Uracil incorporation and photopigment synthesis in Rhodospirillum rubrum

322

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96209

URACIL INCORPORATION AND P H O T O P I G M E N T SYNTHESIS IN

RHODOSPIRILLUM R U B R U M J I N P E I Y A M A S H I T A AND M A R T I N D. K A M E N

Department of Chemistry, University o] California at San Diego, La Jolla, Calif. (U.S.A.) and Laboratoire de Photosynth~se, Centre Nationale de la Recherche Scientifique, Gif-sur-Yvette (France) (Received March 3rd, 1969)

SUMMARY

The relationships between bacteriochlorophyll and RNA synthesis in nongrowing cells of Rhodospirillum rubrum were studied during transition from dark aerobic to light anaerobic metabolism. I. At high cell densities, uracil incorporation and bacteriochlorophyll synthesis were greatly stimulated by illumination. The light-stimulation of uracil incorporation occurred earlier. Cell mass and protein content did not increase significantly. 2. The incorporated uracil was distributed in a slowly sedimenting ("slow") RNA fraction during I h of incubation. The ribosomal and soluble RNA's were gradually labeled at later times. The rate of synthesis of the slow RNA was greater than those of the other RNA fractions particularly in the light. 3. Sucrose-gradient centrifugation and methylated albumen kieselguhr column chromatography produced the same profiles for the pulse-labeled (3 rain) and 6o-min labeled RNA's. 4. Mitomycin, chloramphenicol, antimycin A, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazone, 2-n-nonylhydroxyquinoline-N-oxide and 5-fluorouracil inhibited both RNA and bacteriochlorophyll synthesis. Puromycin inhibited bacteriochlorophyll synthesis, but not RNA synthesis. 5. It is concluded that under non-growing conditions during transition, the slow RNA fraction produced could contain a messenger RNA and that the possibility it includes a specific light messenger component requires further investigation of this fraction.

INTRODUCTION

Facultative photosynthetic bacteria growing photosynthetically form characteristic sub-cellular organelles ("chromatophores") in which the photoactive pigments and photosynthetic metabolic systems are located 1-4. Little is known about the mechanisms at the molecular level which control light-induced membrane synthesis in these processes. There is abundant evidenceT M that the formation of the photopigments is regulated by interplay of light intensity and 0 2 tension; thus, bacAbbreviation: HQNO, 2-n-nonylhydroxyquinoline-N-oxide.

Biochim. Biophys. Acta, 182 (1969) 322-333

URACIL INCORPORATION AND PHOTOPIGMENT SYNTHESIS

323

teriochtorophyll synthesis can occur in the dark, provided 0 2 tension is not too high. Genetic controls are obviously present, as evidenced by facile production of pigmentdeficient mutants by ultraviolet irradiation 13-15. Moreover, inhibitors of protein and nucleic acid synthesis, such as chloramphenicol, 8-azaguanine, mitomycin, actinomycin, etc., interfere with photopigment formation 7,9,1e-~°. An obvious mechanism for molecular control is at the transcriptional level of protein synthesis, presumably involving a light-specific messenger RNA, the formation of which is initiated by light absorption. Our previous efforts to detect such an RNA species in the facultative photoheterotrophe, Rhodospirillum rubrum, by pulselabeling RNA in short periods of light in the presence of labeled uracil z~ have indicated that if such a messenger RNA exists, it is present in amounts too small to be detected by present methods which are based on specific hybridization with appropriate DNA preparations from the photosynthetic system. However, it is possible that failure to detect a specific light messenger RNA might be owing to heterogeneity associated with the great number of cell components required to be synthesized during growth. It is plausible that during transition from dark aerobic to light anaerobic conditions when no growth or net protein synthesis occurs, that differences between dark and light messenger RNA, of existent, might be accentuated. Hence, to examine further the possibility that light metabolism involves modifications in RNA and DNA synthesis, we have conducted experiments on uracil incorporation into nucleic acids, in relation to photopigment formation in non-growing cells during transition. We report briefly in this communication the results obtained.

