- Email: [email protected]
Comparison between RNA capable of stimulating amino acid incorporation and early-labelled RNA in rat liver nuclei
195
SHORT COMMUNICATIONS
SC 931 O0
Comparison between R N A capable of stimulating amino acid incorporation and early.labelled R N A in rat liver nuclei In the liver of higher animals, RNA's resembling bacterial messenger RNA 1-3, having a rapid turnover and capacity to stimulate amino acid incorporation in vitro have been demonstrated 4,5. In bacterial systems it was found that an increase in a given protein corresponds to an increase in a particular fraction of pulse-labelled RNAe-9; it has not yet been possible to demonstrate such a correspondence in animal systems. Protein starvation causes a significant modification in the protein population of rat liver 1°-15. The purpose of this work was to discover whether these modifications -the loss or decrease of some proteins and the increase of others -- are due to changing patterns in messenger RNA's synthesis. To test this possibility we studied the activity of the various RNA fractions obtained from liver nuclei of protein-depleted and control rats by stimulating the incorporation of 14C-labelled amino acids in an Escherichia coli cell-free systemL assuming that in this system amino acid incorporation is stimulated only by messenger RNA. Early-labelled RNA was also studied. We then a t t e m p t e d to correlate, in corresponding fractions, the variations in the pattern of stimulatory activity with those in the pattern of pulse-labelling.
;0~"
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0
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2.0
8.
=L E
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o
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o E
10 20 Tube number
30
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10
20
30
Tube number
Fig. i. S e d i m e n t a t i o n p a t t e r n a n d s t i m u l a t o r y a c t i v i t y of nuclear R N A from control (A) a n d protein-depleted r a t s (B). R N A was prepared as described and was separated into fractions b y sucrose g r a d i e n t centrifugation. The groupings shown b y the vertical lines of the columns represent Fractions i t h r o u g h 6; R N A from each of these 6 fractions was precipitated with alcohol, dried, a n d assayed for its capacity to s t i m u l a t e a m i n o acid incorporation into protein. Absorbance a t 26o m/z is indicated b y the curve. T h e h e i g h t of t h e columns r e p r e s e n t s / , / , m o l e s of [14C]valine incorporated per tube. This was calculated b y dividing the total incorporation per fraction b y t h e n u m b e r of tubes pooled to m a k e up t h a t fraction. Only t h e values of the linear p a r t of the curve were considered in calculating t h e a m o u n t of s t i m u l a t i o n for each fraction. Under t h e c o l u m n s the total s t i m u l a t i o n for each individual fraction is reported. The d a t a are expressed as if I m g of R N & was stratified on t h e gradient. Similar results were obtained in three separate experiments. I n all figures the valine values represent incorporation above t h e levels observed in t h e absence of added RNA. W h e n t h e reaction was r u n w i t h o u t adding R N A samples t h e / , / , m o l e s of [z4C]valine incorporated were never more t h a n o.12.
Biochim. Biophys. Acta, IX4 (1966) 195-198
196
SHORT COMMUNICATIONS
Experiments were performed on Wistar male rats (2oo g) divided into two groups and fed the following diets for 30 days: Group I, ad libitum, casein-free diet; Group 2, 20 o/ /o casein diet, pair-fed with rats of Group I. All rats were fasted overnight before decapitation. Purification and fractionation of liver nuclear RNA was carried out as described by DI GIROLAMO, HENSHAWAND HIATTs, RNA was determined by the orcinol procedure is, or, if relatively pure, by its absorption at 260 m# ( I / , g of RNA gives an absorbance of o.030 (ref. I7) ). Protein was measured by the procedure of LOWRY et al. is. In Fig. I, a marked difference between the groups of rats is seen on comparing the stimulation by the different RNA fractions. It is evident that the heaviest fractions are more active in protein-depleted animals, while the lightest are more active in the controls. These results clearly demonstrate a shift in the pattern of messenger RNA following protein starvation. From the graphs of Fig. I a difference in the A~s0ma profile is also evident. In control rats the 28 S/I8 S ratio is always less then in protein-depleted animals. The reasons for the increase in 28 S/I8 S ratio are now under investigation. Fig. 2 shows the sedimentation patterns of early-labelled nuclear RNA in a sucrose density gradient after a Io-min [14C]orotic acid pulse. The main difference in 06
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30
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10
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25
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Fig. 2. S e d i m e n t a t i o n p a t t e r n s of n u c l e a r R N A o b t a i n e d f r o m livers of control (A) a n d p r o t e i n d e p l e t e d r a t s (B). A n i m a l s were sacrificed io m i n following t h e a d m i n i s t r a t i o n of [14C]orotic acid (I o # C p e r i oo g of b o d y wt. ). T h e R N A w a s p r e p a r e d as described. T h e n u m b e r s o n t h e a b s o r b a n c e scale are i n d i c a t e d as if 6 A 2So m# u n i t s of R N A was s t r a t i f i e d o n t h e g r a d i e n t . T r i c h l o r o a c e t i c acidp r e c i p i t a b l e r a d i o a c t i v i t y is i n d i c a t e d b y t h e o p e n circles.
