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Synthesis of ribonucleic acid in regenerating rat liver after partial hepatectomy
262
BIOCHIMICA ET BIOPHYSI(A ACTA
BBA 95135
S Y N T H E S I S OF R I B O N U C L E I C ACID IN R E G E N E R A T I N G RAT L I V E R AFTER PARTIAL HEPATECTOMY
W. W E L L I N G ' ,
D. B O O T S M A , E. V A N M U I S W I N K E L
AND C. A. P. B E R G H E G E N
Medical Biological Laboratory o/ the National De/enee Research Organization T.N.O., Rijswijk (The Netherlands) (Received, J u l y I3th, 1964)
SUMMARY
A technique for the fractionation of R N A by column chromatography on calcium phosphate is described. With this technique the incorporation of labeled inorganic phosphate into RNA of regenerating rat liver was studied. In the cytoplasm a R N A fraction was found, with molecular weight intermediate between that of soluble and microsomal RNA, that had a very high incorporation rate. This fraction occurred only in small amounts in the cytoplasm. In the nucleus relatively more of this type of RNA was present but there the specific activity did not differ from that of nuclear ribosomal RNA. Determination of the relative amount of label in the four R N A nucleotides showed that the fraction with intermediate molecular weight is not a breakdown product of ribosomal RNA. [sH]Cytidine incorporation in the nucleolar regions of the hepatocyte nucleus was demonstrated with autoradiography. Lengthening the period between injection of the isotope and death of the animal led to a decrease of nucleolar labeling coupled with an increase in cytoplasmic labeling. These results support earlier observations, indicating the important role of the nucleus in the synthesis of cytoplasmic RNA.
INTRODUCTION
I t has long been known that the synthesis of R N A in regenerating rat liver is considerably enhanced as compared with normal resting liver 1-4. This increase occurs before the onset of DNA synthesis, which starts at about 18 h after partial hepatectomy, reaches a m a x i m u m at about 24 h after operation 4,5 and is followed by an intensive activity of mitosis some hours later 6. Several investigators have found that the incorporation rate of labeled R N A A b b r e v i a t i o n s : i - R N A , i n t e r m e d i a t e R N A ; r - R N A , r i b o s o m a l R N A ; s - R N A , soluble RNA. * P r e s e n t address: L a b o r a t o r y for R e s e a r c h on Insecticides, 22 P r i n s e s Marijkeweg, W a g e n i n g e n (the N e t h e r l a n d s ) .
Biochim. Biophys. Acta, 95 (1965) 262 279
RNA
SYNTHESIS IN REGENERATING RAT LIVER
263
precursors reaches a m a x i m u m within 2o h after the operation, i.e. when DNA synthesis 7-9 is on a lower level. The activity in vitro of the enzyme RNA polymerase (EC 2.7.7.6 ) of the nuclei of regenerating rat liver closely parallels that incorporation rate 10. I t is now generally accepted that in mammalian cells R N A is exclusively synthesized in the cell nucleus. Much less clear however is the role of the cytologicallydistinct parts of the nucleus in that process. On the strength of the elegant investigations of PERRY et al. 11-14 it seems to be most probable that the nucleolus and the extra-nucleolar part of the nucleus are largely independent from each other with respect to RNA synthesis, r-RNA is synthesized in the nucleolus, s-RNA on the chromatin. In this study we report the results of a number of experiments that were performed to obtain a better insight into the pattern of RNA synthesis in regenerating rat liver. During the course of this work other investigators published their results on the same subject. A comparison of all data is given in the DISCUSSION. BIOCHEMICAL
STUDIES
METHODS
The separation of the various R N A fractions was based on their localization in the cell (i.e. nuclear and cytoplasmic RNA) and the variation in their molecular weight (by column chromatography on calcium phosphate). The general outline of the experiments was as follows. Male Glaxo rats (16o-18o g) were partially hepatectomized as described by HIGGINS AND ANDERSON15. At different intervals after operation the animals received an intravenous injection of inorganic radioactive phosphate (300 or 6oo/zC for each animal), and 45 min to 2 h later the animals were killed by decapitation, the remaining liver lobes were excised and immediately cooled in ice-cold 0.25 M sucrose. Tissue [ractionation
The liver residues of 8 rats were pooled and homogenized in o.25 M sucrose, 5" lO-3 M MgC12, 0.025 M KC1, 0.05 M Tris buffer (pH 7.6), first in a Potter-Elvehjem type homogenizer with Teflon pestle, and, after filtration through IOO mesh nylon tissue, in a stainless-steel Dounce type homogenizer with a clearance between ball and wall of o.09 ram. The homogenate was then centrifuged for 15 min at 800 ×g. The supernatant fluid was pipetted off by aspiration and set aside for the preparation of cytoplasmic RNA. The sediment was suspended in IOO ml concentrated sucrose solution-- (58 % by weight) buffered with 0.05 M glycerophosphatO 6 (pH 6.6) - - a n d centrifuged for 20 rain at 20 ooo×g. The resulting nuclear pellet was washed by resuspension in concentrated sucrose solution and centrifugation for 30 min at the speed given above. Microscopal inspection of the isolated nuclei showed the absence of whole cells and erythrocytes; the preparation was still contaminated b y a small amount of mitochondria. The RNA/DNA-ratio was o.16. This technique for the isolation of Biochim. Biophys. Acla, 95 (1965) 262-279
264
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nuclei bears a strong resemblance to that described by SPORN AND I)INGMAN 17, published while our experiments were in progress.
RNA isolation Nuclear RNA. The nuclear pellet was suspended in I. IO-3 M MgC12, o.o5 M Tris buffer (pH 7.6) and incubated with sodium laurylsulfate (i % final concentration) for 15 min at 37 °. The nuclei were lysed by this treatment and gave a very viscous solution. An equal volume of freshly-redistilled water-saturated phenol was then added, and the mixture was shaken in the cold room for 0.5 h, followed b y centrifugation at high speed to separate the phenol and water layers. The phenol layer was extracted with an equal volume of the buffer-sodium larurylsulfate mixture, and the combined water layers were reextracted with half a volume of phenol. The nucleic acids in the water layer were precipitated b y the addition of two volumes of cold ethanol and o.I volume 20 o/ ,,o sodium acetate and stored overnight at --25 ° . The precipitate was washed twice with cold o.14 M NaC1 in 67 o/,,oethanol and suspended in 0.05 M Tris buffer (pH 6.6) containing lO -3 M MgCI~. DNA was broken down with deoxyribonuclease (EC 3.1.4.5) (2 × recrystallized) at a concentration of 5 #g/ml for 30 min at 37 °. Material not dissolved at this step was centrifuged down and again treated with the buffer-deoxyribonuclease mixture. The clear supernatants were combined, and nucleic acids were precipitated with two volumes of cold ethanol in the presence of 2 % sodium acetate. DNA-breakdown products coprecipitate with nuclear R N A and cannot be removed by repeated reprecipitation. However, it proved possible to separate these contaminants b y gel filtration through Sephadex G-5o. The sediment from tile last alcohol precipitation step was to that end dissolved in a small amount of 0.005 M phosphate buffer (pH 6.7) applied to a column 40 cm high and 3 cm diameter of gel filtration material in equilibrium with the same buffer and ehited at a rate of about 12 ml/h. An example of tile elution pattern is given in Fig. I, from which it appears that RNA moved as a rather narrow zone through the column, followed by a heterogeneous mixture of DNA breakdown products. The combined R N A fractions of the first peak contained about 1 % DNA-reacting material (DNA reaction as described by BURTONI8).
In this way an amount of nuclear R N A varying from 2-6 mg/5o g liver tissue was isolated. Cytoplasmic RNA. Cytoplasmic RNA was prepared from the supernatant of the first low speed centrifugation (see above). Mitochondria were removed b y centrifugation at I2 ooo x g for IO rain. The deproteinization with phenol was carried out as described for nuclear R N A in the presence of 1 % sodium laurylsulfate, with the exception that residual phenol in the water layer was removed b y extraction (3 x ) with ether. The material precipitated with alcohol was washed twice with cold o.14 M NaC1 in 67 % ethanol, and dissolved in 0.005 M phosphate buffer (pH 6.7). This produced a strongly opalescent solution, caused b y glycogen. The solution was Biochim. 19iophys. Acta, 95 (1965) 262 279
RNA Z.e-
SYNTHESIS IN REGENERATING RAT LIVER
265
RNA
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Fig. I. Separation of nuclear 1RNA from DNA breakdown products by gel filtration through Sephadex G-5o. Rat-liver-nuclear RNA, isolated with the phenol procedure and digested with deoxyribonuclease as described in the text, was dissolved in o.o0 5 M phosphate buffer (pH 6.7) applied to a column (3 × 4° cm) of Sephadex G-5 o, equilibrated with the same phosphate buffer, and washed through with t h a t buffer at a rate of 12 ml/h.
cleared b y centrifugation at lO 5 ooo ×g for 3o min. Glycogen gives a colorless pellet at the bottom of the tube. Radioactive R N A was dialyzed overnight against 0.005 M phosphate buffer (pH 6.7).