MATERIALS AND METHODS

Cultures

R. rubrum was grown under dark aerobic conditions, as described in our previous report 21. Growth of bacterial cultures were checked by measuring turbidity with the Klett Photometer, using a Coming No. 66 filter ~2. In all experiments reported herein, only such dark-grown cells were used, unless otherwise noted. Transition experiments Cells were collected during the first two-thirds of the logarithmic phase of growth in the dark, and resuspended in o.oi M Tris-HC1 buffer (pH 7.2) to make the protein concentration about 2.5 or 3.0 mg per ml. The suspension was sealed with liquid paraffin to prevent the diffusion of 0 2 into the cells. In somes cases, IO/,g/ml of glucose oxidase (D-glucose :02 oxidoreductase, EC I.I.3.4), 5oo#g/ml of D-glucose and 5 #g/ml of beef liver catalase (H20 2 :HzO oxidoreductase, EC 1.11.1.6) were added to the suspension. However, no significant difference was observed in results whether the additions noted above were made or not. No gas was sparged into the suspension. After supplementation with 0.5/~g/ml of unlabeled uracil, the suspension was subjected to a preliminary incubation at 3 °0 for 60 min under dark conditions. The experiment was started by the addition of appropriate amounts of radioactive uracil, with, or without, exposure to light of 7 ° ft-candles. In dark control experiments, the reaction vessels were completely covered with heavy aluminum foil. At various times, aliquots of the suspensions were removed for estimations, performed as noted below. Cells in transition under illumination will be referred to hereafter as Biochim. Biophys. Acta, 182 (1969) 322-333

324

j . YAMASHITA, M. D. KAMEN

"light-treated" and control cells kept in dark throughout the test period will be indicated as "dark-treated".

Estimation o[ uracil incorporation, bacteriochlorophyll, protein and cell mass Uracil incorporation. Uracil incorporation was started by the addition of 0.2-0. 5 #C/ml of [3Hluracil , or 0.02-0.05 #C/ml of E14C~uracil. At the appropriate time, a I-ml aliquot was withdrawn by pipette and mixed with 0.5 ml of cold 30 % trichloroacetic acid. After the mixture was kept for IO min in an ice-water bath, the precipitate was collected on a Millipore filter and washed with IO ml of cold 5 % trichloroacetic acid. The dried filter was assayed with a scintillation counter in the usual manner. Bacteriochlorophyll. 5-ml aliquots were removed from the cell suspensions and centrifuged at IO ooo × g for IO rain. The supernatants were discarded and bacteriochlorophyll was extracted from the residues by shaking with 2.5 ml acetone-methanol (7:2, by vol.). The mixture was centrifuged at 5000 × g for 5 rain. Bacteriochlorophyll in the supernatant fluid was estimated from its absorbance at 775 nm (ref. 23). Protein. I ml of cell suspension was mixed with 0.5 ml of cold 30 % trichloroacetic acid and allowed to stand for IO rain in an ice-water bath. The mixture was centrifuged at io ooo × g for io rain and the precipitate was washed two times with cold 5 % trichloroacetic acid. The washed precipitate was suspended in 2 ml of I M KOH and incubated at 9°o for 9 ° rain. The resultant alkaline suspension was centrifuged and 0.2 ml of supernatant containing the completely dissolved protein was assayed according to the method of LOWRY et al. 24. In this procedure, newly synthesized photopigment did not affect the estimation. Cell mass. The absorbance at 660 nm of cell suspensions, diluted to yield from 0.3-0.6 absorbance with Tris-HC1 buffer, was the basis for the estimate of cell mass. Double-labeling o / R N A with [3Hl- and [14C~uracil The harvested cells were suspended in o.oi M Tris-HCl buffer (pH 7.2) containing 0.5/,g/ml of [12C]uracil. At zero time, o.oi/~C/ml of E14Cluracil was added and the reaction was carried out for 57 min in the light at 3 o°. Then I/~C/ml of [3HI uracil was added to the suspension and the reaction was continued for 3 min. The reaction was stopped by the addition of o.I vol. I M NaN 3 and excess of crushed ice for the RNA extraction, or by addition of 0.5 vol. cold 30 % trichloroacetic acid for the estimation of uracil incorporation.

