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The effect of light on RNA and nucleotide synthesis in synchronous cultures of chlorella
Plant Science Letters, 1 (1973) 129--135 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
THE EFFECT OF LIGHT ON RNA AND NUCLEOTIDE SYNTHESIS IN SYNCHRONOUS CULTURES OF CHLORELLA
F. WANKA and J.M.A. AELEN
Department of Chemical Cytology, University of Nijmegen, Nijmegen (The Netherlands) (Received November llth, 1972)
SUMMARY
The increase of the soluble nucleotide content ceased immediately when young Chlorella cells were removed from the light. Later in the cell cycle (14 h after the beginning of the light period) the soluble nucleotide content also increased significantly upon transfer to the dark. The nucleotide triphosphate levels remained constant or even increased noticeably when the RNA synthesis ceased in the dark. It is concluded, therefore, that the regulation of RNA synthesis by light does not occur at the level of the nucleoside triphosphates. The appropriate rate of nucleotide biosynthesis is apparently regulated to a great extent by feedback control.
INTRODUCTION
When Chlorella cultures are synchronized by alternating periods of 16 h light and 8 h dark the liberation of the autospores from the mother cells takes place at the end of the dark period. Under photoautotrophic conditions there is a strong R N A increase in young cells during most part of the light period which is completely dependent on the illumination '. On the other hand, there is also a marked light-independent R N A increase during the dark period '- 3. This is obviously supported by the mobilization of starch deposits, in particular of the pyrenoid, which have been formed during the preceding light period 2,4. It has been suggested that the immediate cessation of R N A synthesis when young cellsare transferred to the dark might be due to a lack of formation of R N A precursors '. This problem was further pursued in the present study. Abbreviation: a.l., after the. beginning of the light period; MAK, methylated albumin kieselgur.
129
MATERIAL AND METHODS
(a) Culture conditions The Chlorella strain 211/8b (GSttingen) was grown in 400-ml culture tubes (3 cm diameter) immersed in a 30 ° water bath. The tubes were illuminated from two opposite directions each with 6 Philips fluorescence lamps t y p e TL 40 W/32 and TL 40 W/55, respectively. The light intensity at the tube level was 10 000 lux from each side. The cell growth was synchronized by alternating 16 h light and 8 h dark. The suspensions were aerated with air containing 1% CO2 at a flow rate of 250 ml/min. Continuous s y n c h r o n y was maintained by dilution to about 1/16 at the end of each light period, in order to obtain a starting density of 1 to 1.5'106 cells/ml. Cells were c o u n t e d with a Coulter counter. The nutrient solution was composed as follows (moles/l): KNO3 2.10-2, KH:PO4 10 -3 , MgSO4 10 -3 , H3BO3 10 - s , CaC12 10 - s , FeC13 7.5.10 -o , MnSO4 10 -6 , ZnSO4 1.5"10 -7 , CuSO4 6"10 -s , Co(NO3 )2 4 "10-s , (NH4)6Mo7024 2.5"10 -9 and EDTA 1.5.10 -6 .
(b) Determination o f nucleic acids and proteins Nucleic acids were extracted from appropriately pretreated cell samples by hydrolysis for 6 h at 45 ° in 0.5 N HCIO4 and the absorbance of the supernatant was measured at 260 nm (ref. 5). Proteins were extracted from the cell residue for 10 min at 100 ° with 1 N NaOH. After appropriate dilution of the extract the protein was determined by the m e t h o d of Lowry et al. 6 adapt~ ed for the high alkali content.
