118

Biochimica et Biophysica Acta, 519 (1978) 118--124 Q Elsevier/North-Holland Biomedical Press

BBA 99172

RIBOSOMAL DNA IN SPORES OF PHYSARUM POL YCEPHALUM

HANS-URS A F F O L T E R and RICHARD BRAUN

Institute of General Microbiology, University of Bern, Altenbergrain 21, CH-3013 Bern (Switzerland) (Received July l l t h , 1977)

Summary DNA was isolated from plasmodia, spores and newly hatched amoebae of the slime mould Physarum polycephalum. The DNA preparations were fractionated in CsCl gradients and each fraction hybridised to combined 19 S + 26 S rRNA. In all three DNA preparations hybridisation was found to be limited to satellite DNA (p = 1.714 g/cm 3) and at saturation was found to reach a level of 0.16-0.18% of total DNA. The main band of nuclear DNA (p = 1.702 g/cm 3) did not hybridise appreciably. Further experiments using analytical CsC1 gradients revealed that the ratio of satellite to main band DNA was similar in all three preparations. It is concluded that the genes for ribosomal RNA are equally reiterated in spores, hatching amoebae and in plasmodia. They appear to be similarly organised in all stages of the life cycle examined so far.

Introduction The life cycle of the true slime mould Physarum polycephalum comprises a unicellular amoebae stage, a multinucleate plasmodial stage and a sporulation stage [1]. The a m o u n t of rDNA in some of the different stages has been investigated previously [2--4] and found to be the same in spherules, plasmodia and amoebae. Most, if not all the genes coding for rRNA in amoebae and plasmodia are located on linear extrachromosomal DNA molecules of 3 8 . 1 0 6 daltons, each containing two genes for 19 S and 26 S rRNA, palindromically arranged [4--6]. All detectable rDNA is localised in nucleoli and each nucleolus on the average contains 75--150 such rDNA molecules. The extrachromosomal localisation suggests that the multiplicity may have arisen by a process of amplification at some specific stage of the life cycle. Since spores are undergoing meiosis and are metabolically much less active than the vegetative amoebae and plasmodial stages, it seemed conceivable that spore rDNA may not be amplified and that additional copies may be made by amplification during more favorable

119 growth conditions in hatching spores. In fact, no evidence for such an amplification was found. Materials and Methods The plasmodial strain used in these experiments LU 648 X LU 688, kindly provided by Dr. J. Mohberg, was chosen because of its high sporulation and germination rates. Microplasmodia were grown in shake culture and macroplasmodia on filter papers [7,8]. To obtain spores, microplasmodia were fused on agar plates containing half strength medium, incubated for 4 days in the dark and then illuminated in a day-night rhythm by natural light, carefully avoiding direct sunlight and temperatures above 28°C. Using this method, more than 95% of the plasmodia sporulated and melanisation of the spores was achieved 10 days after inoculation. Nuclei from macroplasmodia were isolated by the method of Mohberg and Busch [9] with minor modifications. Total DNA from mature spores was obtained by suspending sporangia from 20 agar plates, 10 days after melanisation, in 5 ml of 0.5 M NaC1, 10 mM EDTA, 10 mM Tris • HC1, pH 7.8, containing 500 gg/ml proteinase K (Boehringer Mannheim) and 2% dodecyl sulphate in a mortar (on a --20°C ice/salt mixture} and grinding the suspension with 15 g alumina for 10 min. Another 500 pg/ml proteinase K were added and the homogenate was then incubated for 30 min at 37°C. Alumina, intact spores and spore debris were pelleted, at 5000 X g for 10 min (0°C). The DNA in the supernatant was then purified according to Hall and Braun [4]. Sporangia from 20 agar plates were ruptured in 10 ml of BTC medium [10] with five strokes in a Potter-Elvehjem homogeniser and then incubated at 26°C to let the spores hatch. After 1 h proteinase K (500 #g/ml) and 2% dodecyl sulphate (w/v) were added, and the mix was incubated for 1 h at 37°C to lyse the amoebae. The solution was then centrifuged and the amoebal DNA isolated from the supernatant as described above. Total nuclear DNA from late G2 surface plasmodia and [3H]rRNA were purified as described by Hall and Braun [4]. RNA-DNA hybridisation was performed either on filters according to Hall and Braun [4], or in solution as follows. 10 #g of DNA were denatured at 85°C for 10 min in 0.5 ml of a buffer containing 0.12 M NaH2PO4 + 0.12 M Na2HPO4 (pH 6.8) in 50% (v/v) formamide plus 0.01% (w/v} dodecyl sulphate, 6.2 mM EDTA, 100 #g Escherichia coli tRNA and 0.5 pg of Physarum [3H]rRNA (ref. 3, and Stalder, personal communication}. Hybridisation was then performed at 47°C and at suitable times the samples were removed and diluted with 4.5 ml standard saline citrate (0.15 M NaC1, 0.015 M sodium citrate, pH 7.0}. 50/~1 of a ribonuclease solution (preboiled for 7 min) containing 4000 units/ml Tl and 2 mg/ml A were then added. The mixture was incubated for 1 h at 30°C with the addition of a further 50 pl of the ribonucleases after 30 min. Hybrids were then precipitated in the presence of 250/~g of calf thymus DNA with 5% trichloroacetic acid (final concentration) for 1 h at 0°C and counted on Whatman GF/C filters. All reactions were carried out in sterile, acid cleaned screw-capped 10-ml Pyrex vials. The DNA content of spores was determined as described by Mohberg et al.

