Planta (Berl.) 111, 157--166 (1973) 9 by Springer-Verlag 1973
Incorporation of Different Labelled Precursors into Chloroplast RNA of Chlordla Volker S s y m a n k Pflanzenphysiologisches Institut der Universitiit, D-3400 G6ttingen, Federal Republic of Germany Received January 17, 1973
Summary. Radioactive uridine is incorporated by Chlorella strain 211-8b/p into ribosomal subunits and their rapidly labelled RNA comigrates with chloroplast RNA on polycrylamide gels. Ribosomal particles which can be labelled by short pulses of orotic acid cosediment with the particles labelled by uridine pulses and contain the same RNA species as these when separated either on sucrose gradients or on polycrylamide gels. This incorporation is, like that of uridine, sensitive to rifampin and chloramphenicol, but insensitive to cycloheximide. A comparative study of short-time incorporation of uridine, orotic acid and guanosine into the RNA of Chlorella showed that all three precursors were incorporated mainly into RNA of chloroplastic origin. However, guanosine was also partly incorporated into cytoplasmic rRNA. Nitrogen-deficient cells always incorporated part of all three precursors into cytoplasmic rRNA, but the proportions of these were different anaong the different precursors. These results are consistent with the hypothesis that the described differences in the incorporation of the above mentioned precursors into I~NA of different cellular compartments are largely attributable to effects of pool sizes. Introduction I n cultures of the green alga Chlorella, evidence has been presented for a preferential incorporation of radioactive uridine into chloroplast ribosomM particles during short pulses (Galling and Ssymank, 1970). This selective incorporation could be affected in p a r t b y changing the physiological conditions for the algal cultures, for example b y growing t h e m in a nitrogen-deficient m e d i u m (Ssymank, 1972). On the other hand, short-time labelling of algal nucleic acids with labelled phosphate does not result in a preference for chloroplast rt~NA (Galling and Ssymank, 1970). One possible explanation of such a preferential labelling could be the fact t h a t uridine itself does not occur a m o n g the natural precursors of I~NA, b u t is a usual degradation intermediate. Thus the local separation of enzymes used for t~NA synthesis and I~NA degradation between cytoplasm and nucleus could result in a delay in the labelling of cytoplasmic rI~NA b y uridine. Another i m p o r t a n t factor is the relative sizes of uridine pools in the cytoplasm, the nucleus and the chloroplast, which will considerably affect the rate of incorporation into I~NA. Influences of precursor pools on the rate
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of i n c o r p o r a t i o n h a v e been r e p o r t e d for r a t liver (Bucher a n d Swaffield, 1969). There is evidence t h a t Chlorella cells c o n t a i n r e l a t i v e l y large nucleotide pools ( I w a m u r a st al., 1963). I n t h e case of t h e first e x p l a n a t i o n described above, n i t r o g e n deficiency m i g h t a c t b y reducing t h e a m i n o acid pools a n d t h u s switch on t h e s t r i n g e n t control m e c h a n i s m of R N A synthesis in t h e chloroplast described b y S u r z y c k i a n d H a s t i n g s (1968). This has a l r e a d y been discussed ( S s y m a n k , 1972). I n t h e case of t h e second e x p l a n a t i o n , this effect of n i t r o g e n deficiency would be covered b y t h e direct influence on nucleotide pool sizes. I n t h e p r e s e n t p a p e r , t h e i n c o r p o r a t i o n of orotic acid, a n a t u r a l precursor to I~NA, is c o m p a r e d with t h a t of uridine. This m i g h t p r o v i d e evidence concerning t h e v a l i d i t y of t h e first h y p o t h e s i s listed above. To t e s t t h e second hypothesis, a t h i r d R N A precursor, guanosine, was used. This nucleoside like uridine does n o t occur in t h e n a t u r a l p a t h w a y of R N A biosynthesis. H o w e v e r , b o t h t h e p a t h w a y s of f o r m a t i o n of purine or p y r i m i d i n e nucleotides a n d t h e i r i n c o r p o r a t i o n into I~NA arc quite different, so t h a t different influences on guanosine or uridine i n c o r p o r a t i o n m i g h t be expected. I n higher e u k a r y o t e s , different r a t e s of uridine a n d adenine i n c o r p o r a t i o n into R N A were o b s e r v e d ( S t a m b r o o k a n d Sisken, 1972). Materials and ~Iethods Culture and Labelling o/ Algae. The green alga Chlorella pyrenoidosa (strain 211-8b/p from the algal collection of the Institute of Plant Physiology, GSttingen) was used throughout. Culture conditions, incubations with antibiotics and labelling procedures employed were as described previously (Ssymank, 1972). The following radioactive substances were used: [5-3H]uridine (30Ci/mmole), [2-14C]uridine (60 mCi/mmole), [5-3H]orotic acid (25 Ci/mmole), and [8-3H]guanosine (t0 Ci/mmole); all were obtained from the Radiochemical Centre, Amersham, U.K. Preparation o/ Cellular Fractions and o] Nucleic Acids. Cellular fractions were prepared as described previously (Ssymank, 1972) ; 0.05 M Tris buffer pH 7.6 containing 10 mM MgC12was used. For isolation of intact nucleic acids cell homogenates in this buffer or in 0.03 M acetate buffer pH 5.0 containing 10 mM Mg2+ and 20 rag/1 polyvinyl sulfate or ribosome suspensions were extracted with an equal volume of a phenol-cresol mixture (140 ml m-cresol and 0.5 g 8-hydroxy quinoline per 1 water saturated phenol). The phenol phase was removed and the aqueous phase reextracted. The nucleic acids were precipitated out of the aqueous phase by three volumes of ethanol in the cold (modification of the method of Parish and Kirby, 1966). Sucrose JDensity Gradients. Lineor sucrose gradients of ribosome preparations (P105) were carried out and evaluted as described elsewhere (Ssymank, 1972). In the experiment described in Fig. 1, a gradient of 10 to 30% sucrose in 0.05 M Tris buffer pH 7.6 with a volume of 60 ml was prepared; this was sampled automatically using an ISCO DG Fractionator. Polyacrylamide Gel Electrophoresis. :Nucleic acids were further fractionated by electrophoresis on 2.4% polyacrylamide gels according to the method of Loening (1967) using the following buffer: 36 mM Tris, 30 mM phosphate, 1 mM EDTA, 0.2% SDS, pSI 7.8. Electrophoresis was conducted at 0 ~ C for 30 rain at 2 mA/gel and about 150 rain at 5 mA/gel. The u.v. absorption of the gels was monitored in a Joyce-Loebl Chromoscan.