Estimation o / D N A Radioactivity of DNA fractions was estimated by the following method: the washed cells were hydrolyzed with 0.5 M KOH for 18 h at 37 ° and then DNA was precipitated by IO % trichloroacetic acid. The precipitate was collected on a filter paper, dried, and counted.

Extraction o/RATA The cells were collected by centrifugation and washed with a mixture of o.oi M Tris-HC1 (pH 7.2) and I mM MgC12 (Tris-Mg 2+ buffer), and then suspended in IO times (v/w) the same buffer containing 5/~g/ml of deoxyribonuclease (deoxyribonucleate oligonucleotidohydrolyase, EC 3.I.4.5) and I o/~g/ml of potassium polyvinyl sulfate. The cells were disrupted with a Sorval Ribi Cell Fractionator, Model RF-I, at a pressure of 2000 lb.inch -~ at 15 °. From the resultant lysate, RNA was extracted by the sodium dodecyl sulfate-phenol method, as described in a previous report 21. Radioactivity in the RNA fraction was determined as the difference between total cell counts and those found in the DNA fraction.

Biochim. Biophys. Acta, 182 (I969) 322-333

URACIL INCORPORATION AND PHOTOPIGMENI" SYNTHESIS

325

Acid-soluble nucleotides were extracted from the washed cells with IO % cold HCI04. The extract was neutralized with I M KOH and the precipitate of KC104 was removed by centrifugation. The radioactivity of the supernatant fluid was estimated by drying on a planchet, followed by assay with a gas-flow counter.

Sucrose gradient centri/ugation Sucrose gradient centrifugation of extracted RNA was performed as previously described~k

Methylated albumen kieselguhr column chromatography Chromatography of extracted RNA on methylated albumen kieselguhr columns was performed as previously reported ~x.

Reagents [3HI- and I14C~uracil were obtained from the New England Nuclear Co., Boston, Mass. Puromycin and mitomycin were purchased from Nutritional Biochemical Co., Cleveland, Ohio. Deoxyribonuclease, glucose oxidase, catalase and carbonylcyanide m-chlorophenylhydrazone were supplied by Sigma Chemical Co., St. Louis, Mo. and Calbiochem, Los Angeles, Calif.

RESULTS

Incorporation o~ uracil during transition/rom dark aerobic to light anaerobic conditions In previous experiments ~1 it was found that RNA's from light-grown cells of

R. rubrum had greater specific radioactivities than those from dark-grown cells, indicating that cells growing in the light could incorporate uracil more rapidly than cells grown in the dark. As shown in Fig. I, the stimulation of uracil incorporation by light in non-growing cells could be noted as soon as IO min after onset of illumination at 7 ° if-candles. On the other hand, synthesis of bacteriochlorophyll showed a lag L >-

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Fig. I. E f f e c t of t r a n s i t i o n on u r a c i l i n c o r p o r a t i o n , b a c t e r i o c h l o r o p h y l l s y n t h e s i s , p r o t e i n cont e n t a n d cell m a s s of R. rubrum. The cell s u s p e n s i o n w a s t r a n s f e r r e d f r o m d a r k t o l i g h t c o n d i t i o n a t t h e t i m e i n d i c a t e d b y arrow. - - , i n t h e l i g h t ; - - -, i n t h e d a r k ; O , u r a c i l i n c o r p o r a t i o n ; × , b a c t e r i o c h l o r o p h y l l ; A, p r o t e i n ; [~, cell mass.

Biochim. Biophys. Acta, 182 (1969) 322-333

326

j . YAMASHITA, M. D. KAMEN

period which was variable with different batches of cells. The marked light dependence of stimulation of uracil incorporation and induction of bacteriochlorophyll formation is shown in Figs. I and 2. We also observed (not shown) that, during repeated cycles of light and dark periods, immediate cessation of bacteriochlorophyll synthesis and diminution of uracil incorporation occurred upon entering the dark period; these processes resumed upon illumination. These observations are consistent with results of previous researches on bacteriochlorophyll synthesis under anaerobic, light conditions s,8. Also, in this connection, we m a y not that HIGUCHI et al. ~8 have reported that Rhodopseudomonas spheroides could incorporate considerable amounts of uracil into RNA during synthesis of bacteriochlorophyll under semi-aerobic conditions.