Chromatography on calcium phosphate Calcium phosphate for the column chromatography of RNA was prepared as described by MAIN, V~ILKINS AND COLE19. The modification denoted as counts/min b y these authors was used in all experiments because it permits a high flow rate. Before use the calcium phosphate, packed in columns of IO × 1. 4 cm, was washed with I M phosphate buffer (pH 6.7) until no more ultraviolet-absorbing material could be eluted; then it was reconditioned b y washing with a large amount of 0.005 M phosphate buffer (pH 6.7). R N A dissolved in 0.005 M phosphate buffer (pH 6.7) was adsorbed on the top layer of the column. The maximal amount of RNA used was 4 mg with columns of 1. 4 cm diameter. Elution was carried out with a phosphate buffer gradient (pH 6.7) prepared by adding o.15 M buffer to a closed mixing vessel of 250 ml entirely filled with 0.07 M buffer. Fractions of 4 ml were collected. After 60 fractions had been collected the concentration of the buffer added to the mixing vessel was raised to 0.3 M to elute high molecular weight RNA. The flow rate was 18 ml/h. The concentration of RNA in the fractions was determined by ultraviolet absorbancy at 260 my. With nuclear RNA, when little material was available the scale of the experiments was reduced to one quarter of the given dimensions. Biochim. Biophys. Acta, 95 (1965) 262-279
266
w . WELI_IN(; el al.
Determination o/ the ratio o~ 32p in the/our nucleotides o/ R N A Selected fractions from the column (see under RESULTS) were pooled and frozen dry. The dry material was dissolved in 3 ml water and dialyzed against water to remove salts. The dialyzed fluid was then brought to o.5 M KOH, and RNA was hydrolyzed by incubation at 37 ° for I8 h. Next the p H of the mixture was decreased to about 7 with o.5 M HC10~. Precipitated KC1Q was spun down and the volume of the supernatant was reduced by freeze-drying. The 3'-nucleotides were separated on paper by high-voltage electrophoresis in pyridine acetate buffer (pH 3.6). The spots of the nucleotides were located by ultraviolet contact photography and counted on the paper with a gasflow counter.
RESULTS AND DISCUSSION
Chromatography on calcium phosphate Fig. 2 shows the elution pattern of cytoplasmic RNA from a calcium phosphate column, Soluble RNA elntes at about 0.07 M phosphate buffer and microsomal RNA at about o.12 M. The identity of two peaks was checked in two ways; first cytoplasmic RNA was freed of soluble RNA by adding an equal volume of 3 M NaC1 and leaving overnight in the refrigerator. Under these conditions only microsomal RNA precipitates. The precipitated material gave only one peak, corresponding with the peak eluting at the higher phosphate buffer concentration in Fig. 2. Soluble RNA was then prepared from a particle-free rat-liver supernatant (centrifuging I h at
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Biochim. Biophys. Acta, 95 (1965) 262 279
RNA SYNTHESIS IN REGENERATING RAT LIVER
267
lO5 ooo ×g). This RNA also gave only one peak corresponding with the first one in Fig. 2. Between 0.07 and o.12 M phosphate buffer the ultraviolet absorption at 260 m# of the eluate did not drop to zero, but small amounts of RNA were continnuously eluting from the column. The identity of this material is not clear at present. Tentatively it will be referred to as i-RNA. The recovery of applied 1RNA from the column was complete. Microsomal RNA gives only one peak, although it has been shown to contain two components with sedimentation coefficients of 16-2o S and 26-30 S. Attempts to resolve the microsomal R N A peak b y using a less steep gradient were not successful. BURNESS AND VISOZO20 claim to have separated 18- and 3o-S r-RNA on calcium phosphate. Inspection of their data however leads us to presume that they have interpreted soluble RNA as I8-S r-RNA. Nuclear RNA gave an elution pattern qualitatively identical with that of cytoplasmic RNA; relatively more material appeared in the i-fraction. It has been reported b y TAKAI et al. 21 that high-molecular weight RNA isolated from Escherichia coli can be separated from the 16 and 23-S component b y chrom a t o g r a p h y on a column of Celite coated with esterified bovine serum albumin. W'e confirmed this observation with E. coli RNA, but rat-liver microsomal RNA behaved differently;this material was eluted as one component from the column,and no separation could be obtained.This is in contrast to a report OfPHILIPSON 22, who describes at least a partial separation of high-molecular weight RNA of HeLa-cells. Using the same technique for the fractionation of nuclear RNA from rat kidney and liver, REVEL et al. ~a mention that it was possible to separate both components only "under the best conditions". However, these authors do not state what these best conditions are.
Incorporation studies o/ asp into rat-liver R N A
When 3ep was injected into animals with a regenerating liver 24 h after operation, and the animals were killed three quarters of an hour later, it was found that cytoplasmic R N A was only weakly labeled. Chromatography of this RNA showed that there were two fractions that had incorporated s~p: s-RNA and i-RNA. Microsomal RNA was unlabeled (Fig. 3A). A large amount of radioactivity in the s-RNA fraction had however to be attributed to 3~p labeled inorganic phosphate, present in the preparation due to insufficient dialysis. Reprecipitation with alcohol reduced the radioactivity in this fraction to about 20 %; we m a y therefore conclude that the specific activity in Fig. 3A was highest in tile i-RNA fraction. Fig. 3B shows that b y extending the incorporation time from 45 min to 2 h microsomal RNA became labeled too. The specific activity however of i-IRNA was greater than that of microsomal RNA. The picture at 18 h after operation shows a clear resemblance to t h a t at 24 h after operation. In Figs. 4 A and 4 B the partition of radioactivity over the three cytoplasmic RNA fractions at one hour is compared with that at 2 h after injection of the label. I h is too short a period for microsomal RNA to become labeled, but Biochim. Biophys. Acta, 95 (1965) 262-279
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Fig. 3. Column chromatography on calcium phosphate of 3aP-labeled R N A in vivo, isolated by the phenol procedure Irom regenerating rat liver. In all experiments 300 or 600/*C of an isotonic saline solution of 3~P-labeled phosphate was intravenously injected. - - , extinction at 260 m#; - - - , radioactivity (counts/min). A, Cytoplasmic R N A labeled w i t h 300/*C 8zp from 24-24.75 h after partial hepatectomy; t3, as in A, but labeling from 24-26 h after operation.
s-RNA and i-RNA do so. In both examples i-RNA has the greatest specific activity. For the experiment of Fig. 4 B we used twice-reprecipitated RNA to remove the contamination of s-RNA with asp labeled inorganic phosphate. Comparison of the three radioactive peaks in Fig. 4 B shows that the order of increasing specific activity is microsomal RNA < s-RNA < i-RNA. Biochim. Biophys. Acta, 95 (1965) 262-279
RNA
269
SYNTHESIS IN REGENERATING RAT LIVER
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Fig. 4- Conditions as in Fig. 3: A, cytoplasmic R N A labeled with 3oo/*C 32p from 18-19 h after operation; B, c y t o p l a s m i c R N A labeled with 60o #C 82p from 18-2o h after partial h e p a t e c t o m y .
There is however one difference between the situations at 18 and 24 h after operation: the radioactive i-RNA fraction at 18 h e l u t e s - r e p r o d u c i b l y - at a lower salt concentration from the column than at 24 h. This points to a difference in molecular weight of both fractions. The appearance of radioactive i-RNA in the cytoplasm is not typical for the 18 and 24-h period after operation. Only 4 h after partial hepatectomy it was already present, as m a y be seen in Fig. 5C. The specific activity of i-RNA was again much higher than that of microsomal RNA. We also studied the incorporation of ~zP into nuclear R N A at the same regeneration time. Figs. 5A and 5 B give the chromaBiochim. Biophys. Acta, 95 (I965) 262-279
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Fig. 5. Conditions as in Fig. 3: A, nuclear IZNA labeled w i t h 600 ffC 32 P from 4-4.5 h after operation B, as in A, b u t t h e labeling period e x t e n d e d to I h; C, c y t o p l a s m i c R N A labeled with 600 # C 3,p from 4 - 6 h after partial h e p a t e c t o m y . Biochim.