Labelling of Chloroplast RNA in Chlorella
159
Determination el t~adioactivity. Polyaerylamide gels were frozen with solid COg and sliced by means of a Joyce-Loebl Gel Slicer into 1 mm fractions; these were solubilized in 0.1 ml It202 (30%) for 15 h at 60 ~ C and counted after addition of 2 ml Aquasol (NEN Chemicals) in a Nuclear Chicago Mark I liquid scintillation counter. Other radioactivity determinations were performed as described previously (Ssymank 1972). Results
In a previous paper (Galling and Ssymank, 1970), we showed that short-time incorporation of [aH]uridine leads to labelled particles with sedimentation constants of 50S and 30S; their nucleic acids sedimented at 23S and between 8S and 16S, respectively. Since sucrose gradients only poorly resolve cytoplasmic and chloroplast rRNA, we conducted the experiment separating the labelled RNA on polyacrylamide gels. This is shown in Fig. 1. Due to the lack of Mg~+ in the gradient buffer, the bulk of the ribosomes had dissociated and so the main u.v. peaks represented sedimentation values of 60S and 40S ; the radioactivity peaks occured at 50S, 30S and in a lighter region (Fig. 1 a). The fractions of each of these three peaks were pooled and subjected to a procedure to prepare the nucleic acids. These were electrophoresed on polyacrylamide gels. Figs. l b - d show that the RNA of the 50S peak migrates like chloroplast 23S rRNA and the RNA of the 50S peak migrates like chloroplast 23S rRNA and the RNA of the 30S peak like chloroplast 16S rRNA; this peak also contains some faster migrating material which may be a contamination as the third labelled peak of the particle gradient also contains similar faster migrating RNA. In previous investigations with orotic acid (Galling, 1972), it was not possible to obtain unequivocal evidence that orotic acid is incorporatiod in the same way as uridine, although this seemed verylikely. Two reasons could be given in an attempt to explain this uncertainty; the 14C-labelled orotic acid then available had an extremely low specific activity and was incorporated at a very low rate. Since this investigation, highly labelled [5-3H] orotie acid has become available, and the present experiments were carried out with this. In Fig. 2, the result of short-time incorporation of orotic acid into ribosomal particles and into rRNA of Chlorella is shown. The labelling pattern is similar to those obtained with uridine (e. g. Galling and Ssymank, 1970, Fig. 1). This correspondence between short-time labelling of ribosomal particles with uridine or with orotie acid was shown directly in the following experiment. Two parallel Chlordla cultures were labelled for 15 rain,one with 2.5 ~Ci [5-SH]orotic acid per ml of culture, the other with 0.5 ~Ci/ml [2-14C]uridine. The ribosomes were prepared from a mixture of the homogenates of both cultures. Fig. 3 shows the result of a sucrose gradient of the common ribosomal preparation. The positions of the peaks of [3H]orotie acid and [14C]uridine strictly coincide. 11 Planta (Berl.)~ Bd. 111
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E x p e r i m e n t s using t h e a n t i b i o t i c s r i f a m p i n , c h l o r a m p h e n i e o l a n d c y c l o h e x i m i d e s u p p o r t e d t h e chloroplastic n a t u r e of t h e particles which were labelled b y pulses of uridine (Galling, 1971). Consequently, these a n t i b i o t i c s were also e m p l o y e d in i n v e s t i g a t i o n s of t h e i n c o r p o r a t i o n of orotie acid. T h i r t y - r a i n p r e i n c u b a t i o n s w i t h these a n t i b i o t i c s in t h e indic a t e d c o n c e n t r a t i o n s (el. S s y m a n k , 1972) affected 15-rain i n c o r p o r a t i o n of orotie acid as shown in T a b l e 1. T h e a n t i b i o t i c s p r i n c i p a l l y a c t as t h e y d i d in t h e case of uridine i n c o r p o r a t i o n (see S s y m a n k , 1972, T a b l e 2). I n t h e e x p e r i m e n t s d e s c r i b e d above, t h e r e was a l w a y s a m u c h lower i n c o r p o r a t i o n of orotie a c i d t h a n of uridine. T h e specific a c t i v i t y values of nucleic-acid p r e p a r a t i o n s after i n c o r p o r a t i o n of different r a d i o a c t i v e precursors a r e listed in T a b l e 2. T h e precursors used were all-