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Fig. 2. E f f e c t of i l l u m i n a t i o n on uracil incorporation, t w o t y p i c a l e x p e r i m e n t s A a n d B. T h e t r a n s i t i o n s were p e r f o r m e d as s h o w n a t t i m e s b y arrows. O , R u n A; × , R u n ]3. Fig. 3. E f f e c t of i l l u m i n a t i o n o n uracil i n c o r p o r a t i o n into acid-soluble fraction, R N A , a n d D N A . Cells were p r e c i p i t a t e d b y c e n t r i f u g a t i o n a n d w a s h e d w i t h cold o.oi M Tris-HC1 b u f f e r (pH 7.2). F o r details, see t e x t . 7], acid-soluble fraction; A, R N A ; O , D N A .

The results of our examination of the incorporation of uracil into RNA, DNA and the nucleotide pool are shown in Fig. 3. About io % of the total radioactivity in the acid-insoluble fraction was found in DNA. This DNA incorporation was also stimulated b y illumination. The effect of light on the rate of RNA labeling was greater than that on DNA labeling. Appearance of labeled uracil in the acid-soluble RNA was initially more rapid than in the trichloroacetic acid-precipitable RNA. In some experiments, as shown in Fig. I, uracil incorporation in the light reached a plateau during transition in about 12o min, but this period was variable, depending on the amount of cold uracil added as a carrier and the growth phase of the cells. Biochim. Biophys. Acta, 182 (1969) 322-333

327

URACIL INCORPORATION AND PHOTOPIGMENT SYNTHESIS

E[[ect o[ inhibitors on the incorporation o[ uracil It has been reported that inhibitors for protein synthesis 17-19 and for oxidationreduction reactions or energy-conservation reactions 25 inhibit synthesis of bacteriochlorophyll. Likewise, we found that the incorporation of uracil in R. rubrum was suppressed by a number of inhibitors, i 0 / , g / m l of mitomycin or chloramphenicol inhibited both uracil incorporation and bacteriochlorophyll synthesis. However, IO/,g per ml of puromycin inhibited bacteriochlorophyll synthesis, but not uracil incorporation. The degrees of inhibition by other compounds are shown in Table I and Fig. 4. Parallelism between the incorporation of uracil and synthesis of bacteriochlorophyll appeared to be established for all the inhibitors tried, except for puromycin. TABLE I INHIBITION OF URACIL INCORPORATION AND BACTERIOCHLOROPHYLL SYNTHESIS BY VARIOUS REAGENTS. Cells g r o w n aerobically as described in the t e x t were illuminsted w i t h the addition of 0. 5/~C/ml of [SH]uracil for 12o min at 3o°. Then, the cell s u s p e n s i o n was divided into t w o parts. One w a s for the e s t i m a t i o n of uracil i n c o r p o r a t i o n and the o t h e r for a s s a y of bacter~ochlorophyll synthesis. O t h e r details of e x p e r i m e n t were as described in text.

Inhibitors

None Puromycin (io/,g/ml) Mitomycin ( I o H g / m l ) Chloramphenicol (io#g/ml) A n t i m y c i n A (12.5 Hg/ml) H Q N O (62.sHg/ml) 2.4-Dinitrophenol (2.5 raM) Carbonylcyanide m-chlorophenylhydrazone (6.2/~M)

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Uracil incorporation in phenol-purified R N A We found previously that cells grown and labeled in the light with radioactive uracil for 60 or 12o min yielded RNA fractions among which the radioactivity was distributed homogeneously 2I. Thus, the distribution profile obtained by sucrose gradient centrifugation showed coincidence between the three peaks of m a x i m u m absorbance at 260 nm and the appearance of radioactivity. To examine the labeled uracil distribution in RNA obtained from non-growing cells, as in the studies now reported, RNA was extracted from anaerobic cells labeled with [SHluracil for 60 min, as described in MATERIALSAND METHODS. At least 99 % of the total RNA obtained in pure form could be hydrolyzed completely b y treatment with i M K O H at 37 ° for 18 h. In contrast to the results obtained previously with RNA from growing cells, the RNA from non-growing cells exhibited a major fraction of its radioactive uracil Biochim. Biophys. Acta, 182 (1969) 322-333

