold and with standard deviations of as much as 4-60 %. This variability m a y stem from the fact that in these incorporation experiments, the cells were removed from the air-agar interface and the dispersed cell masses were submerged in buffer and incubated with the labeled precursor under conditions which totally disrupt the fruiting process, and at a cell density about Io-fold higher than the maximum density attained by liquid cultures of D. discoideum. In the present work as well as that of INSELBURG AND SUSSMAN ~ the amoebae were maintained on the filters and exposed to the labeled precursor simply by shifting the filter to a fresh support pad. Thus normal development continued during the period of incorporation. Furthermore, D. discoideum must be grown with a bacterial associate and when as little as IO-4(w/w) of the latter contaminate the former during developmental sequence, the level of incorporation is exceedingly variable and the major proportion of RNA synthesized turns out to be bacterial rather than amoeboid as revealed by methylated albuminkieselguhr chromatography. Hence special precautions must be taken to rid the amoebae of the associate prior to this kind of determination. The fact that in PANNBACKER's experiments the specific radioactivity of the intracellular UTP pool rose for the first 6-8 min of exposure to labeled uracil and. then decreased b y 30 % in the next IO min can be interpreted in many ways. One would be that the size of the pool changed, but according to the author, its composition did not vary significantly. Therefore, either the external [14C]uracil was not in excess, in which case the uptake of [14C]uracil into RNA after 20 min is not meaningful, or there was a dilution of the pool due to breakdown of unlabeled RNA from the cells. In any case, it is not valid to relate incorporation rates to a diminishing pooh

Net changes in R N A constitution during development During fruiting body construction, P. pallidum amoebae lose at lea.st twothird of the RNA that they had as vegetative cells. The remaining one-third is diluted Biochim. Biophys. Acta, 149 (1967) 4o7-421

RNA

METABOLISM IN CELLULAR SLIME MOLD

421

RNA but rendered nascent RNA more labile when examined either in terms of incorporated radioactivity or the subsequent fabrication of a specific protein after removal of the drug. These results were also interpreted as reflecting the need for association of nascent messenger RNA with newly formed ribosomal components before migration through the nuclear envelope. Finally, it should be noted that experiments performed with levorphanol (unpublished results) which preferentially inhibits ribosome synthesis 32 indicate that the continued fabrication of new ribosomes is required for normal slime mold differentiation and morphogenesis.

ACKNOWLEDGEMENTS I wish to acknowledge the helpful assistance of Miss ELLEN P. RAYNER. This work was supported b y a grant (GB 5976X) from the National Science Foundation.

REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 I8 19 20 21 22 23 24 25 26 27 28 29 3° 31 32

W. D. PHILLIPS, A. RICH AND R. R. SUSSMAN, Biochim. Biophys. Acta, 80 (1964) 508. M. SUSSMAN, Biochem. Biophys. Res. Commun., 18 (1965) 763 . R. ROTH AND 1V[. SUSSMAN, Biochim. Biophys. Acta, I22 (1966) 225. J. M. ASHWORTH AND ~V[. SUSSMAN, J. Biol. Chem., 242 (1907) 1696. D. R. SONNEBORN, M. SUSSMAN AND L. LEVlNE, J. Bac.teriol., 87 (1964) 1321. J. INSELBERG AND M. SUSSMAN, J. Gen. Microbiol., 46 (1967) 59. M. SUSSMAN AND R. R. SUSSMAN,Biochim. Biophys. Acta, lO8 (1965) 463 . M. SUSSMAN, Proc. Natl. Acad. Sci. U.S., 55 (1966) 813. M. SUSSMAN, W . F. LOOMIS, JR., J. M. ASHWORTH AND R. R. SUSSMAN, Biochem. Biophys. Res. Commun., 26 (1967) 353. M. SUSSMAN, Science, 139 (1963) 338. J. T. BONNER, J. Exptl. Zool., lO6 (1947) i. C. G. KURLAND, J. Mol. Biol., 18 (1966) 90. W . GILBERT, J. Mol. Biol., 6 (1963) 389. W. E. COHN, J. Biol. Chem., 235 (196o) 1488. D. GILLESPIE AND S. SPIEGELMAN, J. Mol. Biol., 12 (1965) 829. O. H. LowRY, N. J. ROSEBROUGH,A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . G. ASHWELL,in S. P. COLOWICK AND N. O. KAPLAN,Methods in Eneymology, Vol. 3, A c a d e m i c Press, N e w York, 1957, p. 84. G. J. WHITE AND M. SUSSMAN, Biochim. Biophys. Acta, 53 (1961) 285. J. M. GEBICKI AND S. FREED, Anal. Biochem., 14 (1966) 253. E. D. SEBRING AND N. P. SALZMAN, Anal. Biochem., 8 (1964) 126. R. G. PANNBACKER, Biochem. Biophys. Res. Commun., 21 (1966) 34 o. M. L. PETERMANN, The Physical and Chemical Properties o] Ribosomes, Elsevier, N e w York, 1964, p. lO5. F. M. RITOSSA AND S. SPIEGELMAN,Proc. Natl. Acad. Sci. U.S., 53 (1965) 737S. A. YANKOFSKY AND S. SPIEGELMAN,Proc. Natl. Acad. Sci. U.S., 48 (I962) 1466. D. NIERLICH, Proc. 66th Ann. Meeting Am. Soc. Microbiol., Los Angeles, I966, W i l l i a m s Wilkins, B a l t i m o r e , in t h e press. M. HAYASHI AND S. SPIEGELMAN,Proc. Natl. Acad. Sci. U.S., 47 (1961) 1564. A. S. SPIRIN AND M. NEMER, Science, 15 ° (1965) 214. M. A. AJTKHOSHIN, N. V. BELITSINA AND A. S. SPIRIN, Biokhimiya, 29 (1964) 169. D. D. BROWN AND E. LITNA, J. Mol. Biol., 8 (1964) 669. D. H. SHIN AND I{. MOLDAVE, J. Mol. Biol., 21 (1966) 231. M. K. BACH AND H. G. JOHNSON, Nature, 209 (1966) 893. E. J. SIMON AND D. VAN PRAAG, Proc. Natl. Acad. Sci. U.S., 51 (1964) 1151.