Biophys,
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(1965) 2 6 2 - 2 7 9
RNA SYNTHESIS IN REGENERATING RAT LIVER
271
tographic patterns for this RNA after respective contacts of o.5 and 2 h with 8~p. In contrast to cytoplasmic RNA, nuclear RNA did not show a clear-cut radioactive peak of i-RNA; high-molecular weight RNA and i-RNA had the same specific activity, even after the shorter labeling period. I t seems that the specific activity of lowmolecular weight RNA is somewhat lower than that of the two other fractions. In an experiment analogous to that of Fig. 5A, but 8 h after operation, the same labeling pattern was encountered. I t is not clear what type of RNA the highly labeled i-RNA-fraction in the cytoplasm is. GEORGIEV AND SAMARINA24 found in rat-liver cytoplasm three RNA fractions after 3o-6o min labeling with 82p; one high polymer that was very weakly labeled, one low polymer with a specific activity io times that of the high polymer fraction and a (with phenol) non-extractable one, 2-3 times more strongly labeled than the low polymer fraction. The latter was present only in small amounts. One m a y suppose that this non-extractable fraction corresponds with our i-RNA fraction, assuming that their non-extractable fraction can be solubilized when a detergent (sodium laurylsulfate) is added to the phenol. In m a n y types of mammalian-cells RNA of relatively low molecular weight with high turnover has been reported. MONIER25 finds that after short incubation of hepatome ascites cells with ES-14Clguanine most of the labeled RNA extractable with phenol only has a sedimentation constant in between that of 4- and 2o-S RNA. Extraction however with phenol+detergent gives RNA that is very polydisperse (sedimentation constants ranging from 4-30 S with a peak value between 4 and 20 S). More or less the same situation was encountered by MARKS et al. 2~ with white blood cells and MUNRO AND KORNER27, 2s, LANG AND SEKERIS 29, and DI GIROLAMO et al. a° with rat liver. The three above-mentioned groups of investigators all incline to identify the RNA having high turnover with messenger RNA, shown to have sedimentation constants of 8-12 S by GROS et al. 31 for E . coll. These low sedimentation constants for messenger IRNA, however, were questioned b y TAKAI et al. 21, who put forward the idea that these low values pointed to a considerable breakdown of this messenger RNA by cellular RNAase (EC 2.7.7.16), not inactivated by phenol. However, when the material was carefully isolated, four peaks of rapidly-labeled RNA could be shown b y chromatography on a esterified albumin-coated Celite column, characterized b y sedimentation constants of 8, 12, 19 and 26 S and a nucleotide composition strongly resembling that of E . coli DNA. The objection that low values for the sedimentation constants of messenger RNA are an indication of breakdown does not hold true for those experiments in which Bentonite was used in the isolation of RNA26m. The results of DI GIROLAMO et al. a° and LANG AND SEKERIS29 firmly sustain the hypothesis concerning the possible messenger character of rapidly-labeled low-molecular weight RNA. They showed that in rat-liver-cytoplasmic RNA the fractions with the highest specific stimulating activity towards amino acid incorporation had sedimentation constants of 18 S or lower. There is however also rapidly-labeled RNA of high molecular weight present in mammalian cells. SCHERRER AND ~DARNELL32 found two components of this type of R N A with sedimentation constants of about 45 and 33 S. PERRY14, using a combination of autoradiography and sedimentation of isolated RNA in sucrose Biochim. Biophys. Acta, 95 (1965) 262-279
272
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gradients, was able to demonstrate this high molecular "messenger" RNA after a 3o-min pulse with [3Hlcytidine exclusively in the nucleus of L-cell fibroblasts, and most of it in the nucleolus. This pulse-labeled material was very heterogeneous with sedimentation constants down to 4 S. In rat liver and kidney nuclei rapidly-labeled RNA of high molecular weight was found by HIATT33 and REVEL et al. 2a. Both investigations sustain the idea that this type of RNA is of nucleolar origin. The results of HIATT indicate that the material with the highest sedimentation constants (larger than that of ribosomal RNA) is not transferred to the cytoplasm. This does not speak in favor of a possible messenger role in cytoplasmic protein synthesis, unless we must assume that it breaks down to a limited number of smaller pieces before it reaches the cytoplasm. Comparing our results with those cited above we see that they are analogous in many respects. Just as in the experiments of MONIER 25, MARKS et al. 2n, MUNRO AND KORNER27, 28, LANG AND SEKERIS 29 and DI GIROLAMO et al. 3°, we find a RNA fraction in the cytoplasm characterized by a high incorporation rate of radioactive precursor and a molecular weight between those of s-RNA and microsomal RNA. The latter has the greatest metabolic stability. In the nuclei there is much less variation in the specific activity of the three R N A fractions than in the cytoplasm. This confirms the observations of GEORGIEV AND SAMARINA24. \Ve have no evidence for a rapidly-labeled R N A with sedimentation constants larger than that of ribosomal R N A in the nuclei, as was found by othersl4,~3,32,~'~. An explanation for this discrepancy cannot be given, but it may be that it is not possible to distinguish between ribosomal and higher molecular weight RNA by chromatography on calcium phosphate.
Relative amount o[ 32p in R N A nucleotides To exclude definitely the possibility that i-RNA is a breakdown product of microsomal RNA we determined the relative amounts of 32p in each of the four R N A nucleotides in some experiments. The results are given in Table I. The relative amounts of radioactivity are in general not identical with base ratios; only in the event that the specific activity of tile four direct precursors are the same can one say that the relative amount of radioactivity in the nucleotides is identical to the base ratio. The results in the Table clearly show that i-RNA is not a breakdown product of microsomal RNA, because the ratios of guanylic plus cytidylic acid to adenylic plus uridylic acid ( G + C ) / ( A + U ) differ significantly from each other. Moreover i-RNA seems to be heterogeneous in contrast to microsomal RNA. The ( G + C ) / ( A + U ) ratio for ribosomal RNA of rat liver has been reported to be 1.4-1. 5 . Our "radioactive" ratio does not differ very much from that value. Assuming that this means that our "radioactive" base ratio is a good approximation to true base ratio, we see in Table I that none of the studied fractions has a DNAlike base composition (in rat tissue the ( G + C ) / ( A + T ) ratio of DNA is 0.75 ). Such a R N A fraction has been found b y CxEORGIEVAND ~-4.NTIEVA~ in the nucleolus of rat liver. If in mammalian cells too, messenger R N A has a DNA-like base composition, we must conclude that none of the fractions studied is a likely candidate to play Biochim. Biophys. Acta, 95 (1965) 262-279
RNA
273
SYNTHESIS IN REGENERATING RAT LIVER
TABLE I RELATIVE AMOUNTS OF RADIOACTIVITYIN THE NUCLEOTIDES OF SEVERAL mlWA FRACTIONS OBTAINED BY COLUMN CHROMATOGRAPHY OF REGENERATING RAT-LIVER R N A The R1WA nucleotides were o b t a i n e d b y alkaline hydrolysis of pooled c o l u m n fractions and sepa r a t e d b y high-voltage electrophoresis. I n the c o l u m n denoted RN,4 /raction are represented consecutively, the t i m e of injection of the label in h o u r s after operation, the origin of the R N A fraction (i.e. nuclear or cytoplasmic), the time in h o u r s b e t w e e n injection and decapitation of the a n i m a l and t h e t y p e of R N A fraction (i.e. soluble, i n t e r m e d i a t e or ribosomal). I n some determ i n a t i o n s the c o l u m n fractions were pooled in such a m a n n e r t h a t the c o m b i n e d material represented the first a n d second half of the respective R N A peak. C, A, G and U denote cytidylic, adenylic, guanylic and uridylic acids respectively.
RNA #action
C
4 N 0. 5 S 4N o.5I 4 N 0. 5 R 4 N 2 R1 4 N 2 IRz 81N 0. 5 I 1 8 IW 0. 5 I s 8 N 0. 5 tZ 18C 2 I
21.5 24.1 25.9 26. 5 26.0 23. 9 25.0 25.6 27.3
A
32.6 24. 5 23.2 20.6 20. 7 22. 7 19.9 19.2 21.5
G
18.2 27. 3 31.1 32. 4 33.6 30.3 32.5 35.6 31.5
U
27.8 24.1 19.9 20. 5 19.6 23.1 22.7 19.2 19.6
(C+G)/(A + U) Mean value
Individual determinations *
o.66 1.o6 1.32 1.44 1.48 1.19 1.35 1.58 1.44
o.66 1.o6, 1.32, 1.43 , 1.48, 1.16, 1.34, 1.54, 1.42,
1.o6 1.32, 1.32 1.44 1.48 1.22 1.36 1.62 1.45
" D e t e r m i n a t i o n s were performed on the same material.
that role, unless it forms part of the heterogeneous i-RNA fraction and its nucleotide composition is obscured by other fractions having a higher ( G + C ) / ( A + U ) value.
AUTORADIOGRAPHIC STUDIES METHODS
A few experiments were carried out to study the sequences of events after partial hepatectomy with autoradiography. At different intervals after operation (designated as regeneration time) rats were injected intravenously with either 50 #C [SH~cytidine (Schwarz Bio Research, specific activity I C/mmole) or 50 #C [SH]thymidine (Schwarz Bio Research, specific activity 0.36 C/mmole) in 0.5 ml saline. Animals were killed 0.25, 0.5, I, 2 or 3 h later by decapitation (for details see Table II.) Time between injection and killing is designated as incubation time. The remaining liver lobes were excised and fixed in acetic acid-ethanol (I :3) for I h, and embedded in paraffin through the usual histological procedures. Sections of 4 # thickness were mounted on glass slides previously coated with gelatin and dewaxed. Autoradiograms were prepared with Kodak AR-IO stripping-film (exposure time 2-1o weeks). Hematoxylin and eosin staining was employed after autoradiography. The number of grains was counted separately over nucleolus, chromatin (minus nucleolus associated chromatin) and cytoplasm. Since it was impossible to recognize the cell boundary clearly, a micrometer disc was used (which had 400 equal squares, each encompassing an 8.6 × 8 . 6 # area in the microscope used) to Biochim. Biophys. Acta, 95 (1965) 262-279
274 TABLE
w.
WEIA.IN(;
e[ ~l[,.
ii
LABELING DIFFERENT
INDEN
OF t t E P A T O C Y T E
NUCLEI
AND NUCLEOLI
AFTER INJECTION
OF
iI3HIcYTIDINE
Ar
TIMES AFTER PARTIAL HEPATECTOMY
Time (h) a/ter operation (regeneration time)
Time (h) between Exposure isotope injection time o/ and death autoradiograph (incubation time) (~eeks)
Percentage labeled hepatocyle n,clei
Percentage labeled hepatocyte m*eleoli
6 6 6 12 15 15 18 18
0.25 2 2 3 0-5 3 0.25 0.25
io 4 8 4 4 4 1.5 4
34 7° 82 80 95 79 90 91
33 75 75 42 81 65 88 85
18 18 21 21 24 24
0.25 2 0.5 3 0.5 3
8 4 4 4 4 4
83 58 9I 7° 94 88
89 5I 77 44 91 80
determine cytoplasmic labeling. The number of squares (unit area) with I, 2, 3 etc. grains was counted over cytoplasm and compared with those over cell-free areas (background).