328

J. YAMASHITA M. D. KAMEN ^

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content in slowly sedimenting ("slow") components, rather than showing a homogeneous distribution of labeled uracil in the usual three components (note, as an example, the uppermost curve for SH labeling in Fig. 6 compared to the distribution shown in the lowest curve obtained b y 260 nm absorbance assay). To explain this distribution of radioactivity in RNA, one could assume either that the rate of RNA synthesis was reduced or that RNA was decomposed into smaller molecule fractions during preparation. Further experiments bearing on these possibilities and the nature of the RNA were required. We began by performing a chase experiment. Cells were labeled with ~ H luracil for 9 ° rain in the light, than the cell suspension was divided into two parts. RNA was extracted from one; the other, supplemented with 50 yg/ml of [z~C]uracil was incubated for another 25 ° min in light, and then the RNA was extracted. Each of these RNA fractions, after purification, was subjected to centrifugation in a linear sucrose gradient. As can be seen in Fig. 5, uracil was incorporated in a slow component 90

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Fig. 5. Chase e x p e r i m e n t of i n c o r p o r a t e d u r a c i l i n R. rubrum. E x p e r i m e n t a l c o n d i t i o n s w e r e as d e s c r i b e d in t h e t e x t . /x, r a d i o a c t i v i t y ; O , a b s o r b a n c e a t 26o rim.

Biochim. Biophys. Mcta, 182 (1969) 322-333

32 9

URACIL INCORPORATION AND PHOTOPIGMENT SYNTHESIS

of RNA in the first 9o-min incubation. However, this radioactive uracil was transferred and conserved in the usual three fractions, two ribosomal and one soluble RNA, during further incubation with unlabeled uracil. Therefore, it was not likely that the slow RNA was a product of degradation during RNA preparation, but rather that it was either some intermediate in RNA synthesis or even messenger RNA. We had already noted that R. rubrurn could synthesize messenger RNA, as demonstrated by the pulse-labeling technique2L Cells were double-labeled, using [14C]uracil for a prolonged time and I3H]uracil for a short time (see MATERIALSAND METHODS), then RNA was extracted. The profiles of the sucrose gradient centrifugation and methylated albumen kieselguhr column chromatography of this RNA are shown in Figs. 6 and 7- The pulse-labeled [3H]RNA seen to be distributed heterogeneously

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Fig. 6. Profile of s u c r o s e g r a d i e n t c e n t r i f u g a t i o n of d o u b l y - l a b e l e d R N A f r o m l i g h t - t r e a t e d cells. R N A w a s e x t r a c t e d f r o m cells d o u b l y labeled w i t h [z4C]- a n d [SH]uracil as described in t h e t e x t . × , z4C r a d i o a c t i v i t y ; /X, 3H r a d i o a c t i v i t y ; O , absorbaalce a t 260 n m . Fig. 7- M e t h y l a t e d a l b u m e n l d e s e l g u h r c o l u m n c h r o m a t o g r a p h y of l i g h t d o u b l y - l a b e l e d R N A f r o m l i g h t - t r e a t e d cells. R N A w a s p r e p a r e d as in Fig. 6. × , 14C r a d i o a c t i v i t y ; &, 8H r a d i o a c t i v i t y ; O , absorbaalce a t 260 n m . -- - - - , NaC1 e l u t i o n gradient.