depleted rats is a decrease of the radioactivity in the 18 S- 4 S region and corresponds to the differences of their stimulatory activity in vitro; we have therefore considered these as unstable messenger RNA's*. In order to clarify this result we have performed experiments using actinomycin D. Table I compares, for protein-depleted and control rats, the ratios between * T h e p o s s i b i l i t y t h a t t h e differences o b s e r v e d in t h e p a t t e r n of r a d i o a c t i v i t y a l o n g t h e g r a d i e n t could be p r o d u c e d b y d i f f e r e n t n u c l e a s e a c t i v i t i e s i n t h e two sets of r a t s w a s r u l e d o u t b y t h e following e x p e r i m e n t : D e p l e t e d a n d c o n t r o l r a t s were pulse-labelled w i t h [14C]orotic acid. T o t a l liver h o m o g e n a t e s f r o m d e p l e t e d r a t s were t h e n m i x e d w i t h cold h o m o g e n a t e s f r o m c o n t r o l a n i m a l s a n d vice versa. R N A w a s e x t r a c t e d a n d r a d i o a c t i v i t y w a s m e a s u r e d i n a s u c r o s e g r a d i e n t . T h e differences in t h e p a t t e r n of r a d i o a c t i v i t y f o u n d were as s h o w n in Fig. 2 (A a n d B), a n d are t h e r e f o r e real differences. Biochim. Biophys. Acta, 114 (1966) 195-198
197
SHORT COMMUNICATIONS TABLE I
EFFECT OF ACTINOMYCIN D ON STIMULATORY ACTIVITY OF NUCLEAR ~RNA FROM CONTROL A N D PROTEIN-DEPLETeD RATS R N A was p r e p a r e d f r o m r a t s injected i n t r a p e r i t o n e a l l y w i t h a c t i n o m y c i n D (i.5 m g per kg b o d y wt.) 13 h before sacrifice a n d s e p a r a t e d into 6 fractions, as described in Fig. i. The n u m b e r s are r a t i o s b e t w e e n s t i m u l a t i o n of R N A e x t r a c t e d f r o m liver nuclei of a c t i n o m y c i n D - t r e a t e d r a t s a n d n o n - t r e a t e d rats.
Fractions
Aproteic r a t s Pair-fed r a t s
>28S
28S
<28S >I8S
I8S
4 S
4 S
0.40 0.35
0.38 0.35
0.40 0.40
0.35 0.20
0.30 o.15
0.55 0.25
the total stimulation of each fraction from rats treated with actinomycin D and the stimulation of the corresponding fraction from untreated animals. The results show significant decreases in stimulation b y all fractions. In both groups of animals stimulation decreases most in Fraction 5, which corresponds to an RNA with a sedimentation rate between 18 and 4 S. Moreover, there is less decrease in stimulatory activity in depleted than in control rats. These results confirm the presence in this gradient region of unstable messenger RNA's. These fractions of messenger RNA's are relatively less in protein- depleted rats. In protein-depleted rats, we found an increase in the stimulatory activity in heavy regions of the gradient, but there is no corresponding increase in pulse- labelling. These results are interpreted as reflecting the presence, in heavy regions, of species of messenger RNA's which are more stable; in protein-depleted animals, there is an increase of these messenger RNA's. The presence of these more stable messengers could be accounted for b y two hypotheses: (I) We are really dealing with a large messenger molecule or (2) the apparently large "messenger" molecule is actually an aggregate of messenger RNA strongly bound to some other type of molecule. Since, in the latter case, these messenger RNA's are affected b y nutritional conditions, they must either preexist in vivo, or their large size is an artifact due to physicochemical differences between the RNA's, in vivo, in protein-depleted and control rats. Of the two hypotheses the first seems to be substantiated b y the results of SIDRANSKY, STAEHELIN AND VERNEYTMwho called attention to a shift from lighter to heavier polysomes in rats on a threonine-free diet. This work was partially carried out under the Association Euratom-C.N.R.C.N.E.N., Contract No. o12-61-12 BIAI. We gratefully acknowledge the skilled technical assistance of Mr. E. BUSIELLO. Actinomycin D was a gift from Merck, Sharpe and Dohme.