(c) Labeling and separation o f nucleic acids For labeling experiments the cells were grown axenically. [2-' 4 C] Uracil (spec. act. 56.3 Ci/mole; New England Nuclear Corp.) was used for the experiments. The labeled cells were collected by centrifugation and homogenized by shaking with glass beads. The nucleic acids were extracted from the homogenate, deproteinized with phenol 7 and separated on MAK columns ~ The radioactivity was determined in a Packard Tri-Carb scintillation counter model 3375, using a dioxane-based scintillation fluid 7
(d) Nucleotide analysis The methods used are described in more detail elsewhere 9. Cell samples were collected by centrifugation for 2 min at 2000 X g and extracted with boiling 70% ethanol. For determination of the total nucleotide c o n t e n t the extracts were purified by adsorption on Dowex-1 × 8 and re-elution with 1 M NaC1 in 0.1 N HC1. The composition of the nucleotide pool was analysed by chromatography on Dowex-1 × 8 columns according to Ingle ,0
130
RESULTS
(a) RNA synthesis Most of the experiments were carried o u t early in the light period when light intensity is n o t significantly reduced by self-absorption. Therefore, concentrations of 2--3"106 cells/ml were mostly used. During the first 10 h, RNA formation accounts for more than 99% of the total nucleic acid increase, the remainder being due to the synthesis of satellite DNA 1,7. Table I shows that between the 4th and 6th hour a.l. the nucleic acid content increased b y a b o u t 50%. The increase ceased completely, however, when the culture was removed from the light after the 4th hour. The soluble nucleotide content showed a very similar behaviour and also the protein content remained constant as soon as the illumination was terminated {Table I). Since it is known that r R N A -but not t R N A -- synthesis requires continuing cytoplasmic protein synthesis 11, the possibility had to be considered that t R N A synthesis might continue in the dark b u t not be detected because of the small contribution which it makes to the total R N A content 3. Therefore, RNA synthesis between the 4th and 6th hour a.1. was measured b y labeling with [ 14 C] uracil. In the light the label was incorporated rapidly into all major RNA components (Fig. 1). If the same experiment was performed in the dark, the label was reduced to a b o u t 7%. The reduction was essentially uniform over the whole elution pattern, indicating that the overall R N A synthesis in young cells is dependent on the light.
(b) Nucleotide synthesis During the light period the soluble nucleotide pool increased in a somewhat irregular manner 9 {Fig. 2, Table I). The stronger increase during the last 4 h of the light period may be caused in part by mobilization of the pyrenoid starch 4. I n y o u n g cells the increase ceased as soon as the cultures were reTABLEI DEPENDENCE OF NUCLEOTIDE, NUCLEIC ACID AND PROTEIN SYNTHESIS ON LIGHT A Chlorella culture (3.105 cells/ml) was divided 4 h a.l. and one half remained in the light while the other was placed in the dark. Samples for the determination of soluble nucleotides (200 ml) and nucleic acids (50 ml) were taken in duplicate at the beginning of the experiment and 2 h later. Sample
Soluble nucleotides (A26o nm/ml)
Nucleic acids (A26o nm/ml)
Total (A260 nm/ml)
Protein (ug/ml)
Initial Final dark Final light
0.018 0.021 0.030
0.255 0.247 0.366
0.273 0.268 0.396
39 39 56
131
16 I I I
ght
14
I
12
I 10
o2
t RNA o~,
DNA
I
~A~~_~ 2._~E c-
o
I
10
20
30
40
50
fraction number
Fig. 1. I n c o r p o r a t i o n of [14C ] uracil into different R N A c o m p o n e n t s in the light and dark. A Chlorella culture (2.9.106 cells/ml) was divided 3 h a.1. into 2 o f 300 ml each. One remained in the light and the o t h e r was placed in the dark after addition of 10 uCi [ 14C]uracil to each o f them. T h e cells were collected 2 h later and the nucleic acids were extracted and separated o n M A K c o l u m n s ?,s. 49.5 and 32.5 absorbance units o f nucleic acids were o b t a i n e d f r o m the light and dark culture respectively. • •, absorbance at 260 nm, o-----% counts/min.
m o v e d from the light. In the dark the level remained constant for several hours and then started to decrease (Fig. 2). When the illumination was terminated later in the light period, however, the nucleotide content continued to increase at a markedly reduced b u t still significant rate. Also, a significant increase at the beginning of the normal dark period has been reported 9. It is n o t possible, therefore, to explain the cessation of the R N A synthesis by a depletion of the nucleotide pool. It might be argued, though, that the R N A synthesis is limited by rapid consumption of one of the nucleoside triphosphates which, like CTP for example, are present in very small amounts 9,12,13. An analysis of the nucleotide composition revealed, however, that u p o n transfer of the cultures
132
A6
4 33 c
o
I
I
I
I
8
10 12 14 time after beginning of the Light (h)
i
16
Fig. 2. Increase of the cellular nucleotide contents in the light and in the dark. A Chlorella culture (1.9.105 cells/ml) was divided 7 h a.l. One part was left in the light and the other was placed in the dark. A t 14 h a.l. the light culture was again divided and half o f it was placed in the dark. 200-ml aliquots for the determination of the soluble nucleotides were withdrawn at the times indicated, o, light; e, dark.