120 [11], with the following modifications: Sporangia were homogenised in 0.5 M perchloric acid with 10 strokes in a Potter-Elvehjem homogeniser, an aliquot counted in a h a e m a c y t o m e t e r and the DNA hydrolysed at 70°C for 1 h. The solution was then centrifuged at 5000 × g and the DNA analysed by the method of Burton [12], modified according to Giles and Myers [13].

Results

Buoyant density o f rRNA genes Total DNA from spores, hatched amoebae and from nuclei of late G2-phase plasmodia was isolated and fractionated on isopynic CsC1 gradients. Each fraction was hybridised with [3H]rRNA [4] as described above. Fig. 1 shows that in all cases 19 S and 26 S RNA hybridised with DNA at the heavy satellite position (p = 1.714 g/cm 3) and not with the bulk of the nuclear DNA (p = 1.702 g/cm3). Phage lambda [14C]DNA was used as an internal marker (p = 1.709 g/cm3). This shows that in all three stages of the life cycle examined here the ribosomal genes are present in a satelite DNA. In order to obtain a rough quantitative estimate of the a m o u n t of satellite DNA, total DNA from spores and nuclear DNA from plasmodia was studied in analytical CsC1 gradients. Fig. 2 shows that both DNA preparations contain the typical heavy satellite in approximately equal proportion to the main band DNA. In Fig. 2 a shoulder can also be seen on the lighter side of the main peak in the case of total spore DNA. Its density and its absence from nuclear DNA preparations indicate that it is mitochondrial DNA. Reiteration o f rDNA genes in spores It can be seen from Fig. 1 that spores must contain numerous ribosomal genes, but these experiments do not allow an accurate estimation of the gene number. Consequently we determined the saturation values for total DNA of spores and hatched amoebae and compared them with values obtained with nuclear DNA from plasmodial surface cultures. Due to the extreme mechanical resistance of the spore wall and the harsh method that had to be used for extraction, the resulting DNA had a rather small and heterogeneous size. It ranged in size from 0.5 • 106 to 15 • 106 daltons as determined by alkaline sucrose gradient centrifugation (data not shown). Whilst total, double-stranded DNA from plasmodial nuclei can be isolated with a molecular weight over 50 • 106. To get comparable results the DNA preparations from amoebae and plasmodia were sonicated to a similar size and hybridisations were carried out in solution as described in Materials and Methods, since preliminary experiments had revealed that the use of a filter hybridisation m e t h o d led to losses of DNA. Although the saturation value (H s) may be obtained from the plateau of a plot reporting the extent of hybridisation (H) versus time (t), a more accurate value is obtained from a reciprocal transformation, where t/H is plotted against time (see insets to Fig. 3). This transformation leads to a linear function, where H, is derived from the reciprocal of the slope. This can be computed by linear regression analysis o f the data [3]. It can be seen t h a t at saturation very similar