Fig. 1 a--d. Nucleic acids from ribosomal particles labelled by uridine pulses. From a ChloreUa culture (15th h of light-dark change) incubated for 15 rain with 0.8 ~Ci/ ml[5-SH]uridine the ribosomes were prepared and centrifuged for 15 h at 17 000 rev./ rain in the SW23 rotor of the Christ Omega ultracentrifuge through a sucrose gradient 10 to 30% in 0.05 M Tris buffer pR 7.6; the sedimentation is from right to left (a). Fractions were pooled as indicated, unlabelled ribosomes added and the nucleic acids prepared and eleetrophoresed on 2.4 % polyaerylamide gels; b fractions 35-38, c fractions 43-46, d fractions 49-52. absorbance at 254 nm (a) or 265 nm (b--d) ; o---o, o--. radioactivity 11 9
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Fig. 3. Comparison of particles pulse-labelled with uridinc or with orotic acid. Fifty ml ChlorelIa culture (16th h of light-dark change) were labelled for 15 rain with 0.025 mCi[2-1~C]uridine and other 50 ml for 15 rain with 0.125 mCi[5-SH]orotic acid. Both cultures were worked up jointly and their common P105 was centrifuged through a sucrose gradient as described in Fig. 2 a. Sedimentation is from right to left. - - absorbance at 260 nm; o---o all-radioactivity (orotie acid); ~---~14C-radioactivity (uridine) Table 1. Effect of antibiotics on the incorporation of orotic acid into ribosomes of Chlorella 100-ml cultures of Chlorella (16th h of light-dark change) were preincubated with the amounts of antibiotics mentioned below and then incubated with 0.1 mCi [5-ah]orotic acid for 15 min in the presence of the antibiotic. The specific activities of ribosomal preparations (P105) were determined. Antibiotic added
Specific activity of P105 (per cent of control)
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labelled uridine, orotic acid a n d guanosine. T h e r e are i m m e n s e q u a n t i t a t i v e differences b e t w e e n t h e i n c o r p o r a t i o n s of these t h r e e precursors a n d these are influenced b y n i t r o g e n deficiency t o different extents. T h e y also d e p e n d on t h e stage in t h e cell cycle. I n order t o characterize t h e s e differences a n d to e x a m i n e w h e t h e r q u a n t i t a t i v e differences are a c c o m p a n i e d b y q u a l i t a t i v e ones, parallel Ghlorella cultures were labelled for 15 rain w i t h uridine, orotic a c i d a n d
Labelling of Chloroplas$ I~NA in Chlorella
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Table 2. Specific activities of nucleic acids from Chlorella after 15-rain labelling with different RNA precursors Chlo~'ella cultures were incubated for 15 min with the labelled substances mentioned below (1 ~Ci/ml). The specific activities of total nucleic acids were calculated as pmoles incorporated per mg nucleic acids and then related to the specific activity after uridine incorporation ( ~ 1.0). Precursor offered
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guanosine, respectively. Their nucleic acids were then separated electrophoretically on polyacrylamide gels. Results for cultures from the onset of the dark period are presented in Fig. 4, those for 17-h old N-deficient cultures in Fig. 5. As already reported (Ssymank and Galling, 1972), uridine is incorporated into peaks which migrate like ch]orop]ast 23S and 16S r R N A and a third one which migrates slightly faster t h a n cytoplasmic 18S rRNA. I n N-deficient cultures, there is an indication of some incorporation into 26S rRNA, which in normal cells can prominently be seen with guanosine as precursor. I n N-deficient cultures, 26S r R N A is labelled b y orotic acid or guanosine to a comparable extent, as is 23S rRNA. The high specific activity of a preparation of total nucleic acids from guanosine-labelled Chlorella cells from the end of the light phase (see Table 2) can be explained b y a high incorporation of guanosine into D~TA which is synthesized at t h a t time. Polyacrylamide gels of t h a t period exhibit a very high [sH]guanosine peak a~ ~he DNA position, but are entirely equal to t h a t shown in Fig. 4 over the rest of the gel.