t o t h e d a r k 4 h a.l., t h e t r i p h o s p h a t e l e v e l s r e m a i n e d e i t h e ~ c o n s t a n t o r inc r e a s e d n o t i c e a b l y ( F i g . 3, T a b l e I I ) . T h e i n c r e a s e o f U T P a n d C T P u p o n TABLE II RELATIVE AMOUNTS OF NUCLEOTIDES A F T E R 2 h OF D A R K COMPARED TO CONTINUOUS LIGHT The ratios of corresponding nucleotides in the light and dark (for 2 h) have been determined from the peak areas of the experiment shown in Fig. 3 (Expt. 1). Expt. 2 w~'carried out identically except that the culture density was 2.9.106 cells/ml. CTP was calculated from the absorbances at 260 and 280 nm of the respective fractions of the ATP peak, using the formula: 1.96 A2s0nm(CTP) ffi 1.96--0.17 ' ( A 2 s 0 n m - 0.17"A260nm), where 1.96 and 0.17 are theA2sonm/A26onm ratios o f CTP and ATP, respectively, in 4 N formic acid + 1 M ammonium formate. Nucleotide
Total absorbance per peak
in dark in light
Expt. 1
Expt. 2
ATP GTP CTP UTP AMP + ADP + ATP
1.13 1.36 2.16 1.91 0.84
0.73 0.73 1.54 1.04 0.59
Total extract
0.87
0.73
133
NADP
[[[I.
CTP
GTP UTP I
dark
0
10
20
60
70
80
90 100 tube number
i
120
I 130
J " 140 150
I
160
Fig. 3. N u c l e o t i d e c o m p o s i t i o n s in t h e light a n d in t h e dark. A Chlorella c u l t u r e ( 1 . 7 . 1 0 6 c e l l s / m l ) was divided 4 h a.l. i n t o t w o o f 2 1 each. O n e r e m a i n e d in t h e light a n d o n e was p l a c e d in t h e dark. T h e cells were c o l l e c t e d a n d e x t r a c t ed 2 h later a n d t h e n u c l e o t i d e s were a n a l y s e d b y c h r o m a t o g r a p h y o n D o w e x c o l u m n s . T h e f o r m a t e g r a d i e n t was s t a r t e d at f r a c t i o n 95. T h e f r a c t i o n size was 5.5 ml. C o n t i n u o u s lines i n d i c a t e t h e a b s o r b a n c e at 260 n m a n d d a s h e d lines at 280 nm.
2 h exposure to dark was even stronger than in the light controls. The lower nucleotide pool size in the dark {Fig. 2) was mainly due to a loss of AMP and ADP. DISCUSSION
The main conclusion to be drawn from these results is that the cessation of RNA synthesis in young Chlorella cells is not caused by a depletion of the nucleotide pool. It is likely that beyond the regular 4 ribonucleoside triphosphates one or more additional compounds are required for a measurable RNA increase. In young cells the light, possibly via the photosynthetic metabolism ,4, plays a d o m i n a n t role in the formation of such compounds, while later in the cell cycle intermediates of the starch degradation 4 seem to contribute significantly. It can not be excluded at present, however, that qualitatively different regulating principles may control the RNA synthesis in young and old cells respectively. The immediate cessation or strong reduction of the increase of the nucleotide pool upon termination of the light could simply be explained by assuming an insufficient precursor supply. The stability of the nucleotide levels is surprising, however, if one considers the fact that the amounts of guanosine
134
a n d c y t i d i n e n u c l e o t i d e s p r e s e n t 9,, 5,~ 3 m i g h t n o t b e s u f f i c i e n t t o s u p p o r t t h e R N A s y n t h e s i s in t h e early light p e r i o d f o r even 5 min. This s u p p o r t s o u r p r e v i o u s suggestion t h a t t h e p o o l size a n d c o m p o s i t i o n in Chlorella is controlled b y a d d i t i o n a l r e g u l a t o r y processes 9. T h e slight increase o f t h e nucleoside t r i p h o s p h a t e c o n t e n t s in t h e d a r k indicates t h a t f e e d b a c k c o n t r o l b y these c o m p o u n d s ' 5,, 6 m a y be a m a j o r e l e m e n t in t h e r e g u l a t i o n o f t h e size a n d c o m p o s i t i o n o f t h e cellular n u c l e o t i d e p o o l . REFERENCES