RESULTS
AND DISCUSSION
In one experiment the mitotic activity at 22-32 h after partial hepatectomy was investigated b y counting at least 5000 hepatocytes per time interval (Fig. 6). 22 h after operation the mitotic index had increased above the normal level (in resting liver the mitotic index was less than one division per 2000 cells). Peak values were obtained 28-30 h after operation, which is in close agreement with findings of CATER et al. ~. [aHlCytidine is incorporated into both RNA and DNA. In this system DNA synthesis starts at about 18 h after partial hepatectomy 4& I h after injection of tritiated thymidine, 0.08 Yo of the hepatocyte nuclei were labeled when injection was carried out 6 h after operation; this value was 0.22 O//o 18 h after operation (Table III). These results indicate that [3Hlcytidine was only incorporated into RNA, at least when regeneration periods up to 18 h were employed. ~sH~Cytidine injection caused a rapid and heavy labeling located over the nucleoli especially 15-24 h after operation and during short incubation periods (0.25 or 0.5 h). By lengthening this time between injection and killing a decrease of nucleolus labeling was observed in most of the regeneration times investigated. This decrease was reflected in the percentage of labeled nucleoli after long incubation periods (2 or 3 h) compared with short periods (Table II). For instance, 15 rain after injection at 18 h after operation, in three separate slides 85, 88 and 89 % of the nucleoli were labeled. 2 h after injection only 51 °/o were scored as labeled nucleoli. Biochim. Biophys. Acta, 9 5 ( 1 9 6 5 ) 2 6 2 2 7 9
RNA
SYNTHESIS
IN R E G E N E R A T I N G
275
RAT LIVER
&O-
o
35-
o •~
lO-
i
E 20--
Tetophase Anaphase Metaphase Prometaphase
10~
Late prophase
5-
Earty prophase I
I
hours after partiat hepatectorny F i g . 6. T h e m i t o t i c i n d e x a t d i f f e r e n t i n t e r v a l s of t i m e a f t e r p a r t i a l h e p a t e c t o m y . divided into 6 phases.
TABLE
Mitosis was
III
LABELING INDEX OF HEPATOCYTE NUCLEI AFTER INJECTION OF [aHJTHYMIDINE AT DIFFERENT TIMES AFTER PARTIAL HEPATECTOMY
Regeneration period (h)
Incubation period (h)
Exposure time o/ autoradiograph (weehs)
Percentage labeled hepatocytes
6 18
i I
3 3
0.08 0.22
This decrease was less 24 h after operation, but this m a y have been caused b y incorporation of label into DNA being synthesized at that moment. The decrease of nucleolus labeling found after longer incubation times is also shown by the number of grains above the nucleoli. In Fig. 7 tile number of grains per nucleolus was plotted against the number of nucleoli in a cumulative way. Short incubation times of 0.25 or 0.5 h resulted in a higher grain count when injection was carried out 18 h (Fig. 7 b) and 24 h (Fig. 7c) after partial hepatectomy. The opposite was true 6 h after operation (Fig. 7a). At this time the nucleoli were more heavily labeled during the longer incubation period, which is also reflected in the percentage of labeled nucleoli (Table II). This difference m a y be caused b y a slower rate of RNA synthesis 6 h after operation, and at the same time a slower rate of transport of nuclear R N A to the cytoplasm. An indication of this transport was obtained by comparing the labeling pattern of the nucleoli with that of the cytoplasm. In Fig. 8 the number of grains per unit area (see M E T H O D S ) w a s Biochim. Biophys. Acta,
95 (1965) 262-279
276
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number of grains per nucleotus
Fig. 7" Labeling index of nucleoli after injection of [3H]cytidine at different intervals of time after partial hepatectomy (regeneration time), and different intervals of time between injection and death (incubation time), x - × , "short" incubation time, O- - -O, "long" incubation time.
plotted against the area in a cumulative way. This figure represents the results based on the same slides as those given in Fig. 7. After short incubation periods the cytoplasm was not, or only very slightly, labeled. By lengthening this period an increase in cytoplasmic labeling was observed (the curves shift to the right). From Figs. 7 and 8 it is evident that a decrease in nucleolar labeling coincided with an increase in cytoplasmic labeling. This labeling pattern is explained by assuming a transport of nuclear (possibly nucleolar) RNA to the cytoplasm. However, the possibility of a nucleus-independent RNA synthesis in the cytoplasm with a very slow rate 35 cannot be ruled out. Since only one rat per observation time was used in these experiments, the present results have only qualitative significance. Quantitative interpretations will require more extensive observations. These autoradiographical results do not permit a distinction between the different types of RNA which have caused the observed labeling pattern. The present experiments indicate that the nucleolus (and perhaps the nucleolus-associated chromatin) plays an important role in the metabolism of nuclear RNA; they also support the findings of other investigators11,86, sT.
Relation between 3iochemical and autoradiographic results The labeling results of the biochemical and autoradiographic studies may provide some information on the transport of the different types of I~NA from the Biochim. Biophys. Acta, 95 (1965) 262-279
RNA SYNTHESIS IN REGENERATING RAT LIVER A
~ too4
regeneration time: 6 h
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~ ~ .~ ._~c
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Fig. 8. Labeling of cytoplasm after injection of [3Hlcytidine, using different regeneration and incubation times. Unit area: see METHODS. O " " - O, grains over tissue-free areas (background), x - X , grains over cytoplasmic areas.
nucleus to the cytoplasm. However, in trying to interpret these combined results it should be kept in mind that biochemical data were obtained with 32P-labeled phosphate, and autoradiographic data with [~H]cytidine; these substances may be incorporated at different rates. In regenerating liver 24 h after hepatectomy, biochemical investigations showed no labeling of cytoplasmic ribosomal RNA at a 45-min incubation time (Fig. 3A). Intermediate and soluble RNA fractions, however, were labeled. Thus the labeling found with the autoradiographic technique in the cytoplasm at 30 min incubation time must be due to intermediate and s-RNA. After longer incubation Biochim. Biophys. Acta, 95 (1965) 262-279
278
w. WELLING el al..
times the increase in RNA labeling observed in the cytoplasm autoradiographically (Fig. 8C) is probably due to transport of ribosomal RNA from the nucleus to the cytoplasm, since the increase of labeling in the cytoplasm is principally caused by an increase in ribosomal R N A (Fig. 3A and B). At 18 h after partial hepatectomy a combined interpretation of the biochemical and autoradiographic results indicates that the first labeling of cytoplasmic RNA occurs between 15 and 60 min after injection of the radioisotope (Figs. 4-A and 8B). This RNA is exclusively i-RNA and s-RNA (Fig. 4A). The increase of the isotope at two hours after labeling in the cytoplasmic RNA found b y autoradiography (Fig. 8B) must have been caused by all three types of [RNA (Fig. 4B). The appearance of labeled ribosomal cytoplasmic RNA occurs between I and 2 h after injection of the isotope. The incorporation of the labeled compounds at a short time after hepatectomy was studied biochemically at 4 h, and autoradiographically at 6 h, after operation. In view of the publication of HOPE McARDLE AND CREASER38, who found a decrease in the incorporated activity between 4 and 8 h after hepatectomy, a comparison of our autoradiographie and biochemical data at a short interval after hepatectomy seems too difficult. The autoradiographically observed decrease in nuclear, and increase in cytoplasmic, labeling after 24 h regeneration time and longer incubation periods --biochemically shown to be due to transfer of ribosomal R N A from the nucleus to the cytoplasm -- is in good agreement with ~PERRY'Sfindings with L cell fibroblasts in tissue culture. He also inferred that the cytoplasmic autoradiographically demonstrable RNA observed 4 h after labeling corresponded for 75-80 O//oto ribosomal RNA. However, our observations indicate that under our conditions the transport of r-RNA from the nucleus to the cytoplasm is preceded by a transport of i-RNA and s-RNA. At least one of these compounds will be detected autoradiographically at the shorter incubation times.
REFERENCES I N. A. ELIASSON, E. HAMMERSTEN, P. REICHARD, S. ~kQUIST, g . THORELL AND G. EHRENSVARD, Acta Chem. Scan&, 5 (1951 ) 431. 2 R. M. JOHNSON AND S. ALBERT, Arch. Biochem. Biophys., 35 (1952) 34 °. 3 A. NOVlKOFF AND V. R. POTTER, J. Biol. Chem., 173 (1948 ) 223. 4 0 . I~YGAARD AND H. P. RUSCH, Cancer Res., 15 (1955) 24o5 L. I. HECHT AND V. R. POTTER, Cancer Res., 16 (1956) 988. 6 ]). B. CATER, B. E. HOLMES AND L. K. MEE, Acta Radiol., 46 (1956) 655. 7 E. P. ANDERSON AND S. AQUIST, Acta Chem. Scan&, io (1956) 1576. 8 D. J. HOLBROOK, J. H. EVANS AND J. ~¢V. IRVIN, Exptl. Cell Res., 28 (1962) i2o. 9 W. WELLING AND J. A. COHEN, Bioehim. Biophys. Aeta, 42 (196o) 181. IO S. BUSCH, P. CHAMBON, P. MANDEL AND J. D. WEILL, Bioehem. Biophys. Res. Commun., 7 (1962) 255. I i R. P. PERRY, Exptl. Cell Res., 20 (196o) 216. 12 R. P. PERRY, M. ERRERA, A. HELL AND H. DORWALD, J. Biophys. Biochem. Cytol., I I (1961) 1. 13 R. P. PERRY, A. HELL AND M. ERRERA, Biochim. Biophys. Acta, 49 (1961) 47. 14 R. P. PERRY, Proe. Natl. Acad. Sci. U.S., 48 (1962) 2179. 15 G. M. HIGGINS AND R. M. ANDERSON, Arch. Pathol., 12 (1931) 186. 16 G. P. GEORGIEV, L. P. ERMOLAEVA AND I. B. ZHARSKII, Biochimia, 25 (196o) 318. 17 M. B. SPURN AND W. DINGMAN, Biochim. Biophys. Acta, 68 (1963) 387 . 18 K. BURTON, Biochem. J., 62 (1956) 315 . 19 R. K. MAIN, 2V[. J. WILKINS AND L. J. COLE, J. A~n. Chem. Sue., 81 (1959) 6490.