Labelling of Chloroplast RNA in Chlorella
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Discussion The ribosomal particles into which labelled uridine is incorporated during short pulses in Chlorella could be characterized as chloroplastie by several criteria (Galling and Ssymank, 1970; Galling, 1971; and this paper, Fig. 1). A further peak of radioactivity besides the RNA of the chloroplast ribosomes, which migrated slightly faster than cytoplasmic 18S rRNA on polyacrylamide gels (e.g. Fig. 4a), may be a high molecular precursor of 16S rRNA. The main purpose of this paper is to discuss various possibihties which might explain this selective incorporation of uridine during pulse experiments in Chlorella. In comparative studies, [3H]uridine, [3H]orotic acid and [SH]guanosine were incorporated to appreciably different extents (el. Table 2) which might be explained by assuming different capacities of entry into the cell for different precursors. The alterations in the incorporation capacities observed after nitrogen deficiency might be due to alterations in the permeability of the cell surface. But this could only explain quantitative differences and not qualitative ones which were observed (compare Fig. 5 with Fig. 4). Therefore, different extents of incorporation for different precursors may be partially due to uptake rates, but also partially to other mechanisms. The question that arises is, is the selective incorporation of uridine into chloroplast rl~NA due to the fact that it has to be phosphorylated by an enzyme not employed in normal RNA biosynthesis before incorporation (Galling and Ssymalnk, 1970). On the other hand, orotie acid, which is like uridine incorporated into I~NA as UTP, occurs in the biosynthetic pathway leading to t~NA. Thus a comparison of orotic acid and uridine might provide an answer to the above question. The results presented here show that orotie acid is incorporated preferentially into chloroplast rRNA. This means that there are at least other mechanisms involved in the selctivity of incorporation other than the compartmenration of enzymes. The assumption of different precursor pool sizes in chloroplasts and nuclei is fully consistent with the results obtained in this and previous papers. It is assumed that Chlorella contains rather large nuclcotide pools in the nucleus, but not in the chloroplast. This would result in a strong dilution of the labelled precursor in the nucleus, but less dilution in the chloroplast. In this way, the preferential incorporation into chloroplast rRNA in pulse labelling experiments may be explained. It is to be expected that different precursors have different pool sizes and that the ratio of the pools in ~he chloroplast to the pools in the nucleus will vary for different precursors. This could explain the higher proportion of labelled cytoplasmic rRNA which could be observed with guanosine (:Fig. 4c). Nitrogen deficiency effectively reduces nucleotide pool sizes (Hewmark and Fujimoto, 1959) and it is likely that larger pools are affected
166
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to a greater extent t h a n smaller ones. This would result in some labelling of cytoplasmic r R N A under conditions of nitrogen deficiency as seen in Fig. 5. However, this effect could also be explained b y a stringent control mechanism; this kind of regulation of r R N A synthesis b y the sizes of amino acid pools, which are reduced b y nitrogen deficiency, was demonstrated for chloroplasts of Chlamydomonas reinhardii b y Surzycki and Hastings (1968). Presumably, these two effects work together and are thus indistinguishable. The m o s t likely explanation for a selective incorporation of externally supplied nucleic acid precursors into chloroplast r R N A of Chlorella is to assume an influence of the pool sizes at the sites of tCNA synthesis. This assumption has to be considered whenever different precursors lead to different results or whenever nucleic acids of one c o m p a r t m e n t are preferentially labelled. I wish to thank Dr. G.Galling for many useful discussions and his permanent inf~rest in my work. For technical assistance I want to thank Miss E. tteuer. This work was supported by the Deutsche Forschungsgemeinschaft.
References Bucher, N. L. R., Swaffield, M. N. : Ribonucleic acid synthesis in relation to precursor pools in regenerating rat liver. Biochim. biophys. Acta (Amst.) 174, 491-502 (1969). Galling, G. : Der EinfluB yon Rifampicin, Chloramphenicol und Cycloheximid auf den Uridin-Einbau in chlorplastid~re Ribosomenvorstufen yon Chlorella. Planta (Berl.) 98, 50--62 (1971). Galling, G. : Einbau yon Uridin und Orots~ure in plastid~re ribosomale RNA yon Chlorelta nach Antibiotica-Behandlung. Arch. Mikrobiol. 81, 245-259 (1972). Galling, G., Ssymank, V. : Bevorzugter Einbau markierten Uridins in die Vorli~ufer yon Chloroplasten-Ribosomen in Algenzellen. Planta (Berl.) 94, 203-212 (1970). Iwamura, T., Kanazawa, T., Kanazawa, K. : Nucleotide metabolism in Chlorella. In: Studies on microalgae and photosynthetic bacteria, p. 577-596. ,Tap. Soc. Plant Physiologists, ed. Tokyo: The University of Tokyo Press 1963. Loening, U. E. : The fractionation of high molecular weight ribonucleic acid by polyacrylamide gel electrophoresis. Biochem. J. 102, 251-257 (1967). ~ewmark, P., Fujimoto, Y. : Nucleic acids of nitrogen deficient Chlorella pyrenoldo8a. Fed. Proc. 18, 293 (1959). Parish, J. tL, Kirby, K. S.: Reagents which reduce interactions between ribosomal RNA and rapidly labelled RNA from rat liver. Biochim. biophys. Acta (Amst.) 129, 554-562 (1966). Ssymank, V.: Influence of nitrogen deficiency on uridine incorporation into ribosomes in the green alga Chlorella. Arch. Mikrobiol. 82, 311-324 (1972). Ssymank, V., Galling, G.: Labelling of chloroplast ribosomes by uridine in Chlorella. Abstr. Commun. Meet. Fed. Eur. Biochem. Soc. 8, No 496 (1972). Stambrook, P. J., Sisken, J. E.: The relationship between rates of 3tt-uridine and 3H-adenine incorporation into RNA and the measured rates of RNA synthesis during the cell cycle. Biochim. biophys. Acta (Amst.) 281, 45-54 (1972). Surzycki, S., Hastings, P. J.: Control of chloroplast RNA synthesis in Chlamydomonas reinhardii. Nature (Lond.) 220, 786-787 (1968).