1 F. Wanka, Bet. Dtsch. Bot. Gesellsch., 75 (1962) 457. 2 H.G. Ruppel, Flora, 152 (1962) 113. 3 F. En6ckl, Z. Pflanzenphysiol., 58 (1968) 240. 4 F. Wanka, M.M.J. Joppen and Ch.M.A. Kuyper, Z. Pflanzenphysiol., 62 (1970) 146. 5 F. Wanka, J. Moors and F.N.C.M. Krijzer, Biochim. Biophys. Acta, 269 (1972) 153. 6 0 . H . Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 7 F. Wanka, H.F.P. Joosten and W.J. de Grip, Arch. Mikrobiol., 75 (1970) 25. 8 J.D. Mandell and A.D. Hershey, Analyt. Biochem., 1 (1960) 66. 9 F. Wanka, P.H. Vereijken, W. Weijers and J.M.A. Aelen, Proc. Koninkl. Akad. v. Wetensch. Ser. C, 75 (1972) 441. 10 J. Ingle, Biochim. Biophys. Acta, 61 (1962) 147. 11 F. Wanka and P.J.A. Schrauwen, Biochim. Biophys. Aeta, 254 (1971) 237. 12 T. Iwamura, T. Kanazawa and K. Kanazawa, Plant and Cell Physiol. (Special issue on Microalgae and Photosynthetic Bacteria), ( 1963 ) 577. 13 T. Iwamura, T. Kanazawa and K. Kanazawa, Plant Cell Physiol. (Special issue on Microalgae and Photosynthetic Bacteria), (1963) 587. 14 H. Senger and N.I. Bishop, Plant CellPhysiol., 7 (1966) 441. 15 S.C. Hartman, Purines and pyrimidines, in D.M. Greenberg (Ed.), Metabolic Pathways, Vol. 4, Academic Press, New York, 1970, p. 1. 16 S, Kit, Nucleotides and nucleic acids, in D.M. Greenberg (Ed.), Metabolic Pathways, Vol. 4, Academic Press, New York, 1970, p. 69.
135
THE EFFECT OF LIGHT ON RNA AND NUCLEOTIDE SYNTHESIS IN SYNCHRONOUS CULTURES OF CHLORELLA
F. WANKA and J.M.A. AELEN
Department of Chemical Cytology, University of Nijmegen, Nijmegen (The Netherlands) (Received November llth, 1972)
SUMMARY
The increase of the soluble nucleotide content ceased immediately when young Chlorella cells were removed from the light. Later in the cell cycle (14 h after the beginning of the light period) the soluble nucleotide content also increased significantly upon transfer to the dark. The nucleotide triphosphate levels remained constant or even increased noticeably when the RNA synthesis ceased in the dark. It is concluded, therefore, that the regulation of RNA synthesis by light does not occur at the level of the nucleoside triphosphates. The appropriate rate of nucleotide biosynthesis is apparently regulated to a great extent by feedback control.
INTRODUCTION
When Chlorella cultures are synchronized by alternating periods of 16 h light and 8 h dark the liberation of the autospores from the mother cells takes place at the end of the dark period. Under photoautotrophic conditions there is a strong R N A increase in young cells during most part of the light period which is completely dependent on the illumination '. On the other hand, there is also a marked light-independent R N A increase during the dark period '- 3. This is obviously supported by the mobilization of starch deposits, in particular of the pyrenoid, which have been formed during the preceding light period 2,4. It has been suggested that the immediate cessation of R N A synthesis when young cellsare transferred to the dark might be due to a lack of formation of R N A precursors '. This problem was further pursued in the present study. Abbreviation: a.l., after the. beginning of the light period; MAK, methylated albumin kieselgur.