ln/,'rnational Laboratory o/ Genetics amt l¢iophysics, Naples (Italy) and IslilHIo Nazionale della Nutrizione, /~',,;;t,: (II,dy)
A. DI GIROLAMO M. DI GIROLAMO S. GAETANI M.A. SPADONI Biochim. Biophys. Acta, 114 (1966) 195-198
198
SHORT COMMUNICATIONS
1 F. GRos, H. HIATT, W. GILBER'r, C. C. KURLAND, }{. W. I{ISEBROUGH AND J. D. WATSON, Nature, 19o (1961) 581. 2 M. W. NIREMBERG AND J, H. MATTItAEI, JProe. Natl. Acad. Sci. U.S., 47 (1961) 1588. 3 A. T]SSII~RES AND J. W. HOPKINS, Proc. Natl. Acad. Sci. U.S., 47 (1961) 2oi 5. 4 H. H. HIATT, .]. ]~lol. Biol., 5 ( 1 9 6 2 ) 217. 5 A. DI GIROLAMO, E. C. HENSHAW AND H. H. HIATT, J. Mo!. Biol., 8 (1964) 479. 6 H. H. HIATT, F. GROS AND I~. JACOB, Biochim. Biophys. Acta, 72 (1963) 15. 7 G. ATTARDI, S. NAONO, J. t{OUVIERE, F. JACOB AND F. GROS, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 363 . 8 B. S. GUTTMAN AND A. NOVlCK, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 373. 9 M. HAYASHI, S. SPIEGELMAN, N. FRANKLIN AND S. E. LURIA, Proc. Natl. Acad. Sci. U.S., 49 (I963) 729. Io W. E. KNox, W. H. AUERBACK AND E. C. LIN, Physiol. Rev., 36 (1956) 164. I I J. C. WATERLOW, Federation -Proc., 18 (1959) 1143. I2 H. C. PITOT, VV. IR. POTTER AND H. P. MORRlS, Cancer Res., 21 (1961) i o o i . 13 J. B. ALLISON, R. W. WANNEMACHER, W. L. BANKS, W. H. WUNNER AND R. A. COMEZBRENES, J. Nutrition, 78 (1962) 333. 14 J. N. WILLIAMS, J. Nutrition, 82 (1964) 51 . 15 S. GAETANI, A. M. I)AOLUCCI, M. A. SPADON1 AND G. TOMASS1, J. Nutrition, 84 (1964) 173. I6 H. G. ALBAUM AND W. W. UMBREI% J. Biol. Chem., 167 (I947) 369 . 17 A. B. PARDEE, K. PAIGEN AND L. PRESTIDGE, Biochim. Biophys. Acta, 23 (1957) 162. 18 O. H. LOWRY, INT.J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J . Biol. Chem., 193 (1951) 265 . I9 H. SIDRANSKY, T. STAEHELIN AND E. VERNEY, Science, 146 (1964) 766.
Received April 2oth, 1965 Revised manuscript received August 2nd, 1965
BBA 93106
Patterns of mRNA synthesis during rat-liver carcinogenesis induced by
diethylnitrosamine and thioacetamide Changes in the m R N A patterns on counter-current distribution were observed during isolation of the m R N A - D N A complex from rat livers when rats were fed a diet containing 4'-fluoro-4-dimethylaminoazobenzene 1. The changes were reversible during the first month by removing the carcinogen and refeeding the control diet. After this time the patterns on counter-current distribution became irreversible and a complete change in the pattern had appeared by the time the liver tumour arose. Upon feeding the weakly carcinogenic dye 4-aminoazobenzene ~ the counter-current distribution patterns remained reversible up to 4 months. Since the incorporation of orotic acid had greatly decreased prior and subsequent to tumour induction we have examined 2 different carcinogens to determine how general the change is during the carcinogenic process. We have chosen diethylnitrosamine 3 and thioacetamide 4 since, while these are both hepatocarcinogens, it seemed possible that each m a y have different biochemical reactions within the hepatic cells and hence induce different biochemical lesions. Adult Wistar rats (3 months old) of both sexes were maintained on a 20 % protein diet with normal supplements. In one group a solution of 0.006 % (w/v) of diethylnitrosamine in darkened drinking bottles was supplied in place of drinking water (c/. ref. 3). The diet of the second group was supplemented with thioacetamide (320 mg per kg diet) and a third group received the 20 % protein control diet. Biochim. Biophys. dcta, 114 (I966) 198-2oo
SHORT COMMUNICATIONS
SC 931 O0
Comparison between R N A capable of stimulating amino acid incorporation and early.labelled R N A in rat liver nuclei In the liver of higher animals, RNA's resembling bacterial messenger RNA 1-3, having a rapid turnover and capacity to stimulate amino acid incorporation in vitro have been demonstrated 4,5. In bacterial systems it was found that an increase in a given protein corresponds to an increase in a particular fraction of pulse-labelled RNAe-9; it has not yet been possible to demonstrate such a correspondence in animal systems. Protein starvation causes a significant modification in the protein population of rat liver 1°-15. The purpose of this work was to discover whether these modifications -the loss or decrease of some proteins and the increase of others -- are due to changing patterns in messenger RNA's synthesis. To test this possibility we studied the activity of the various RNA fractions obtained from liver nuclei of protein-depleted and control rats by stimulating the incorporation of 14C-labelled amino acids in an Escherichia coli cell-free systemL assuming that in this system amino acid incorporation is stimulated only by messenger RNA. Early-labelled RNA was also studied. We then a t t e m p t e d to correlate, in corresponding fractions, the variations in the pattern of stimulatory activity with those in the pattern of pulse-labelling.