in a sucrose gradient or in the methylated albumen kieselguhr column fractions could be the messenger RNA noted in our previous report ~1. The molecular weight distribution of the [z4C]RNA was found to be quite similar to that of the pulse-labeled [all]uracil. Upon high-speed centrifugation, a major portion of [z4C]RNA exhibited sedimentation constants between 6 and 16 S. However, a small portion of radioactive uracil was incorporated into ribosomal and soluble RNA. The sedimentationvelocity constants for the slow RNA fraction are in the same range (8-30 S) as those reported by others 31,82 for Escherichia coli. It appears that further fractionation of the slow RNA produced during transition should be attempted before concluding that no specific light messenger RNA exists for the bacterial chromatophore system. To further characterize this RNA, its stability in vivo was examined. GRAY2e and COST AND GRAY27 found that the rapidly-formed RNA of R. spheroides was rapidly degraded in the presence of proflavine, an inhibitor of RNA synthesis. We exploited this finding in the following experiment. The cell suspension simultaneously labeled with [SH]- and [14C]uracil, as described above, was divided into two parts. Biochim. Biophys. Acta, 182 (1969) 322-333

330

j . YAMASHITA, M. D. KAMEN

Then, one was mixed with 200 pg/ml of proflavine. Both samples were incubated in the light and the amount of radioactivity was estimated at various times. The results are shown in Fig. 8. In agreement with the investigations of the results of GRAY AND COST, 3H radioactivity was found to decrease rapidly as soon as proflavine was added. Likewise, 14C radioactivity also decreased rapidly. After 12o min, 3H and t4C radioactive materials were degraded to 36 and 33 % of the original level, respectively. On the other hand, the controls without proflavine maintained radioactivity and even continued to incorporate uracil. From both the chase experiment and the experiment with proflavine, it could be concluded that this labeled slow RNA was metabolically active. 4

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Fig. 8. E f f e c t of p r o f l a v i n e o n short- a n d l o n g - t i m e uracil incorporations. E x p e r i m e n t a l conditions were as described in t e x t . O , 3H r a d i o a c t i v i t y ; Z~, 1'C r a d i o a c t i v i t y . • a n d A, c o r r e s p o n d i n g t e s t cells in p r e s e n c e of proflavine. Fig. 9. Profile of sucrose g r a d i e n t c e n t r i f u g a t i o n of R N A f r a c t i o n s a f t e r uracil i n c o r p o r a t i o n in light a n d d a r k for vaxious periods. 1RNA's were e x t r a c t e d f r o m cells a t t h e t i m e i n d i c a t e d in light or d a r k a f t e r b e g i n n i n g of t r a n s i t i o n period, as described in t h e t e x t . A, r a d i o a c t i v i t y of R N A f r o m l i g h t - t r e a t e d cells; O , r a d i o a c t i v i t y of R N A f r o m d a r k - t r e a t e d cells; - - - , a b s o r b a n c e at 26o n m .

The following experiments were performed to elucidate further the nature of light stimulation of uracil uptake. A concentrated suspension of cells grown aerobically in the dark was divided into two parts. One was incubated with [SH]uracil in the light and the other with [14C]uracil in the dark, after which RNA's were extracted from each batch and purified. The profile of sucrose gradient centrifugation of these two RNA's showed 62 % of total radioactivity of light RNA and 53 % of dark RNA were present in the slow fraction. This incorporation into the slow fraction continued for a relatively long period. As can be seen in Fig. 9, for early periods of transition, uracil was incorporated mainly in the slow fraction. But, 2 or 2.5 h later, the rate of incorporation in this component (Fractions 15-24) gradually fell and peaks of radioactivity distinctly appeared at positions corresponding to ribosomal RNA. After Biochim. Biophys. Acta, 182 (1969) 322-333