Incorporation of Different Labelled Precursors into Chloroplast RNA of Chlordla Volker S s y m a n k Pflanzenphysiologisches Institut der Universitiit, D-3400 G6ttingen, Federal Republic of Germany Received January 17, 1973
Summary. Radioactive uridine is incorporated by Chlorella strain 211-8b/p into ribosomal subunits and their rapidly labelled RNA comigrates with chloroplast RNA on polycrylamide gels. Ribosomal particles which can be labelled by short pulses of orotic acid cosediment with the particles labelled by uridine pulses and contain the same RNA species as these when separated either on sucrose gradients or on polycrylamide gels. This incorporation is, like that of uridine, sensitive to rifampin and chloramphenicol, but insensitive to cycloheximide. A comparative study of short-time incorporation of uridine, orotic acid and guanosine into the RNA of Chlorella showed that all three precursors were incorporated mainly into RNA of chloroplastic origin. However, guanosine was also partly incorporated into cytoplasmic rRNA. Nitrogen-deficient cells always incorporated part of all three precursors into cytoplasmic rRNA, but the proportions of these were different anaong the different precursors. These results are consistent with the hypothesis that the described differences in the incorporation of the above mentioned precursors into I~NA of different cellular compartments are largely attributable to effects of pool sizes. Introduction I n cultures of the green alga Chlorella, evidence has been presented for a preferential incorporation of radioactive uridine into chloroplast ribosomM particles during short pulses (Galling and Ssymank, 1970). This selective incorporation could be affected in p a r t b y changing the physiological conditions for the algal cultures, for example b y growing t h e m in a nitrogen-deficient m e d i u m (Ssymank, 1972). On the other hand, short-time labelling of algal nucleic acids with labelled phosphate does not result in a preference for chloroplast rt~NA (Galling and Ssymank, 1970). One possible explanation of such a preferential labelling could be the fact t h a t uridine itself does not occur a m o n g the natural precursors of I~NA, b u t is a usual degradation intermediate. Thus the local separation of enzymes used for t~NA synthesis and I~NA degradation between cytoplasm and nucleus could result in a delay in the labelling of cytoplasmic rI~NA b y uridine. Another i m p o r t a n t factor is the relative sizes of uridine pools in the cytoplasm, the nucleus and the chloroplast, which will considerably affect the rate of incorporation into I~NA. Influences of precursor pools on the rate
158
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of i n c o r p o r a t i o n h a v e been r e p o r t e d for r a t liver (Bucher a n d Swaffield, 1969). There is evidence t h a t Chlorella cells c o n t a i n r e l a t i v e l y large nucleotide pools ( I w a m u r a st al., 1963). I n t h e case of t h e first e x p l a n a t i o n described above, n i t r o g e n deficiency m i g h t a c t b y reducing t h e a m i n o acid pools a n d t h u s switch on t h e s t r i n g e n t control m e c h a n i s m of R N A synthesis in t h e chloroplast described b y S u r z y c k i a n d H a s t i n g s (1968). This has a l r e a d y been discussed ( S s y m a n k , 1972). I n t h e case of t h e second e x p l a n a t i o n , this effect of n i t r o g e n deficiency would be covered b y t h e direct influence on nucleotide pool sizes. I n t h e p r e s e n t p a p e r , t h e i n c o r p o r a t i o n of orotic acid, a n a t u r a l precursor to I~NA, is c o m p a r e d with t h a t of uridine. This m i g h t p r o v i d e evidence concerning t h e v a l i d i t y of t h e first h y p o t h e s i s listed above. To t e s t t h e second hypothesis, a t h i r d R N A precursor, guanosine, was used. This nucleoside like uridine does n o t occur in t h e n a t u r a l p a t h w a y of R N A biosynthesis. H o w e v e r , b o t h t h e p a t h w a y s of f o r m a t i o n of purine or p y r i m i d i n e nucleotides a n d t h e i r i n c o r p o r a t i o n into I~NA arc quite different, so t h a t different influences on guanosine or uridine i n c o r p o r a t i o n m i g h t be expected. I n higher e u k a r y o t e s , different r a t e s of uridine a n d adenine i n c o r p o r a t i o n into R N A were o b s e r v e d ( S t a m b r o o k a n d Sisken, 1972). Materials and ~Iethods Culture and Labelling o/ Algae. The green alga Chlorella pyrenoidosa (strain 211-8b/p from the algal collection of the Institute of Plant Physiology, GSttingen) was used throughout. Culture conditions, incubations with antibiotics and labelling procedures employed were as described previously (Ssymank, 1972). The following radioactive substances were used: [5-3H]uridine (30Ci/mmole), [2-14C]uridine (60 mCi/mmole), [5-3H]orotic acid (25 Ci/mmole), and [8-3H]guanosine (t0 Ci/mmole); all were obtained from the Radiochemical Centre, Amersham, U.K. Preparation o/ Cellular Fractions and o] Nucleic Acids. Cellular fractions were prepared as described previously (Ssymank, 1972) ; 0.05 M Tris buffer pH 7.6 containing 10 mM MgC12was used. For isolation of intact nucleic acids cell homogenates in this buffer or in 0.03 M acetate buffer pH 5.0 containing 10 mM Mg2+ and 20 rag/1 polyvinyl sulfate or ribosome suspensions were extracted with an equal volume of a phenol-cresol mixture (140 ml m-cresol and 0.5 g 8-hydroxy quinoline per 1 water saturated phenol). The phenol phase was removed and the aqueous phase reextracted. The nucleic acids were precipitated out of the aqueous phase by three volumes of ethanol in the cold (modification of the method of Parish and Kirby, 1966). Sucrose JDensity Gradients. Lineor sucrose gradients of ribosome preparations (P105) were carried out and evaluted as described elsewhere (Ssymank, 1972). In the experiment described in Fig. 1, a gradient of 10 to 30% sucrose in 0.05 M Tris buffer pH 7.6 with a volume of 60 ml was prepared; this was sampled automatically using an ISCO DG Fractionator. Polyacrylamide Gel Electrophoresis. :Nucleic acids were further fractionated by electrophoresis on 2.4% polyacrylamide gels according to the method of Loening (1967) using the following buffer: 36 mM Tris, 30 mM phosphate, 1 mM EDTA, 0.2% SDS, pSI 7.8. Electrophoresis was conducted at 0 ~ C for 30 rain at 2 mA/gel and about 150 rain at 5 mA/gel. The u.v. absorption of the gels was monitored in a Joyce-Loebl Chromoscan.