Biochim. Biophys. Acta, 95 (1965) 262 279
RNA 20 21 22 23 24 25 26 27 28 29 3o 3I 32 33 34 35 36 37 38
SYNTHESIS IN REGENERATING RAT LIVER
279
A. T, H. BURNESS AND A. D. VIsozo, Biochim. Biophys. Acta, 49 (1961) 225. M. TAKAI, M. KONDO AND S. OSAWA, Biochim. Biophys. Acta, 55 (1962) 416. L. PHILIPSON, J. Gen. Physiol., 44 (1961) 899. M. REVEL, M. DELEMAN AND P. MANDEL, Biochim. Biophys. Aeta, 68 (1963) 547. G. P. GEORGIEV AND O. P. SAMARINA,Biochimia, 26 (1961) 454. R. MONIER, Biochim. Biophys. Acta, 55 (1962) i o o i . P. A. MARKS, C. WILSON, J. KRUH AND F. GROS, Biochem. Biophys. Res. Commun., 8 (1962) 9. A. J. MUNRO AND A. KORNER, Biochem. J,, 85 (1962) 37 p. A. J. MUNRO AND A. KORNER, Nature, 2Ol (1964) 1194. N. LANG AND C. E. SNKERIS, Li/e Sci., 3 (1964) 161. A. DI GIROLAMO, E. C, HENSHAW AND H. H. HIATT, J. Mol. Biol., 8 (1964) 479. F. Gl~os, H. H. HIATT, W. GILBERT, C. G. KURLAND, l~. W. I~ISEBROUGH AND J. D. WATSON, Nature, 19° (1961) 581. K. SCHERRER AND J. E. DARNELL, Biochem. Biophys. Res. Commun., 7 (1962) 486. H. H. HIATT, jr. Mol. Biol., 5 (1962) 217' G. P. GEORGIEV AND V. L. MANTIEVA, Biochim. Biophys. Acta, 61 (1962) 153. H. HARRIS AND L. V. LACOUR, Nature, 200 (1963) 227. M. AMANO AND C. P. LEBLOND, Exptl. Cell Res., 20 (196o) 25 o. L. E. FEINENDEGEN, V. P. BOND, W. W. SCHREEVE, R. B. PAINTER, Exptl. Cell Res., I9 (196o) 443. A. HOPE MCARDLE AND E. H. CREASER, Biochim. Biophys. Acta, 68 (1963) 561.
Biochim. Biophys. Acta, 95 (1965) 262-279
BIOCHIMICA ET BIOPHYSI(A ACTA
BBA 95135
S Y N T H E S I S OF R I B O N U C L E I C ACID IN R E G E N E R A T I N G RAT L I V E R AFTER PARTIAL HEPATECTOMY
W. W E L L I N G ' ,
D. B O O T S M A , E. V A N M U I S W I N K E L
AND C. A. P. B E R G H E G E N
Medical Biological Laboratory o/ the National De/enee Research Organization T.N.O., Rijswijk (The Netherlands) (Received, J u l y I3th, 1964)
SUMMARY
A technique for the fractionation of R N A by column chromatography on calcium phosphate is described. With this technique the incorporation of labeled inorganic phosphate into RNA of regenerating rat liver was studied. In the cytoplasm a R N A fraction was found, with molecular weight intermediate between that of soluble and microsomal RNA, that had a very high incorporation rate. This fraction occurred only in small amounts in the cytoplasm. In the nucleus relatively more of this type of RNA was present but there the specific activity did not differ from that of nuclear ribosomal RNA. Determination of the relative amount of label in the four R N A nucleotides showed that the fraction with intermediate molecular weight is not a breakdown product of ribosomal RNA. [sH]Cytidine incorporation in the nucleolar regions of the hepatocyte nucleus was demonstrated with autoradiography. Lengthening the period between injection of the isotope and death of the animal led to a decrease of nucleolar labeling coupled with an increase in cytoplasmic labeling. These results support earlier observations, indicating the important role of the nucleus in the synthesis of cytoplasmic RNA.
INTRODUCTION
I t has long been known that the synthesis of R N A in regenerating rat liver is considerably enhanced as compared with normal resting liver 1-4. This increase occurs before the onset of DNA synthesis, which starts at about 18 h after partial hepatectomy, reaches a m a x i m u m at about 24 h after operation 4,5 and is followed by an intensive activity of mitosis some hours later 6. Several investigators have found that the incorporation rate of labeled R N A A b b r e v i a t i o n s : i - R N A , i n t e r m e d i a t e R N A ; r - R N A , r i b o s o m a l R N A ; s - R N A , soluble RNA. * P r e s e n t address: L a b o r a t o r y for R e s e a r c h on Insecticides, 22 P r i n s e s Marijkeweg, W a g e n i n g e n (the N e t h e r l a n d s ) .
Biochim. Biophys. Acta, 95 (1965) 262 279
RNA
SYNTHESIS IN REGENERATING RAT LIVER
263
precursors reaches a m a x i m u m within 2o h after the operation, i.e. when DNA synthesis 7-9 is on a lower level. The activity in vitro of the enzyme RNA polymerase (EC 2.7.7.6 ) of the nuclei of regenerating rat liver closely parallels that incorporation rate 10. I t is now generally accepted that in mammalian cells R N A is exclusively synthesized in the cell nucleus. Much less clear however is the role of the cytologicallydistinct parts of the nucleus in that process. On the strength of the elegant investigations of PERRY et al. 11-14 it seems to be most probable that the nucleolus and the extra-nucleolar part of the nucleus are largely independent from each other with respect to RNA synthesis, r-RNA is synthesized in the nucleolus, s-RNA on the chromatin. In this study we report the results of a number of experiments that were performed to obtain a better insight into the pattern of RNA synthesis in regenerating rat liver. During the course of this work other investigators published their results on the same subject. A comparison of all data is given in the DISCUSSION. BIOCHEMICAL
STUDIES
METHODS
The separation of the various R N A fractions was based on their localization in the cell (i.e. nuclear and cytoplasmic RNA) and the variation in their molecular weight (by column chromatography on calcium phosphate). The general outline of the experiments was as follows. Male Glaxo rats (16o-18o g) were partially hepatectomized as described by HIGGINS AND ANDERSON15. At different intervals after operation the animals received an intravenous injection of inorganic radioactive phosphate (300 or 6oo/zC for each animal), and 45 min to 2 h later the animals were killed by decapitation, the remaining liver lobes were excised and immediately cooled in ice-cold 0.25 M sucrose. Tissue [ractionation
The liver residues of 8 rats were pooled and homogenized in o.25 M sucrose, 5" lO-3 M MgC12, 0.025 M KC1, 0.05 M Tris buffer (pH 7.6), first in a Potter-Elvehjem type homogenizer with Teflon pestle, and, after filtration through IOO mesh nylon tissue, in a stainless-steel Dounce type homogenizer with a clearance between ball and wall of o.09 ram. The homogenate was then centrifuged for 15 min at 800 ×g. The supernatant fluid was pipetted off by aspiration and set aside for the preparation of cytoplasmic RNA. The sediment was suspended in IOO ml concentrated sucrose solution-- (58 % by weight) buffered with 0.05 M glycerophosphatO 6 (pH 6.6) - - a n d centrifuged for 20 rain at 20 ooo×g. The resulting nuclear pellet was washed by resuspension in concentrated sucrose solution and centrifugation for 30 min at the speed given above. Microscopal inspection of the isolated nuclei showed the absence of whole cells and erythrocytes; the preparation was still contaminated b y a small amount of mitochondria. The RNA/DNA-ratio was o.16. This technique for the isolation of Biochim. Biophys. Acla, 95 (1965) 262-279
264
\v. WELLING e[ aZ.
nuclei bears a strong resemblance to that described by SPORN AND I)INGMAN 17, published while our experiments were in progress.
RNA isolation Nuclear RNA. The nuclear pellet was suspended in I. IO-3 M MgC12, o.o5 M Tris buffer (pH 7.6) and incubated with sodium laurylsulfate (i % final concentration) for 15 min at 37 °. The nuclei were lysed by this treatment and gave a very viscous solution. An equal volume of freshly-redistilled water-saturated phenol was then added, and the mixture was shaken in the cold room for 0.5 h, followed b y centrifugation at high speed to separate the phenol and water layers. The phenol layer was extracted with an equal volume of the buffer-sodium larurylsulfate mixture, and the combined water layers were reextracted with half a volume of phenol. The nucleic acids in the water layer were precipitated b y the addition of two volumes of cold ethanol and o.I volume 20 o/ ,,o sodium acetate and stored overnight at --25 ° . The precipitate was washed twice with cold o.14 M NaC1 in 67 o/,,oethanol and suspended in 0.05 M Tris buffer (pH 6.6) containing lO -3 M MgCI~. DNA was broken down with deoxyribonuclease (EC 3.1.4.5) (2 × recrystallized) at a concentration of 5 #g/ml for 30 min at 37 °. Material not dissolved at this step was centrifuged down and again treated with the buffer-deoxyribonuclease mixture. The clear supernatants were combined, and nucleic acids were precipitated with two volumes of cold ethanol in the presence of 2 % sodium acetate. DNA-breakdown products coprecipitate with nuclear R N A and cannot be removed by repeated reprecipitation. However, it proved possible to separate these contaminants b y gel filtration through Sephadex G-5o. The sediment from tile last alcohol precipitation step was to that end dissolved in a small amount of 0.005 M phosphate buffer (pH 6.7) applied to a column 40 cm high and 3 cm diameter of gel filtration material in equilibrium with the same buffer and ehited at a rate of about 12 ml/h. An example of tile elution pattern is given in Fig. I, from which it appears that RNA moved as a rather narrow zone through the column, followed by a heterogeneous mixture of DNA breakdown products. The combined R N A fractions of the first peak contained about 1 % DNA-reacting material (DNA reaction as described by BURTONI8).