URACIL INCORPORATIONAND PHOTOPIGMENTSYNTHESIS

331

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Fig. io. Incorporation of uracil into various fractions of RNA. Experimental conditions were the same as in Fig. 9. - - - , RNA from light-treated cells; - - -, RNA from dark-treated cells; A, "slow" RNA; C), faster-moving ribosomal RNA; VI, slower-moving ribosomal RNA. 4-h incubation, the radioactivity of ribosomal RNA was greater than that of the slow fraction. Labeling of the three fractions of RNA in the dark proceeded continuously at constant rates, which were slower than those in the light. Considerable amounts of the slow RNA were synthesized even when the cells were not making significant amounts of bacteriochlorophyll. To elaborate this observation, the specific radioactivities of the various fractions of RNA were computed from data of Fig. 9 and plotted in Fig. I0. The two fractions of ribosomal RNA were synthesized at similar rates, but the rate of synthesis of the third, or slow, fraction of RNA was greater than those of other fractions, particularly in the light. DISCUSSION The fact that uracil incorporated for 6o or 9 ° min in non-growing cells appeared mainly in the slow component of RNA was in sharp contrast to results in our previous experiments ~1 which had demonstrated that three fractions of RNA purified from growing cells of R. rubrum in both light and dark were homogeneously labeled within 60 min incubation and labeled messenger RNA was only observed when bacterial cells were pulse-labeled for short periods (about 3 min). In general, however, the rate of RNA synthesis in animal cells which grow relatively slowly is known to be slower than that in bacterial cells ~8,~s. GNANAM AND KHANa° also reported slow synthesis of messenger RNA in Euglena gracilis under non-growing condition. Therefore, the low labeling rate of RNA in the present experiments was probably owing to the cessation of growth. Lack of coincidence of peaks between the radioactivity and the absorption at 260 nm indicated that uracil was first incorporated into species of RNA molecules other than the main ribosomal and soluble components. These RNA's were quickly degraded when RNA synthesis was inhibited, and also metabolized into the ribosomal and soluble RNA's upon prolonged incubation. Furthermore, the similar

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profiles of both prolonged- and puLe-labeled RNA's in sucrose gradient centrifugation and methylated albumen kieselguhr column chromatography indicated that these two RNA's might be made up of similar components (i.e., precursors of ribosomal and transfer RNA, or messenger RNA, or both). The apparently prior appearance of label in the acid-soluble p0ol of nucleotides (Fig. 3) poses the possibility that the increased rate of uracil uptake seen in the light resulted from increased accessibility of the cells to exogenous uracil which, in turn, produced elevated levels of precursors for ribosomal RNA. Thus, the increase of uracil labeling in the slow RNA fraction could have reflected increase in levels of precursors for ribosomal RNA, rather than in levels of messenger RNA synthesis. However, the possibility that the slow RNA contained messenger RNA remains so that further investigation of this fraction is warranted. In this connection, the reader's attention is directed again to the experiments summarized in Fig. I. It is seen that in transition for several hours, uracil incorporation into RNA occurs in dark-treated as well as light-treated cells, even after 60 min of prior incubation under dark anaerobic conditions. Thus, it appears that endogenous reserves present in the cells from previous dark, aerobic growth can be mobilized by fermentative processes to provide energy needed for uracil incorporation (and, by inference, Amino acid redistribution and incorporation into enzyme proteins required to preceed such uptake). It is well-known that facultative heterotrophes can utilize fermentation to drive a number of synthetic processes but not net cell synthesis (for a review see ref. 33). A particular example is the well-documented carboxylation of acetone to form acetoacetate ~t (see for a discussion ref. 35). Thus, it is not surprising that some uracil uptake is observed in the dark control (dark-treated) cells during the transition experiments. However, it will be noted that no bacteriochlorophyll synthesis occurs in dark-treated cells, while the RNA synthesis occurs at a rate which is appreciable compared to that in the light-treated cells. In both cases, the RNA labeling by uracil appears to reach a plateau after a few hours, that for the darktreated cells being almost as high as for that of light-treated cells. It remains to be determined if the two levels would eventually coincide. However, it seems certain that the initial light-stimulated incorporation of uracil into RNA during the first 2 h of transition is real, and may represent the additional presence of messenger RNA associated with synthesis of bacteriochlorophyll and other photosynthetically functional membrane components. ACKNOWLEDGMENTS

This research was supported by grants-in-aid from the National Institutes of Health (HD-oI262), the National Science Foundation (GD-7o33X) and a Special Research Award of the C. F. Kettering Foundation to one of us (M.D.K.) who also wishes to record his indebtedness for aid in the form of a travel subsidy given by the Centre Nationale de la Recherche Scientifique, France. REFERENCES

I H. K. SCHACHMANN, A. B. PARDEE AND R. Y. STANIER, Arch. Bio6hem. Biophys., 38 (i952) 245. 2 A. W. FRENKEL, J. Am. Chem. Soc., 76 (1954) 5568.