129
MATERIAL AND METHODS
(a) Culture conditions The Chlorella strain 211/8b (GSttingen) was grown in 400-ml culture tubes (3 cm diameter) immersed in a 30 ° water bath. The tubes were illuminated from two opposite directions each with 6 Philips fluorescence lamps t y p e TL 40 W/32 and TL 40 W/55, respectively. The light intensity at the tube level was 10 000 lux from each side. The cell growth was synchronized by alternating 16 h light and 8 h dark. The suspensions were aerated with air containing 1% CO2 at a flow rate of 250 ml/min. Continuous s y n c h r o n y was maintained by dilution to about 1/16 at the end of each light period, in order to obtain a starting density of 1 to 1.5'106 cells/ml. Cells were c o u n t e d with a Coulter counter. The nutrient solution was composed as follows (moles/l): KNO3 2.10-2, KH:PO4 10 -3 , MgSO4 10 -3 , H3BO3 10 - s , CaC12 10 - s , FeC13 7.5.10 -o , MnSO4 10 -6 , ZnSO4 1.5"10 -7 , CuSO4 6"10 -s , Co(NO3 )2 4 "10-s , (NH4)6Mo7024 2.5"10 -9 and EDTA 1.5.10 -6 .
(b) Determination o f nucleic acids and proteins Nucleic acids were extracted from appropriately pretreated cell samples by hydrolysis for 6 h at 45 ° in 0.5 N HCIO4 and the absorbance of the supernatant was measured at 260 nm (ref. 5). Proteins were extracted from the cell residue for 10 min at 100 ° with 1 N NaOH. After appropriate dilution of the extract the protein was determined by the m e t h o d of Lowry et al. 6 adapt~ ed for the high alkali content.
(c) Labeling and separation o f nucleic acids For labeling experiments the cells were grown axenically. [2-' 4 C] Uracil (spec. act. 56.3 Ci/mole; New England Nuclear Corp.) was used for the experiments. The labeled cells were collected by centrifugation and homogenized by shaking with glass beads. The nucleic acids were extracted from the homogenate, deproteinized with phenol 7 and separated on MAK columns ~ The radioactivity was determined in a Packard Tri-Carb scintillation counter model 3375, using a dioxane-based scintillation fluid 7
(d) Nucleotide analysis The methods used are described in more detail elsewhere 9. Cell samples were collected by centrifugation for 2 min at 2000 X g and extracted with boiling 70% ethanol. For determination of the total nucleotide c o n t e n t the extracts were purified by adsorption on Dowex-1 × 8 and re-elution with 1 M NaC1 in 0.1 N HC1. The composition of the nucleotide pool was analysed by chromatography on Dowex-1 × 8 columns according to Ingle ,0
130
RESULTS
(a) RNA synthesis Most of the experiments were carried o u t early in the light period when light intensity is n o t significantly reduced by self-absorption. Therefore, concentrations of 2--3"106 cells/ml were mostly used. During the first 10 h, RNA formation accounts for more than 99% of the total nucleic acid increase, the remainder being due to the synthesis of satellite DNA 1,7. Table I shows that between the 4th and 6th hour a.l. the nucleic acid content increased b y a b o u t 50%. The increase ceased completely, however, when the culture was removed from the light after the 4th hour. The soluble nucleotide content showed a very similar behaviour and also the protein content remained constant as soon as the illumination was terminated {Table I). Since it is known that r R N A -but not t R N A -- synthesis requires continuing cytoplasmic protein synthesis 11, the possibility had to be considered that t R N A synthesis might continue in the dark b u t not be detected because of the small contribution which it makes to the total R N A content 3. Therefore, RNA synthesis between the 4th and 6th hour a.1. was measured b y labeling with [ 14 C] uracil. In the light the label was incorporated rapidly into all major RNA components (Fig. 1). If the same experiment was performed in the dark, the label was reduced to a b o u t 7%. The reduction was essentially uniform over the whole elution pattern, indicating that the overall R N A synthesis in young cells is dependent on the light.