Biochim. Biophys. Acta, 149 (1967) 4o7-421

420

R. R. SuSSMAN

b y an additional quantity of RNA synthesized during the course of morphogenesis equal to at least one-fourth of the amount originally present. Thus in the cells of the mature fruiting body, the net RNA content has fallen to 60 °/o of the original, of which about three-filth is old and two-fifth new. Since at least some of the latter also turns over during developmental sequence, it is clear that RNA synthesis represents major metabolic activity during slime mold development. Data on the net loss of RNA content and the rate of uridine incorporation in D. discoideum ~ are consistent with the above conclusions. The sensitivity of D. discoideum to actinomycin D in respect to morphogenetic topography and cytodifferentiation9 and to the synthesis of at least two developmentally regulated enzymes 7,8 indicates that the fabrication of this RNA is indeed required for normal slime mold development.

Classes o/ R N A synthesized during development Sedimentation analyses of cells labeled during a short pulse of [3H]uracil reveal the synthesis of RNA heterogeneous with respect to physical characteristics and quite unstable. This is presumably messenger RNA and work is in progress to confirm this supposition and to characterize the fraction in greater detail. A second class of RNA is distributed in the gradient coincidentally with ribosomal and transfer RNA and is quite stable. When cells were subjected to a long pulse of [all]uracil or to a short pulse and chase as in Fig. 3, over 9 ° °/o of the remaining radioactivity turned out to be associated with these fractions. Under comparable conditions (i.e. a 'shift down' from a nutrient-rich environment to one which is nutrient-poor or lacking in nutrients), bacteria differentially repress the synthesis of new rRNA and confine themselves to the formation of messenger RNA ~6. Early sea urchin 27, loach zs and amphibian embryos ~9, perform similarly and resume ribosomal synthesis only at the onset of gastrulation. In contrast, the data reported here indicate that the bulk of the RNA synthesized during the morphogenetic sequence is rRNA even at its terminal stages. Why these differentiating cells synthesize an appreciable amount of new ribosomes while destroying an even larger amount of old ribosomes that preexisted in the vegetative amoebae is an intriguing question. One obvious explanation, i.e. that a special class of ribosomes is required for the events attending fruiting body construction receives no support from comparisons of rRNA base composition, protein:RNA ratios, or specific hybridizability with DNA. If any difference does exist it must reside in the ribosomal proteins. However, J. AS~IWORTH (unpublished results) has compared the profiles of protein fractions obtained from isolated ribosomes of D. discoideum by treatment with guanidine and after separation by acrylamide gel electrophoresis. No differences were found between the ribosomal preparations of vegetative ceils and mature fruiting bodies. Another explanation is that newly synthesized messenger RNA must be stripped from the DNA template a° and/or conducted through the nuclear envelope b y ribosomes or ribosomal subunits 31 and that, in this organism at least, the absence of a preformed pool of ribosomes within the nucleus and the inability of old ribosomes to return to the nucleus from the cytoplasm requires continued fabrication of new ribosomes even in the presence of a surplus of old ones. A previous study of D. discoideum 8 revealed that cycloheximide, a reversible inhibitor of protein (and ribosome) synthesis, did not affect the stability of preformed Biochim. Biophys. Acta, 149 (1967) 4o7-421