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A. W. FRENKEL, Ann. Rev. Plant Physiol., 9 (1959) 53. T. HORIO AND M. D. KAMEN, Biochemistry, I (1963) 1141. C. B. VAN NIEL, Bacteriol. Rev., 8 (1944) i. G. COHEN-BAZlRE, W. R. SISTROM AND R. Y. STANIER, J. Cell Comp. Physiol., 49 (1951) 25. J. LASCELLES, Biochem. J., 72 (1959) 508. J. LASCELLES, J. Gen. Microbiol., 23 (196o) 487 . W. R. SISTROM, J. Gen. Microbiol., 28 (1962) 6o 7. S. C. HOLT AND A. G. MARR, J. Bacteriol., 89 (1965) 1421. D. G. PRATT, A. W. FRENKEL AND D. D. HICKMAN, in T. W. GOODWIN AND O. LINDBERG, Biological Structure and Function, Vol. 2, A c a d e m i c Press, N e w York, 1961, p. 295. M. BIEDERMANN, G. DREWS, R. MARX AND J. SCHRODER, Arch. Mihrobiol., 56 (1967) 133. M. GRIFFITH AND R. Y. STANIER, ft. Gen. Microbiol., 14 (1956) 698. W. R. SISTROM AND R. Y. CLAYTON, Biochim. Biophys. Acta, 88 (1964) 61. J. LASCELLES, Biochem. J., IOO (1966) I75. W . R. SISTROM, J. Gen. Microbiol., 28 (1962) 599. M. J. BULL AND J. LASCELLES, Biochem. J., 87 (1963) 15. M. HIGUCHI, K. GOTO, M. FUJIMOTO, O. •AMIKI AND G. KIKUCHI, Biochim. Biophys. Acta, 95 (1965) 94. G. DREWS, Arch. Mikrobiol., 51 (1965) 186. G. DREWS AND J. SCHICK, Z. Natur/orsch., 2I (1966) lO97. J. YAMASHITA AND M. D. KAMEN, Biochim. Biophys. Acta, 161 (1968) 162. S. TANIGUCHI AND M. D. KAMEN, Biochim. Biophys. Acta, 96 (1965) 395G. COHEN-BAZlRE AND W. R. SISTROM, in L. P. VERNON AND G. R. SEELY, Chlorophyll, A c a d e m i c Press, N e w York, 1966, p. 313 . O. H. LOWRY, N. J. ROSEBEOUGH, A. L. FARR AND J. R. RANDELL, J. Biol. Chem., 193 (1951) 265. W. R. SISTROM, in H. GEST, A. SAN PIETRO AND L. P. VERNON, Bacterial Photosynthesis, A n t i o c h Press, Yellow Springs, Ohio, 1963, p. 53. E. D. GRAY, Biochim. Biophys. Acta, 138 (1967) 55o. H. B. COST AND E. D. GRAY, Biochim. Biophys. Aaa, 138 (1967) 6Ol. D. D. BROWN AND J. B. GURDON, Proc. Natl. Acad. Sci. U.S., 51 (1964) 139. G. ATTARDI, H. PARNAS, M. HWANG AND B. ATTARDI, jr. Mol. Biol., 2o (1966). A. GNANAM AND J. S. I{AHN, Biochim. Biophys. Aaa, 142 (1967) 475B. P. SAGIK, M. H. GREEN, M. HAYASHI AND S. SPIEGELMAN, Biophys. J . , 2 (1962) 4o9 . K. ASANO, J. Mol. Biol., 14 (1965) 71. H. GEST AND M. D. KAMEN, Handbuch der P/lanzenphysiologie, Vol. V, 196o, p. 568. J- M. SIEGEL, J. Biol. Chem., 228 (1957) 41. H. GEST, in H. GEST, A. SAN PIETRO AND L. P. VERNON, Bacterial Photosynthesis, A n t i o c h Press, Yellow Springs, Ohio, 1963, p. 13o.

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