Labelling of Chloroplast RNA in Chlorella
159
Determination el t~adioactivity. Polyaerylamide gels were frozen with solid COg and sliced by means of a Joyce-Loebl Gel Slicer into 1 mm fractions; these were solubilized in 0.1 ml It202 (30%) for 15 h at 60 ~ C and counted after addition of 2 ml Aquasol (NEN Chemicals) in a Nuclear Chicago Mark I liquid scintillation counter. Other radioactivity determinations were performed as described previously (Ssymank 1972). Results
In a previous paper (Galling and Ssymank, 1970), we showed that short-time incorporation of [aH]uridine leads to labelled particles with sedimentation constants of 50S and 30S; their nucleic acids sedimented at 23S and between 8S and 16S, respectively. Since sucrose gradients only poorly resolve cytoplasmic and chloroplast rRNA, we conducted the experiment separating the labelled RNA on polyacrylamide gels. This is shown in Fig. 1. Due to the lack of Mg~+ in the gradient buffer, the bulk of the ribosomes had dissociated and so the main u.v. peaks represented sedimentation values of 60S and 40S ; the radioactivity peaks occured at 50S, 30S and in a lighter region (Fig. 1 a). The fractions of each of these three peaks were pooled and subjected to a procedure to prepare the nucleic acids. These were electrophoresed on polyacrylamide gels. Figs. l b - d show that the RNA of the 50S peak migrates like chloroplast 23S rRNA and the RNA of the 50S peak migrates like chloroplast 23S rRNA and the RNA of the 30S peak like chloroplast 16S rRNA; this peak also contains some faster migrating material which may be a contamination as the third labelled peak of the particle gradient also contains similar faster migrating RNA. In previous investigations with orotic acid (Galling, 1972), it was not possible to obtain unequivocal evidence that orotic acid is incorporatiod in the same way as uridine, although this seemed verylikely. Two reasons could be given in an attempt to explain this uncertainty; the 14C-labelled orotic acid then available had an extremely low specific activity and was incorporated at a very low rate. Since this investigation, highly labelled [5-3H] orotie acid has become available, and the present experiments were carried out with this. In Fig. 2, the result of short-time incorporation of orotic acid into ribosomal particles and into rRNA of Chlorella is shown. The labelling pattern is similar to those obtained with uridine (e. g. Galling and Ssymank, 1970, Fig. 1). This correspondence between short-time labelling of ribosomal particles with uridine or with orotie acid was shown directly in the following experiment. Two parallel Chlordla cultures were labelled for 15 rain,one with 2.5 ~Ci [5-SH]orotic acid per ml of culture, the other with 0.5 ~Ci/ml [2-14C]uridine. The ribosomes were prepared from a mixture of the homogenates of both cultures. Fig. 3 shows the result of a sucrose gradient of the common ribosomal preparation. The positions of the peaks of [3H]orotie acid and [14C]uridine strictly coincide. 11 Planta (Berl.)~ Bd. 111
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E x p e r i m e n t s using t h e a n t i b i o t i c s r i f a m p i n , c h l o r a m p h e n i e o l a n d c y c l o h e x i m i d e s u p p o r t e d t h e chloroplastic n a t u r e of t h e particles which were labelled b y pulses of uridine (Galling, 1971). Consequently, these a n t i b i o t i c s were also e m p l o y e d in i n v e s t i g a t i o n s of t h e i n c o r p o r a t i o n of orotie acid. T h i r t y - r a i n p r e i n c u b a t i o n s w i t h these a n t i b i o t i c s in t h e indic a t e d c o n c e n t r a t i o n s (el. S s y m a n k , 1972) affected 15-rain i n c o r p o r a t i o n of orotie acid as shown in T a b l e 1. T h e a n t i b i o t i c s p r i n c i p a l l y a c t as t h e y d i d in t h e case of uridine i n c o r p o r a t i o n (see S s y m a n k , 1972, T a b l e 2). I n t h e e x p e r i m e n t s d e s c r i b e d above, t h e r e was a l w a y s a m u c h lower i n c o r p o r a t i o n of orotie a c i d t h a n of uridine. T h e specific a c t i v i t y values of nucleic-acid p r e p a r a t i o n s after i n c o r p o r a t i o n of different r a d i o a c t i v e precursors a r e listed in T a b l e 2. T h e precursors used were all-