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F i l l 1. B u o y a n t d e n ~ t y o f pbu~nodtRI spore and amoebal r R N A genes. 60--100 ~g o f purified D N A and a s m a l l a m o u n t o f ?,-phage [ 1 4 C ] D N A m a r k e r w e r e d i l u t e d t o 4 m l w i t h s t a n d a r d saline c i t r a t e ( 0 . 1 5 M NaCI, 0 . 0 1 5 M s o d i u m c i t r a t e , p H 7 . 0 ) a n d m i x e d w i t h 5 g o f CsCI. A f t e r a d j u s t i n g t h e r e f r a c t i v e i n d e x t o 1 . 4 0 0 0 a t 1 8 ° C , t h e s o l u t i o n w a s o v e r l a i d w i t h m i n e r a l oil a n d c e n t r i f u g e d in a B e c k m a n T i 5 0 f i x e d - a n g l e rotor at 33 000 rev./min for 55--60 h (18°C). 0.2-ml fractions were collected, diluted to 0.7 ml with w a t e r a n d t h e a b s o r b a n e e at 2 5 8 n m m e a s u r e d . A f t e r d e n a t u r a t i o n o f t h e D N A w i t h alkali a n d n e u t r ~ l i u t i o n , e a c h f r a c t i o n w a s b o u n d t o n i t r o c e l l u l o s e filters a n d h y b r i d i s e d w i t h c o m b i n e d 1 9 S a n d 2 6 S [ 3 H ] r R N A . o, a b s o r b a n c e at 2 5 8 n m ; e, R N A • D N A h y b r i d ; × , b u o y a n t d e n s i t y . T h e a r r o w i n d i c a t e s t h e p o s i t i o n o f t h e ?,-phage D N A m a r k e r (p = 1 . 7 0 9 g / c m 3 ) . ( A ) P l a s m o d i a l n u c l e a r D N A . (B) T o t a l s p o r e D N A . (C) T o t a l a m o e b a ] D N A . Fig. 2. A n a l y t i c a l CsCI c e n t r i f u g a t i o n p r o f i l e s o f t o t a l s p o r e D N A a n d n u c l e a r p l a s m o d i a l D N A . 2 5 - - 3 0 ~ug o f p u r i f i e d D N A w a s t m d i m e n t e d in a n a n a l y t i c a l i t o p y c n i c CsCI g r a d i e n t ( s t a r t i n g r e f r a c t i v e i n d e x : 1 . 4 0 3 0 ) a t 4 4 7 7 0 r e v . / m i n f o r 2 4 h ( 2 5 ° C ) in a S p i n e o M o d e l E u l t r a c e n t r i f u g e , t h e D N A h a n d s b e i n g l o c a t e d b y u l t r a v i o l e t o p t i c s . A n i n t e r n a l Microcoecus lysodeicticua D N A d e n s i t y m a r k e r w a s i n c l u d e d . ( A ) T o t a l s p o r e D N A . (B) P l a m n o d l a l n u c l e a r D N A . ~ M. |ysodeicticum D N A (p = 1 . 7 3 1 g / e r a 3 ) ; b , Physarum h e a v y n u c l e a r m t e l l l t e D N A (p ffi 1 . 7 1 4 g / e r a 3 ) ; c, Physarum m a i n b a n d n u c l e a r D N A (p = 1 . 7 0 2 g / c m 3 ) ; d, P h y ~ r u m m i t o c h o n d r i a l D N A (p = 1 . 6 8 6 g / c m 3 ) . D e n s i t i e s w e r e c a l c u l a t e d b y t h e method of Szybahtki [17] uttng densttometer traeingt of photographs.

122

hybridisation values are obtained for spores, hatching spores and synchronous G2 phase surface cultures o f plasmodia. In order to convert the saturation values to numbers of gene copies per spore, the D N A c o n t e n t of spores was determined. It was found to be 0.68 -+ 0.03 pg (mean of five determinations) of DNA per spore. Using this value together with the saturation value from Fig. 3, it can be calculated that spores contain 340 rRNA genes, a number very close to the 320 copies found by Hall et al. [3] in synchronous surface cultures at mitotic prophase. The data clearly show that there are no siginificant differences in numbers o f rRNA genes between amoebae, spores and plasmodia.