In this way an amount of nuclear R N A varying from 2-6 mg/5o g liver tissue was isolated. Cytoplasmic RNA. Cytoplasmic RNA was prepared from the supernatant of the first low speed centrifugation (see above). Mitochondria were removed b y centrifugation at I2 ooo x g for IO rain. The deproteinization with phenol was carried out as described for nuclear R N A in the presence of 1 % sodium laurylsulfate, with the exception that residual phenol in the water layer was removed b y extraction (3 x ) with ether. The material precipitated with alcohol was washed twice with cold o.14 M NaC1 in 67 % ethanol, and dissolved in 0.005 M phosphate buffer (pH 6.7). This produced a strongly opalescent solution, caused b y glycogen. The solution was Biochim. 19iophys. Acta, 95 (1965) 262 279
RNA Z.e-
SYNTHESIS IN REGENERATING RAT LIVER
265
RNA
~k2. 0-
=o 1.6-
o.e-
o.~-
fraction number
Fig. I. Separation of nuclear 1RNA from DNA breakdown products by gel filtration through Sephadex G-5o. Rat-liver-nuclear RNA, isolated with the phenol procedure and digested with deoxyribonuclease as described in the text, was dissolved in o.o0 5 M phosphate buffer (pH 6.7) applied to a column (3 × 4° cm) of Sephadex G-5 o, equilibrated with the same phosphate buffer, and washed through with t h a t buffer at a rate of 12 ml/h.
cleared b y centrifugation at lO 5 ooo ×g for 3o min. Glycogen gives a colorless pellet at the bottom of the tube. Radioactive R N A was dialyzed overnight against 0.005 M phosphate buffer (pH 6.7).
Chromatography on calcium phosphate Calcium phosphate for the column chromatography of RNA was prepared as described by MAIN, V~ILKINS AND COLE19. The modification denoted as counts/min b y these authors was used in all experiments because it permits a high flow rate. Before use the calcium phosphate, packed in columns of IO × 1. 4 cm, was washed with I M phosphate buffer (pH 6.7) until no more ultraviolet-absorbing material could be eluted; then it was reconditioned b y washing with a large amount of 0.005 M phosphate buffer (pH 6.7). R N A dissolved in 0.005 M phosphate buffer (pH 6.7) was adsorbed on the top layer of the column. The maximal amount of RNA used was 4 mg with columns of 1. 4 cm diameter. Elution was carried out with a phosphate buffer gradient (pH 6.7) prepared by adding o.15 M buffer to a closed mixing vessel of 250 ml entirely filled with 0.07 M buffer. Fractions of 4 ml were collected. After 60 fractions had been collected the concentration of the buffer added to the mixing vessel was raised to 0.3 M to elute high molecular weight RNA. The flow rate was 18 ml/h. The concentration of RNA in the fractions was determined by ultraviolet absorbancy at 260 my. With nuclear RNA, when little material was available the scale of the experiments was reduced to one quarter of the given dimensions. Biochim. Biophys. Acta, 95 (1965) 262-279
266
w . WELI_IN(; el al.
Determination o/ the ratio o~ 32p in the/our nucleotides o/ R N A Selected fractions from the column (see under RESULTS) were pooled and frozen dry. The dry material was dissolved in 3 ml water and dialyzed against water to remove salts. The dialyzed fluid was then brought to o.5 M KOH, and RNA was hydrolyzed by incubation at 37 ° for I8 h. Next the p H of the mixture was decreased to about 7 with o.5 M HC10~. Precipitated KC1Q was spun down and the volume of the supernatant was reduced by freeze-drying. The 3'-nucleotides were separated on paper by high-voltage electrophoresis in pyridine acetate buffer (pH 3.6). The spots of the nucleotides were located by ultraviolet contact photography and counted on the paper with a gasflow counter.
RESULTS AND DISCUSSION
Chromatography on calcium phosphate Fig. 2 shows the elution pattern of cytoplasmic RNA from a calcium phosphate column, Soluble RNA elntes at about 0.07 M phosphate buffer and microsomal RNA at about o.12 M. The identity of two peaks was checked in two ways; first cytoplasmic RNA was freed of soluble RNA by adding an equal volume of 3 M NaC1 and leaving overnight in the refrigerator. Under these conditions only microsomal RNA precipitates. The precipitated material gave only one peak, corresponding with the peak eluting at the higher phosphate buffer concentration in Fig. 2. Soluble RNA was then prepared from a particle-free rat-liver supernatant (centrifuging I h at
/ /./
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fradlonnumber Fig. 2. Column c h r o m a t o g r a p h y of rat-liver-cytoplasmic R N A on calcium p h o s p h a t e . 4 mg RNA, dissolved in o.oo 5 M p h o s p h a t e buffer (pH 6.7) was adsorbed on a column (IO× 1. 4 cm) of calcium p h o s p h a t e and eluted w i t h p h o s p h a t e buffer of increasing strength. After 53 fractions the c o n c e n t r a t i o n of buffer, added to the mixing vessel, was raised to 0. 3 M.
Biochim. Biophys. Acta, 95 (1965) 262 279
RNA SYNTHESIS IN REGENERATING RAT LIVER
267
lO5 ooo ×g). This RNA also gave only one peak corresponding with the first one in Fig. 2. Between 0.07 and o.12 M phosphate buffer the ultraviolet absorption at 260 m# of the eluate did not drop to zero, but small amounts of RNA were continnuously eluting from the column. The identity of this material is not clear at present. Tentatively it will be referred to as i-RNA. The recovery of applied 1RNA from the column was complete. Microsomal RNA gives only one peak, although it has been shown to contain two components with sedimentation coefficients of 16-2o S and 26-30 S. Attempts to resolve the microsomal R N A peak b y using a less steep gradient were not successful. BURNESS AND VISOZO20 claim to have separated 18- and 3o-S r-RNA on calcium phosphate. Inspection of their data however leads us to presume that they have interpreted soluble RNA as I8-S r-RNA. Nuclear RNA gave an elution pattern qualitatively identical with that of cytoplasmic RNA; relatively more material appeared in the i-fraction. It has been reported b y TAKAI et al. 21 that high-molecular weight RNA isolated from Escherichia coli can be separated from the 16 and 23-S component b y chrom a t o g r a p h y on a column of Celite coated with esterified bovine serum albumin. W'e confirmed this observation with E. coli RNA, but rat-liver microsomal RNA behaved differently;this material was eluted as one component from the column,and no separation could be obtained.This is in contrast to a report OfPHILIPSON 22, who describes at least a partial separation of high-molecular weight RNA of HeLa-cells. Using the same technique for the fractionation of nuclear RNA from rat kidney and liver, REVEL et al. ~a mention that it was possible to separate both components only "under the best conditions". However, these authors do not state what these best conditions are.
Incorporation studies o/ asp into rat-liver R N A
When 3ep was injected into animals with a regenerating liver 24 h after operation, and the animals were killed three quarters of an hour later, it was found that cytoplasmic R N A was only weakly labeled. Chromatography of this RNA showed that there were two fractions that had incorporated s~p: s-RNA and i-RNA. Microsomal RNA was unlabeled (Fig. 3A). A large amount of radioactivity in the s-RNA fraction had however to be attributed to 3~p labeled inorganic phosphate, present in the preparation due to insufficient dialysis. Reprecipitation with alcohol reduced the radioactivity in this fraction to about 20 %; we m a y therefore conclude that the specific activity in Fig. 3A was highest in tile i-RNA fraction. Fig. 3B shows that b y extending the incorporation time from 45 min to 2 h microsomal RNA became labeled too. The specific activity however of i-IRNA was greater than that of microsomal RNA. The picture at 18 h after operation shows a clear resemblance to t h a t at 24 h after operation. In Figs. 4 A and 4 B the partition of radioactivity over the three cytoplasmic RNA fractions at one hour is compared with that at 2 h after injection of the label. I h is too short a period for microsomal RNA to become labeled, but Biochim. Biophys. Acta, 95 (1965) 262-279
268
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Fig. 3. Column chromatography on calcium phosphate of 3aP-labeled R N A in vivo, isolated by the phenol procedure Irom regenerating rat liver. In all experiments 300 or 600/*C of an isotonic saline solution of 3~P-labeled phosphate was intravenously injected. - - , extinction at 260 m#; - - - , radioactivity (counts/min). A, Cytoplasmic R N A labeled w i t h 300/*C 8zp from 24-24.75 h after partial hepatectomy; t3, as in A, but labeling from 24-26 h after operation.
s-RNA and i-RNA do so. In both examples i-RNA has the greatest specific activity. For the experiment of Fig. 4 B we used twice-reprecipitated RNA to remove the contamination of s-RNA with asp labeled inorganic phosphate. Comparison of the three radioactive peaks in Fig. 4 B shows that the order of increasing specific activity is microsomal RNA < s-RNA < i-RNA. Biochim. Biophys. Acta, 95 (1965) 262-279
RNA
269
SYNTHESIS IN REGENERATING RAT LIVER
lO *00 --
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Fig. 4- Conditions as in Fig. 3: A, cytoplasmic R N A labeled with 3oo/*C 32p from 18-19 h after operation; B, c y t o p l a s m i c R N A labeled with 60o #C 82p from 18-2o h after partial h e p a t e c t o m y .
There is however one difference between the situations at 18 and 24 h after operation: the radioactive i-RNA fraction at 18 h e l u t e s - r e p r o d u c i b l y - at a lower salt concentration from the column than at 24 h. This points to a difference in molecular weight of both fractions. The appearance of radioactive i-RNA in the cytoplasm is not typical for the 18 and 24-h period after operation. Only 4 h after partial hepatectomy it was already present, as m a y be seen in Fig. 5C. The specific activity of i-RNA was again much higher than that of microsomal RNA. We also studied the incorporation of ~zP into nuclear R N A at the same regeneration time. Figs. 5A and 5 B give the chromaBiochim. Biophys. Acta, 95 (I965) 262-279
27°
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WELLING
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Fig. 5. Conditions as in Fig. 3: A, nuclear IZNA labeled w i t h 600 ffC 32 P from 4-4.5 h after operation B, as in A, b u t t h e labeling period e x t e n d e d to I h; C, c y t o p l a s m i c R N A labeled with 600 # C 3,p from 4 - 6 h after partial h e p a t e c t o m y . Biochim.