Fig. 1 a--d. Nucleic acids from ribosomal particles labelled by uridine pulses. From a ChloreUa culture (15th h of light-dark change) incubated for 15 rain with 0.8 ~Ci/ ml[5-SH]uridine the ribosomes were prepared and centrifuged for 15 h at 17 000 rev./ rain in the SW23 rotor of the Christ Omega ultracentrifuge through a sucrose gradient 10 to 30% in 0.05 M Tris buffer pR 7.6; the sedimentation is from right to left (a). Fractions were pooled as indicated, unlabelled ribosomes added and the nucleic acids prepared and eleetrophoresed on 2.4 % polyaerylamide gels; b fractions 35-38, c fractions 43-46, d fractions 49-52. absorbance at 254 nm (a) or 265 nm (b--d) ; o---o, o--. radioactivity 11 9
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Fig. 3. Comparison of particles pulse-labelled with uridinc or with orotic acid. Fifty ml ChlorelIa culture (16th h of light-dark change) were labelled for 15 rain with 0.025 mCi[2-1~C]uridine and other 50 ml for 15 rain with 0.125 mCi[5-SH]orotic acid. Both cultures were worked up jointly and their common P105 was centrifuged through a sucrose gradient as described in Fig. 2 a. Sedimentation is from right to left. - - absorbance at 260 nm; o---o all-radioactivity (orotie acid); ~---~14C-radioactivity (uridine) Table 1. Effect of antibiotics on the incorporation of orotic acid into ribosomes of Chlorella 100-ml cultures of Chlorella (16th h of light-dark change) were preincubated with the amounts of antibiotics mentioned below and then incubated with 0.1 mCi [5-ah]orotic acid for 15 min in the presence of the antibiotic. The specific activities of ribosomal preparations (P105) were determined. Antibiotic added
Specific activity of P105 (per cent of control)
None 50 ~zg/ml rifampin 500 f~g/ml chloramphenicol 20 ~zg/ml cycloheximide
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labelled uridine, orotic acid a n d guanosine. T h e r e are i m m e n s e q u a n t i t a t i v e differences b e t w e e n t h e i n c o r p o r a t i o n s of these t h r e e precursors a n d these are influenced b y n i t r o g e n deficiency t o different extents. T h e y also d e p e n d on t h e stage in t h e cell cycle. I n order t o characterize t h e s e differences a n d to e x a m i n e w h e t h e r q u a n t i t a t i v e differences are a c c o m p a n i e d b y q u a l i t a t i v e ones, parallel Ghlorella cultures were labelled for 15 rain w i t h uridine, orotic a c i d a n d
Labelling of Chloroplas$ I~NA in Chlorella
163
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Table 2. Specific activities of nucleic acids from Chlorella after 15-rain labelling with different RNA precursors Chlo~'ella cultures were incubated for 15 min with the labelled substances mentioned below (1 ~Ci/ml). The specific activities of total nucleic acids were calculated as pmoles incorporated per mg nucleic acids and then related to the specific activity after uridine incorporation ( ~ 1.0). Precursor offered
[5-aHJuridine [5-aH]orotie acid [8-~H]guanosine
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guanosine, respectively. Their nucleic acids were then separated electrophoretically on polyacrylamide gels. Results for cultures from the onset of the dark period are presented in Fig. 4, those for 17-h old N-deficient cultures in Fig. 5. As already reported (Ssymank and Galling, 1972), uridine is incorporated into peaks which migrate like ch]orop]ast 23S and 16S r R N A and a third one which migrates slightly faster t h a n cytoplasmic 18S rRNA. I n N-deficient cultures, there is an indication of some incorporation into 26S rRNA, which in normal cells can prominently be seen with guanosine as precursor. I n N-deficient cultures, 26S r R N A is labelled b y orotic acid or guanosine to a comparable extent, as is 23S rRNA. The high specific activity of a preparation of total nucleic acids from guanosine-labelled Chlorella cells from the end of the light phase (see Table 2) can be explained b y a high incorporation of guanosine into D~TA which is synthesized at t h a t time. Polyacrylamide gels of t h a t period exhibit a very high [sH]guanosine peak a~ ~he DNA position, but are entirely equal to t h a t shown in Fig. 4 over the rest of the gel.