(b) Nucleotide synthesis During the light period the soluble nucleotide pool increased in a somewhat irregular manner 9 {Fig. 2, Table I). The stronger increase during the last 4 h of the light period may be caused in part by mobilization of the pyrenoid starch 4. I n y o u n g cells the increase ceased as soon as the cultures were reTABLEI DEPENDENCE OF NUCLEOTIDE, NUCLEIC ACID AND PROTEIN SYNTHESIS ON LIGHT A Chlorella culture (3.105 cells/ml) was divided 4 h a.l. and one half remained in the light while the other was placed in the dark. Samples for the determination of soluble nucleotides (200 ml) and nucleic acids (50 ml) were taken in duplicate at the beginning of the experiment and 2 h later. Sample
Soluble nucleotides (A26o nm/ml)
Nucleic acids (A26o nm/ml)
Total (A260 nm/ml)
Protein (ug/ml)
Initial Final dark Final light
0.018 0.021 0.030
0.255 0.247 0.366
0.273 0.268 0.396
39 39 56
131
16 I I I
ght
14
I
12
I 10
o2
t RNA o~,
DNA
I
~A~~_~ 2._~E c-
o
I
10
20
30
40
50
fraction number
Fig. 1. I n c o r p o r a t i o n of [14C ] uracil into different R N A c o m p o n e n t s in the light and dark. A Chlorella culture (2.9.106 cells/ml) was divided 3 h a.1. into 2 o f 300 ml each. One remained in the light and the o t h e r was placed in the dark after addition of 10 uCi [ 14C]uracil to each o f them. T h e cells were collected 2 h later and the nucleic acids were extracted and separated o n M A K c o l u m n s ?,s. 49.5 and 32.5 absorbance units o f nucleic acids were o b t a i n e d f r o m the light and dark culture respectively. • •, absorbance at 260 nm, o-----% counts/min.
m o v e d from the light. In the dark the level remained constant for several hours and then started to decrease (Fig. 2). When the illumination was terminated later in the light period, however, the nucleotide content continued to increase at a markedly reduced b u t still significant rate. Also, a significant increase at the beginning of the normal dark period has been reported 9. It is n o t possible, therefore, to explain the cessation of the R N A synthesis by a depletion of the nucleotide pool. It might be argued, though, that the R N A synthesis is limited by rapid consumption of one of the nucleoside triphosphates which, like CTP for example, are present in very small amounts 9,12,13. An analysis of the nucleotide composition revealed, however, that u p o n transfer of the cultures
132
A6
4 33 c
o
I
I
I
I
8
10 12 14 time after beginning of the Light (h)
i
16
Fig. 2. Increase of the cellular nucleotide contents in the light and in the dark. A Chlorella culture (1.9.105 cells/ml) was divided 7 h a.l. One part was left in the light and the other was placed in the dark. A t 14 h a.l. the light culture was again divided and half o f it was placed in the dark. 200-ml aliquots for the determination of the soluble nucleotides were withdrawn at the times indicated, o, light; e, dark.
t o t h e d a r k 4 h a.l., t h e t r i p h o s p h a t e l e v e l s r e m a i n e d e i t h e ~ c o n s t a n t o r inc r e a s e d n o t i c e a b l y ( F i g . 3, T a b l e I I ) . T h e i n c r e a s e o f U T P a n d C T P u p o n TABLE II RELATIVE AMOUNTS OF NUCLEOTIDES A F T E R 2 h OF D A R K COMPARED TO CONTINUOUS LIGHT The ratios of corresponding nucleotides in the light and dark (for 2 h) have been determined from the peak areas of the experiment shown in Fig. 3 (Expt. 1). Expt. 2 w~'carried out identically except that the culture density was 2.9.106 cells/ml. CTP was calculated from the absorbances at 260 and 280 nm of the respective fractions of the ATP peak, using the formula: 1.96 A2s0nm(CTP) ffi 1.96--0.17 ' ( A 2 s 0 n m - 0.17"A260nm), where 1.96 and 0.17 are theA2sonm/A26onm ratios o f CTP and ATP, respectively, in 4 N formic acid + 1 M ammonium formate. Nucleotide
Total absorbance per peak
in dark in light
Expt. 1
Expt. 2
ATP GTP CTP UTP AMP + ADP + ATP
1.13 1.36 2.16 1.91 0.84
0.73 0.73 1.54 1.04 0.59
Total extract
0.87
0.73
133
NADP
[[[I.
CTP
GTP UTP I
dark
0
10
20
60
70
80
90 100 tube number
i
120
I 130
J " 140 150
I
160
Fig. 3. N u c l e o t i d e c o m p o s i t i o n s in t h e light a n d in t h e dark. A Chlorella c u l t u r e ( 1 . 7 . 1 0 6 c e l l s / m l ) was divided 4 h a.l. i n t o t w o o f 2 1 each. O n e r e m a i n e d in t h e light a n d o n e was p l a c e d in t h e dark. T h e cells were c o l l e c t e d a n d e x t r a c t ed 2 h later a n d t h e n u c l e o t i d e s were a n a l y s e d b y c h r o m a t o g r a p h y o n D o w e x c o l u m n s . T h e f o r m a t e g r a d i e n t was s t a r t e d at f r a c t i o n 95. T h e f r a c t i o n size was 5.5 ml. C o n t i n u o u s lines i n d i c a t e t h e a b s o r b a n c e at 260 n m a n d d a s h e d lines at 280 nm.