Biophys,
A c t a , 95
(1965) 2 6 2 - 2 7 9
RNA SYNTHESIS IN REGENERATING RAT LIVER
271
tographic patterns for this RNA after respective contacts of o.5 and 2 h with 8~p. In contrast to cytoplasmic RNA, nuclear RNA did not show a clear-cut radioactive peak of i-RNA; high-molecular weight RNA and i-RNA had the same specific activity, even after the shorter labeling period. I t seems that the specific activity of lowmolecular weight RNA is somewhat lower than that of the two other fractions. In an experiment analogous to that of Fig. 5A, but 8 h after operation, the same labeling pattern was encountered. I t is not clear what type of RNA the highly labeled i-RNA-fraction in the cytoplasm is. GEORGIEV AND SAMARINA24 found in rat-liver cytoplasm three RNA fractions after 3o-6o min labeling with 82p; one high polymer that was very weakly labeled, one low polymer with a specific activity io times that of the high polymer fraction and a (with phenol) non-extractable one, 2-3 times more strongly labeled than the low polymer fraction. The latter was present only in small amounts. One m a y suppose that this non-extractable fraction corresponds with our i-RNA fraction, assuming that their non-extractable fraction can be solubilized when a detergent (sodium laurylsulfate) is added to the phenol. In m a n y types of mammalian-cells RNA of relatively low molecular weight with high turnover has been reported. MONIER25 finds that after short incubation of hepatome ascites cells with ES-14Clguanine most of the labeled RNA extractable with phenol only has a sedimentation constant in between that of 4- and 2o-S RNA. Extraction however with phenol+detergent gives RNA that is very polydisperse (sedimentation constants ranging from 4-30 S with a peak value between 4 and 20 S). More or less the same situation was encountered by MARKS et al. 2~ with white blood cells and MUNRO AND KORNER27, 2s, LANG AND SEKERIS 29, and DI GIROLAMO et al. a° with rat liver. The three above-mentioned groups of investigators all incline to identify the RNA having high turnover with messenger RNA, shown to have sedimentation constants of 8-12 S by GROS et al. 31 for E . coll. These low sedimentation constants for messenger IRNA, however, were questioned b y TAKAI et al. 21, who put forward the idea that these low values pointed to a considerable breakdown of this messenger RNA by cellular RNAase (EC 2.7.7.16), not inactivated by phenol. However, when the material was carefully isolated, four peaks of rapidly-labeled RNA could be shown b y chromatography on a esterified albumin-coated Celite column, characterized b y sedimentation constants of 8, 12, 19 and 26 S and a nucleotide composition strongly resembling that of E . coli DNA. The objection that low values for the sedimentation constants of messenger RNA are an indication of breakdown does not hold true for those experiments in which Bentonite was used in the isolation of RNA26m. The results of DI GIROLAMO et al. a° and LANG AND SEKERIS29 firmly sustain the hypothesis concerning the possible messenger character of rapidly-labeled low-molecular weight RNA. They showed that in rat-liver-cytoplasmic RNA the fractions with the highest specific stimulating activity towards amino acid incorporation had sedimentation constants of 18 S or lower. There is however also rapidly-labeled RNA of high molecular weight present in mammalian cells. SCHERRER AND ~DARNELL32 found two components of this type of R N A with sedimentation constants of about 45 and 33 S. PERRY14, using a combination of autoradiography and sedimentation of isolated RNA in sucrose Biochim. Biophys. Acta, 95 (1965) 262-279
272
W. }Vt~LL1NC~ gt a[.
gradients, was able to demonstrate this high molecular "messenger" RNA after a 3o-min pulse with [3Hlcytidine exclusively in the nucleus of L-cell fibroblasts, and most of it in the nucleolus. This pulse-labeled material was very heterogeneous with sedimentation constants down to 4 S. In rat liver and kidney nuclei rapidly-labeled RNA of high molecular weight was found by HIATT33 and REVEL et al. 2a. Both investigations sustain the idea that this type of RNA is of nucleolar origin. The results of HIATT indicate that the material with the highest sedimentation constants (larger than that of ribosomal RNA) is not transferred to the cytoplasm. This does not speak in favor of a possible messenger role in cytoplasmic protein synthesis, unless we must assume that it breaks down to a limited number of smaller pieces before it reaches the cytoplasm. Comparing our results with those cited above we see that they are analogous in many respects. Just as in the experiments of MONIER 25, MARKS et al. 2n, MUNRO AND KORNER27, 28, LANG AND SEKERIS 29 and DI GIROLAMO et al. 3°, we find a RNA fraction in the cytoplasm characterized by a high incorporation rate of radioactive precursor and a molecular weight between those of s-RNA and microsomal RNA. The latter has the greatest metabolic stability. In the nuclei there is much less variation in the specific activity of the three R N A fractions than in the cytoplasm. This confirms the observations of GEORGIEV AND SAMARINA24. \Ve have no evidence for a rapidly-labeled R N A with sedimentation constants larger than that of ribosomal R N A in the nuclei, as was found by othersl4,~3,32,~'~. An explanation for this discrepancy cannot be given, but it may be that it is not possible to distinguish between ribosomal and higher molecular weight RNA by chromatography on calcium phosphate.
Relative amount o[ 32p in R N A nucleotides To exclude definitely the possibility that i-RNA is a breakdown product of microsomal RNA we determined the relative amounts of 32p in each of the four R N A nucleotides in some experiments. The results are given in Table I. The relative amounts of radioactivity are in general not identical with base ratios; only in the event that the specific activity of tile four direct precursors are the same can one say that the relative amount of radioactivity in the nucleotides is identical to the base ratio. The results in the Table clearly show that i-RNA is not a breakdown product of microsomal RNA, because the ratios of guanylic plus cytidylic acid to adenylic plus uridylic acid ( G + C ) / ( A + U ) differ significantly from each other. Moreover i-RNA seems to be heterogeneous in contrast to microsomal RNA. The ( G + C ) / ( A + U ) ratio for ribosomal RNA of rat liver has been reported to be 1.4-1. 5 . Our "radioactive" ratio does not differ very much from that value. Assuming that this means that our "radioactive" base ratio is a good approximation to true base ratio, we see in Table I that none of the studied fractions has a DNAlike base composition (in rat tissue the ( G + C ) / ( A + T ) ratio of DNA is 0.75 ). Such a R N A fraction has been found b y CxEORGIEVAND ~-4.NTIEVA~ in the nucleolus of rat liver. If in mammalian cells too, messenger R N A has a DNA-like base composition, we must conclude that none of the fractions studied is a likely candidate to play Biochim. Biophys. Acta, 95 (1965) 262-279
RNA
273
SYNTHESIS IN REGENERATING RAT LIVER
TABLE I RELATIVE AMOUNTS OF RADIOACTIVITYIN THE NUCLEOTIDES OF SEVERAL mlWA FRACTIONS OBTAINED BY COLUMN CHROMATOGRAPHY OF REGENERATING RAT-LIVER R N A The R1WA nucleotides were o b t a i n e d b y alkaline hydrolysis of pooled c o l u m n fractions and sepa r a t e d b y high-voltage electrophoresis. I n the c o l u m n denoted RN,4 /raction are represented consecutively, the t i m e of injection of the label in h o u r s after operation, the origin of the R N A fraction (i.e. nuclear or cytoplasmic), the time in h o u r s b e t w e e n injection and decapitation of the a n i m a l and t h e t y p e of R N A fraction (i.e. soluble, i n t e r m e d i a t e or ribosomal). I n some determ i n a t i o n s the c o l u m n fractions were pooled in such a m a n n e r t h a t the c o m b i n e d material represented the first a n d second half of the respective R N A peak. C, A, G and U denote cytidylic, adenylic, guanylic and uridylic acids respectively.
RNA #action
C
4 N 0. 5 S 4N o.5I 4 N 0. 5 R 4 N 2 R1 4 N 2 IRz 81N 0. 5 I 1 8 IW 0. 5 I s 8 N 0. 5 tZ 18C 2 I
21.5 24.1 25.9 26. 5 26.0 23. 9 25.0 25.6 27.3
A
32.6 24. 5 23.2 20.6 20. 7 22. 7 19.9 19.2 21.5
G
18.2 27. 3 31.1 32. 4 33.6 30.3 32.5 35.6 31.5
U
27.8 24.1 19.9 20. 5 19.6 23.1 22.7 19.2 19.6
(C+G)/(A + U) Mean value
Individual determinations *
o.66 1.o6 1.32 1.44 1.48 1.19 1.35 1.58 1.44
o.66 1.o6, 1.32, 1.43 , 1.48, 1.16, 1.34, 1.54, 1.42,
1.o6 1.32, 1.32 1.44 1.48 1.22 1.36 1.62 1.45
" D e t e r m i n a t i o n s were performed on the same material.
that role, unless it forms part of the heterogeneous i-RNA fraction and its nucleotide composition is obscured by other fractions having a higher ( G + C ) / ( A + U ) value.