Labelling of Chloroplast RNA in Chlorella
165
Discussion The ribosomal particles into which labelled uridine is incorporated during short pulses in Chlorella could be characterized as chloroplastie by several criteria (Galling and Ssymank, 1970; Galling, 1971; and this paper, Fig. 1). A further peak of radioactivity besides the RNA of the chloroplast ribosomes, which migrated slightly faster than cytoplasmic 18S rRNA on polyacrylamide gels (e.g. Fig. 4a), may be a high molecular precursor of 16S rRNA. The main purpose of this paper is to discuss various possibihties which might explain this selective incorporation of uridine during pulse experiments in Chlorella. In comparative studies, [3H]uridine, [3H]orotic acid and [SH]guanosine were incorporated to appreciably different extents (el. Table 2) which might be explained by assuming different capacities of entry into the cell for different precursors. The alterations in the incorporation capacities observed after nitrogen deficiency might be due to alterations in the permeability of the cell surface. But this could only explain quantitative differences and not qualitative ones which were observed (compare Fig. 5 with Fig. 4). Therefore, different extents of incorporation for different precursors may be partially due to uptake rates, but also partially to other mechanisms. The question that arises is, is the selective incorporation of uridine into chloroplast rl~NA due to the fact that it has to be phosphorylated by an enzyme not employed in normal RNA biosynthesis before incorporation (Galling and Ssymalnk, 1970). On the other hand, orotie acid, which is like uridine incorporated into I~NA as UTP, occurs in the biosynthetic pathway leading to t~NA. Thus a comparison of orotic acid and uridine might provide an answer to the above question. The results presented here show that orotie acid is incorporated preferentially into chloroplast rRNA. This means that there are at least other mechanisms involved in the selctivity of incorporation other than the compartmenration of enzymes. The assumption of different precursor pool sizes in chloroplasts and nuclei is fully consistent with the results obtained in this and previous papers. It is assumed that Chlorella contains rather large nuclcotide pools in the nucleus, but not in the chloroplast. This would result in a strong dilution of the labelled precursor in the nucleus, but less dilution in the chloroplast. In this way, the preferential incorporation into chloroplast rRNA in pulse labelling experiments may be explained. It is to be expected that different precursors have different pool sizes and that the ratio of the pools in ~he chloroplast to the pools in the nucleus will vary for different precursors. This could explain the higher proportion of labelled cytoplasmic rRNA which could be observed with guanosine (:Fig. 4c). Nitrogen deficiency effectively reduces nucleotide pool sizes (Hewmark and Fujimoto, 1959) and it is likely that larger pools are affected
166
V. Ssymank: Labelling of Chloroplas~ RNA in Chlorella
to a greater extent t h a n smaller ones. This would result in some labelling of cytoplasmic r R N A under conditions of nitrogen deficiency as seen in Fig. 5. However, this effect could also be explained b y a stringent control mechanism; this kind of regulation of r R N A synthesis b y the sizes of amino acid pools, which are reduced b y nitrogen deficiency, was demonstrated for chloroplasts of Chlamydomonas reinhardii b y Surzycki and Hastings (1968). Presumably, these two effects work together and are thus indistinguishable. The m o s t likely explanation for a selective incorporation of externally supplied nucleic acid precursors into chloroplast r R N A of Chlorella is to assume an influence of the pool sizes at the sites of tCNA synthesis. This assumption has to be considered whenever different precursors lead to different results or whenever nucleic acids of one c o m p a r t m e n t are preferentially labelled. I wish to thank Dr. G.Galling for many useful discussions and his permanent inf~rest in my work. For technical assistance I want to thank Miss E. tteuer. This work was supported by the Deutsche Forschungsgemeinschaft.
References Bucher, N. L. R., Swaffield, M. N. : Ribonucleic acid synthesis in relation to precursor pools in regenerating rat liver. Biochim. biophys. Acta (Amst.) 174, 491-502 (1969). Galling, G. : Der EinfluB yon Rifampicin, Chloramphenicol und Cycloheximid auf den Uridin-Einbau in chlorplastid~re Ribosomenvorstufen yon Chlorella. Planta (Berl.) 98, 50--62 (1971). Galling, G. : Einbau yon Uridin und Orots~ure in plastid~re ribosomale RNA yon Chlorelta nach Antibiotica-Behandlung. Arch. Mikrobiol. 81, 245-259 (1972). Galling, G., Ssymank, V. : Bevorzugter Einbau markierten Uridins in die Vorli~ufer yon Chloroplasten-Ribosomen in Algenzellen. Planta (Berl.) 94, 203-212 (1970). Iwamura, T., Kanazawa, T., Kanazawa, K. : Nucleotide metabolism in Chlorella. In: Studies on microalgae and photosynthetic bacteria, p. 577-596. ,Tap. Soc. Plant Physiologists, ed. Tokyo: The University of Tokyo Press 1963. Loening, U. E. : The fractionation of high molecular weight ribonucleic acid by polyacrylamide gel electrophoresis. Biochem. J. 102, 251-257 (1967). ~ewmark, P., Fujimoto, Y. : Nucleic acids of nitrogen deficient Chlorella pyrenoldo8a. Fed. Proc. 18, 293 (1959). Parish, J. tL, Kirby, K. S.: Reagents which reduce interactions between ribosomal RNA and rapidly labelled RNA from rat liver. Biochim. biophys. Acta (Amst.) 129, 554-562 (1966). Ssymank, V.: Influence of nitrogen deficiency on uridine incorporation into ribosomes in the green alga Chlorella. Arch. Mikrobiol. 82, 311-324 (1972). Ssymank, V., Galling, G.: Labelling of chloroplast ribosomes by uridine in Chlorella. Abstr. Commun. Meet. Fed. Eur. Biochem. Soc. 8, No 496 (1972). Stambrook, P. J., Sisken, J. E.: The relationship between rates of 3tt-uridine and 3H-adenine incorporation into RNA and the measured rates of RNA synthesis during the cell cycle. Biochim. biophys. Acta (Amst.) 281, 45-54 (1972). Surzycki, S., Hastings, P. J.: Control of chloroplast RNA synthesis in Chlamydomonas reinhardii. Nature (Lond.) 220, 786-787 (1968).
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