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123

Discussion The vast majority of rRNA genes in plasmodia and amoebae of Physarum polycephalum are located in nucleoli on linear extra-chromosomal pieces of DNA [4--6]. Each rDNA molecule is a palindrome from which t w o precursors of r R N A are transcribed. Not only their extrachromosomal location, b u t also their m o d e of replication makes them resemble prokaryotic episomes. Previous experiments have shown that the extrachromosomal rDNA is growing plasmodia is metabolically stable at least over several mitotic cycles and that it is capable of replication. On the average each extrachromosomal rDNA molecule replicates once per mitotic cycle [14]. Despite the ability of extrachromosomal rDNA to serve as a template for its own replication during vegetative growth, the extrachromosomal rDNA may conceivably have arisen through a process of amplification of chromosomal genes at a certain stage of the life cycle, as most likely occurs in Tetrahymena [15]. If this were the case also in Physarum, one would have to find a "deamplified" stage and this might well be the spore, which arises through meiosis [16] and is able to survive for long periods of time under adverse conditions. In fact the experiments presented here clearly establish that both mature spores and freshly hatched amoebae contain the same number of copies of rDNA as plasmodia do. This suggests that the ratio of rDNA to total DNA stays constant within a factor of 2 throughout the life cycle of Physarum and that no amplification takes plane. However, this conclusion needs to be qualified. It cannot be excluded that all extrachromosomal rDNA is rapidly degraded at some time in the life cycle and immediately re-synthesised from one or several chromosomal master,copies. A possible time, at which such a rapid degradation and synthesis could occur, is meiosis. Were this the case, then genes fcrr rRNA could be inherited in the usual Mendelian fashion in Physarum as in other organisms. Otherwise non-Mendelian inheritance would be expected as is known for other extrachromosomal genetic elements. It had previously been shown by Mohberg et al. [11] that spores are in a haploid G: phase. This was based on colorimetric assays of the amount of DNA in spores and on chromosome counts in dividing amoebae. In this communication the DNA determinations are confirmed; in addition the hybridisation data show that the ratio of rDNA to total DNA is the same as that found in plasmodia in the late G: phase.

Acknowledgements We wish to thank Dr. L. Hall for critical reading of the manuscript. Supported b y grant No, 3.501.75 of the Swiss National Science Foundation.

References 1 Sauer, H.W. (1973) in Symposia of the Society for General Microbiology (Ashworth, J.M. and Smith, J.E., eds), Vol. 23, pp. 375---405, Cambridge University Press, L o n d o n 2 Ryser, U. and Braun, R. (1974) Binchim. Biophys. Acta 361, 33--36 3 Hall, L., Turnock, B. and Cox, B.J. (1975) Eur. J. Biochem. 51,459---465 4 Hall, L. and Braun, R. (1977) Eur. J. Biochem. 76, 165--174

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5 Vogt, V.M. and B r a u n , R. ( 1 9 7 6 ) J. Mol. Biol. 106, 5 6 7 - - 5 8 7 6 Molgaard, H.V., M a t t h e w s , H.R. and B r a d b u r y , E.M. ( 1 9 7 6 ) Eur. J. B i o c h e m . 68, 5 4 1 - - 5 4 9 7 Daniel, J.W. a n d Baldwin, H.H. ( 1 9 6 4 ) in M e t h o d s in Cell P h y s i o l o g y ( P r e s c o t t , D.M., ed), VoL 1, pp. 9 - - 4 1 , A c a d e m i c Press, New Y o r k 8 G u t t e s , E. a n d G u t t e s , S. ( 1 9 6 4 ) in M e t h o d s in Cell P h y s i o l o g y ( P r e s c o t t , D.M., ed), Vol. 1, pp. 4--8, A c a d e m i c Press, New Y o r k 9 Mohberg, J. and Ruach, H.P. ( 1 9 7 1 ) Exp. Cell Res. 66, 3 1 5 - - 3 1 6 10 McCullough, C.H.R. a n d Dee, J. ( 1 9 7 6 ) J. Gen. Microbiol. 95, 1 5 1 - - 1 5 8 11 M o h b e r g , J., B a b c o c k , K.L., Haugli, F.B. a n d R u s c h , H.P. ( 1 9 7 3 ) Dev. Biol. 34, 2 2 8 - - 2 4 5 12 B u r t o n , K. ( 1 9 5 6 ) B i o c h e m . J. 62, 3 1 5 - - 3 2 3 13 Giles. K.W. and Myers, A. ( 1 9 6 5 ) N a t u r e 206, 9 3 14 Vogt, V.M. and Braun, R. ( 1 9 7 7 ) Eur. J. B i o c h e m . 80, 5 5 7 - - 5 6 6 15 Yao, M.-C. a n d Gall, J.G. ( 1 9 7 7 ) Cell 12, 1 2 1 - - 1 3 2 16 Aldrich, H.C. ( 1 9 6 7 ) M y c o i o g i a 59, 1 2 7 - - 1 4 8 17 Szybalski, W. ( 1 9 6 8 ) in M e t h o d s in E n z y m o l o g y ( G r o a s m a n , L. a n d Moldave, K., eds), Vol. 12B, pp. 3 3 0 - - 3 6 0 , A c a d e m i c Press, New Y o r k