AUTORADIOGRAPHIC STUDIES METHODS
A few experiments were carried out to study the sequences of events after partial hepatectomy with autoradiography. At different intervals after operation (designated as regeneration time) rats were injected intravenously with either 50 #C [SH~cytidine (Schwarz Bio Research, specific activity I C/mmole) or 50 #C [SH]thymidine (Schwarz Bio Research, specific activity 0.36 C/mmole) in 0.5 ml saline. Animals were killed 0.25, 0.5, I, 2 or 3 h later by decapitation (for details see Table II.) Time between injection and killing is designated as incubation time. The remaining liver lobes were excised and fixed in acetic acid-ethanol (I :3) for I h, and embedded in paraffin through the usual histological procedures. Sections of 4 # thickness were mounted on glass slides previously coated with gelatin and dewaxed. Autoradiograms were prepared with Kodak AR-IO stripping-film (exposure time 2-1o weeks). Hematoxylin and eosin staining was employed after autoradiography. The number of grains was counted separately over nucleolus, chromatin (minus nucleolus associated chromatin) and cytoplasm. Since it was impossible to recognize the cell boundary clearly, a micrometer disc was used (which had 400 equal squares, each encompassing an 8.6 × 8 . 6 # area in the microscope used) to Biochim. Biophys. Acta, 95 (1965) 262-279
274 TABLE
w.
WEIA.IN(;
e[ ~l[,.
ii
LABELING DIFFERENT
INDEN
OF t t E P A T O C Y T E
NUCLEI
AND NUCLEOLI
AFTER INJECTION
OF
iI3HIcYTIDINE
Ar
TIMES AFTER PARTIAL HEPATECTOMY
Time (h) a/ter operation (regeneration time)
Time (h) between Exposure isotope injection time o/ and death autoradiograph (incubation time) (~eeks)
Percentage labeled hepatocyle n,clei
Percentage labeled hepatocyte m*eleoli
6 6 6 12 15 15 18 18
0.25 2 2 3 0-5 3 0.25 0.25
io 4 8 4 4 4 1.5 4
34 7° 82 80 95 79 90 91
33 75 75 42 81 65 88 85
18 18 21 21 24 24
0.25 2 0.5 3 0.5 3
8 4 4 4 4 4
83 58 9I 7° 94 88
89 5I 77 44 91 80
determine cytoplasmic labeling. The number of squares (unit area) with I, 2, 3 etc. grains was counted over cytoplasm and compared with those over cell-free areas (background).
RESULTS
AND DISCUSSION
In one experiment the mitotic activity at 22-32 h after partial hepatectomy was investigated b y counting at least 5000 hepatocytes per time interval (Fig. 6). 22 h after operation the mitotic index had increased above the normal level (in resting liver the mitotic index was less than one division per 2000 cells). Peak values were obtained 28-30 h after operation, which is in close agreement with findings of CATER et al. ~. [aHlCytidine is incorporated into both RNA and DNA. In this system DNA synthesis starts at about 18 h after partial hepatectomy 4& I h after injection of tritiated thymidine, 0.08 Yo of the hepatocyte nuclei were labeled when injection was carried out 6 h after operation; this value was 0.22 O//o 18 h after operation (Table III). These results indicate that [3Hlcytidine was only incorporated into RNA, at least when regeneration periods up to 18 h were employed. ~sH~Cytidine injection caused a rapid and heavy labeling located over the nucleoli especially 15-24 h after operation and during short incubation periods (0.25 or 0.5 h). By lengthening this time between injection and killing a decrease of nucleolus labeling was observed in most of the regeneration times investigated. This decrease was reflected in the percentage of labeled nucleoli after long incubation periods (2 or 3 h) compared with short periods (Table II). For instance, 15 rain after injection at 18 h after operation, in three separate slides 85, 88 and 89 % of the nucleoli were labeled. 2 h after injection only 51 °/o were scored as labeled nucleoli. Biochim. Biophys. Acta, 9 5 ( 1 9 6 5 ) 2 6 2 2 7 9
RNA
SYNTHESIS
IN R E G E N E R A T I N G
275
RAT LIVER
&O-
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hours after partiat hepatectorny F i g . 6. T h e m i t o t i c i n d e x a t d i f f e r e n t i n t e r v a l s of t i m e a f t e r p a r t i a l h e p a t e c t o m y . divided into 6 phases.
TABLE
Mitosis was
III
LABELING INDEX OF HEPATOCYTE NUCLEI AFTER INJECTION OF [aHJTHYMIDINE AT DIFFERENT TIMES AFTER PARTIAL HEPATECTOMY
Regeneration period (h)
Incubation period (h)
Exposure time o/ autoradiograph (weehs)
Percentage labeled hepatocytes
6 18
i I
3 3
0.08 0.22
This decrease was less 24 h after operation, but this m a y have been caused b y incorporation of label into DNA being synthesized at that moment. The decrease of nucleolus labeling found after longer incubation times is also shown by the number of grains above the nucleoli. In Fig. 7 tile number of grains per nucleolus was plotted against the number of nucleoli in a cumulative way. Short incubation times of 0.25 or 0.5 h resulted in a higher grain count when injection was carried out 18 h (Fig. 7 b) and 24 h (Fig. 7c) after partial hepatectomy. The opposite was true 6 h after operation (Fig. 7a). At this time the nucleoli were more heavily labeled during the longer incubation period, which is also reflected in the percentage of labeled nucleoli (Table II). This difference m a y be caused b y a slower rate of RNA synthesis 6 h after operation, and at the same time a slower rate of transport of nuclear R N A to the cytoplasm. An indication of this transport was obtained by comparing the labeling pattern of the nucleoli with that of the cytoplasm. In Fig. 8 the number of grains per unit area (see M E T H O D S ) w a s Biochim. Biophys. Acta,
95 (1965) 262-279
276
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Fig. 7" Labeling index of nucleoli after injection of [3H]cytidine at different intervals of time after partial hepatectomy (regeneration time), and different intervals of time between injection and death (incubation time), x - × , "short" incubation time, O- - -O, "long" incubation time.
plotted against the area in a cumulative way. This figure represents the results based on the same slides as those given in Fig. 7. After short incubation periods the cytoplasm was not, or only very slightly, labeled. By lengthening this period an increase in cytoplasmic labeling was observed (the curves shift to the right). From Figs. 7 and 8 it is evident that a decrease in nucleolar labeling coincided with an increase in cytoplasmic labeling. This labeling pattern is explained by assuming a transport of nuclear (possibly nucleolar) RNA to the cytoplasm. However, the possibility of a nucleus-independent RNA synthesis in the cytoplasm with a very slow rate 35 cannot be ruled out. Since only one rat per observation time was used in these experiments, the present results have only qualitative significance. Quantitative interpretations will require more extensive observations. These autoradiographical results do not permit a distinction between the different types of RNA which have caused the observed labeling pattern. The present experiments indicate that the nucleolus (and perhaps the nucleolus-associated chromatin) plays an important role in the metabolism of nuclear RNA; they also support the findings of other investigators11,86, sT.
Relation between 3iochemical and autoradiographic results The labeling results of the biochemical and autoradiographic studies may provide some information on the transport of the different types of I~NA from the Biochim. Biophys. Acta, 95 (1965) 262-279
RNA SYNTHESIS IN REGENERATING RAT LIVER A
~ too4
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Fig. 8. Labeling of cytoplasm after injection of [3Hlcytidine, using different regeneration and incubation times. Unit area: see METHODS. O " " - O, grains over tissue-free areas (background), x - X , grains over cytoplasmic areas.
nucleus to the cytoplasm. However, in trying to interpret these combined results it should be kept in mind that biochemical data were obtained with 32P-labeled phosphate, and autoradiographic data with [~H]cytidine; these substances may be incorporated at different rates. In regenerating liver 24 h after hepatectomy, biochemical investigations showed no labeling of cytoplasmic ribosomal RNA at a 45-min incubation time (Fig. 3A). Intermediate and soluble RNA fractions, however, were labeled. Thus the labeling found with the autoradiographic technique in the cytoplasm at 30 min incubation time must be due to intermediate and s-RNA. After longer incubation Biochim. Biophys. Acta, 95 (1965) 262-279
278
w. WELLING el al..
times the increase in RNA labeling observed in the cytoplasm autoradiographically (Fig. 8C) is probably due to transport of ribosomal RNA from the nucleus to the cytoplasm, since the increase of labeling in the cytoplasm is principally caused by an increase in ribosomal R N A (Fig. 3A and B). At 18 h after partial hepatectomy a combined interpretation of the biochemical and autoradiographic results indicates that the first labeling of cytoplasmic RNA occurs between 15 and 60 min after injection of the radioisotope (Figs. 4-A and 8B). This RNA is exclusively i-RNA and s-RNA (Fig. 4A). The increase of the isotope at two hours after labeling in the cytoplasmic RNA found b y autoradiography (Fig. 8B) must have been caused by all three types of [RNA (Fig. 4B). The appearance of labeled ribosomal cytoplasmic RNA occurs between I and 2 h after injection of the isotope. The incorporation of the labeled compounds at a short time after hepatectomy was studied biochemically at 4 h, and autoradiographically at 6 h, after operation. In view of the publication of HOPE McARDLE AND CREASER38, who found a decrease in the incorporated activity between 4 and 8 h after hepatectomy, a comparison of our autoradiographie and biochemical data at a short interval after hepatectomy seems too difficult. The autoradiographically observed decrease in nuclear, and increase in cytoplasmic, labeling after 24 h regeneration time and longer incubation periods --biochemically shown to be due to transfer of ribosomal R N A from the nucleus to the cytoplasm -- is in good agreement with ~PERRY'Sfindings with L cell fibroblasts in tissue culture. He also inferred that the cytoplasmic autoradiographically demonstrable RNA observed 4 h after labeling corresponded for 75-80 O//oto ribosomal RNA. However, our observations indicate that under our conditions the transport of r-RNA from the nucleus to the cytoplasm is preceded by a transport of i-RNA and s-RNA. At least one of these compounds will be detected autoradiographically at the shorter incubation times.
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