2 h exposure to dark was even stronger than in the light controls. The lower nucleotide pool size in the dark {Fig. 2) was mainly due to a loss of AMP and ADP. DISCUSSION
The main conclusion to be drawn from these results is that the cessation of RNA synthesis in young Chlorella cells is not caused by a depletion of the nucleotide pool. It is likely that beyond the regular 4 ribonucleoside triphosphates one or more additional compounds are required for a measurable RNA increase. In young cells the light, possibly via the photosynthetic metabolism ,4, plays a d o m i n a n t role in the formation of such compounds, while later in the cell cycle intermediates of the starch degradation 4 seem to contribute significantly. It can not be excluded at present, however, that qualitatively different regulating principles may control the RNA synthesis in young and old cells respectively. The immediate cessation or strong reduction of the increase of the nucleotide pool upon termination of the light could simply be explained by assuming an insufficient precursor supply. The stability of the nucleotide levels is surprising, however, if one considers the fact that the amounts of guanosine
134
a n d c y t i d i n e n u c l e o t i d e s p r e s e n t 9,, 5,~ 3 m i g h t n o t b e s u f f i c i e n t t o s u p p o r t t h e R N A s y n t h e s i s in t h e early light p e r i o d f o r even 5 min. This s u p p o r t s o u r p r e v i o u s suggestion t h a t t h e p o o l size a n d c o m p o s i t i o n in Chlorella is controlled b y a d d i t i o n a l r e g u l a t o r y processes 9. T h e slight increase o f t h e nucleoside t r i p h o s p h a t e c o n t e n t s in t h e d a r k indicates t h a t f e e d b a c k c o n t r o l b y these c o m p o u n d s ' 5,, 6 m a y be a m a j o r e l e m e n t in t h e r e g u l a t i o n o f t h e size a n d c o m p o s i t i o n o f t h e cellular n u c l e o t i d e p o o l . REFERENCES
1 F. Wanka, Bet. Dtsch. Bot. Gesellsch., 75 (1962) 457. 2 H.G. Ruppel, Flora, 152 (1962) 113. 3 F. En6ckl, Z. Pflanzenphysiol., 58 (1968) 240. 4 F. Wanka, M.M.J. Joppen and Ch.M.A. Kuyper, Z. Pflanzenphysiol., 62 (1970) 146. 5 F. Wanka, J. Moors and F.N.C.M. Krijzer, Biochim. Biophys. Acta, 269 (1972) 153. 6 0 . H . Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 7 F. Wanka, H.F.P. Joosten and W.J. de Grip, Arch. Mikrobiol., 75 (1970) 25. 8 J.D. Mandell and A.D. Hershey, Analyt. Biochem., 1 (1960) 66. 9 F. Wanka, P.H. Vereijken, W. Weijers and J.M.A. Aelen, Proc. Koninkl. Akad. v. Wetensch. Ser. C, 75 (1972) 441. 10 J. Ingle, Biochim. Biophys. Acta, 61 (1962) 147. 11 F. Wanka and P.J.A. Schrauwen, Biochim. Biophys. Aeta, 254 (1971) 237. 12 T. Iwamura, T. Kanazawa and K. Kanazawa, Plant and Cell Physiol. (Special issue on Microalgae and Photosynthetic Bacteria), ( 1963 ) 577. 13 T. Iwamura, T. Kanazawa and K. Kanazawa, Plant Cell Physiol. (Special issue on Microalgae and Photosynthetic Bacteria), (1963) 587. 14 H. Senger and N.I. Bishop, Plant CellPhysiol., 7 (1966) 441. 15 S.C. Hartman, Purines and pyrimidines, in D.M. Greenberg (Ed.), Metabolic Pathways, Vol. 4, Academic Press, New York, 1970, p. 1. 16 S, Kit, Nucleotides and nucleic acids, in D.M. Greenberg (Ed.), Metabolic Pathways, Vol. 4, Academic Press, New York, 1970, p. 69.
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