;0~"
2.0
N!/ 1.0
0
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2.0
8.
=L E
"{
o
0~
I.C
o E
10 20 Tube number
30
o E
10
20
30
Tube number
Fig. i. S e d i m e n t a t i o n p a t t e r n a n d s t i m u l a t o r y a c t i v i t y of nuclear R N A from control (A) a n d protein-depleted r a t s (B). R N A was prepared as described and was separated into fractions b y sucrose g r a d i e n t centrifugation. The groupings shown b y the vertical lines of the columns represent Fractions i t h r o u g h 6; R N A from each of these 6 fractions was precipitated with alcohol, dried, a n d assayed for its capacity to s t i m u l a t e a m i n o acid incorporation into protein. Absorbance a t 26o m/z is indicated b y the curve. T h e h e i g h t of t h e columns r e p r e s e n t s / , / , m o l e s of [14C]valine incorporated per tube. This was calculated b y dividing the total incorporation per fraction b y t h e n u m b e r of tubes pooled to m a k e up t h a t fraction. Only t h e values of the linear p a r t of the curve were considered in calculating t h e a m o u n t of s t i m u l a t i o n for each fraction. Under t h e c o l u m n s the total s t i m u l a t i o n for each individual fraction is reported. The d a t a are expressed as if I m g of R N & was stratified on t h e gradient. Similar results were obtained in three separate experiments. I n all figures the valine values represent incorporation above t h e levels observed in t h e absence of added RNA. W h e n t h e reaction was r u n w i t h o u t adding R N A samples t h e / , / , m o l e s of [z4C]valine incorporated were never more t h a n o.12.
Biochim. Biophys. Acta, IX4 (1966) 195-198
196
SHORT COMMUNICATIONS
Experiments were performed on Wistar male rats (2oo g) divided into two groups and fed the following diets for 30 days: Group I, ad libitum, casein-free diet; Group 2, 20 o/ /o casein diet, pair-fed with rats of Group I. All rats were fasted overnight before decapitation. Purification and fractionation of liver nuclear RNA was carried out as described by DI GIROLAMO, HENSHAWAND HIATTs, RNA was determined by the orcinol procedure is, or, if relatively pure, by its absorption at 260 m# ( I / , g of RNA gives an absorbance of o.030 (ref. I7) ). Protein was measured by the procedure of LOWRY et al. is. In Fig. I, a marked difference between the groups of rats is seen on comparing the stimulation by the different RNA fractions. It is evident that the heaviest fractions are more active in protein-depleted animals, while the lightest are more active in the controls. These results clearly demonstrate a shift in the pattern of messenger RNA following protein starvation. From the graphs of Fig. I a difference in the A~s0ma profile is also evident. In control rats the 28 S/I8 S ratio is always less then in protein-depleted animals. The reasons for the increase in 28 S/I8 S ratio are now under investigation. Fig. 2 shows the sedimentation patterns of early-labelled nuclear RNA in a sucrose density gradient after a Io-min [14C]orotic acid pulse. The main difference in 06
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Fig. 2. S e d i m e n t a t i o n p a t t e r n s of n u c l e a r R N A o b t a i n e d f r o m livers of control (A) a n d p r o t e i n d e p l e t e d r a t s (B). A n i m a l s were sacrificed io m i n following t h e a d m i n i s t r a t i o n of [14C]orotic acid (I o # C p e r i oo g of b o d y wt. ). T h e R N A w a s p r e p a r e d as described. T h e n u m b e r s o n t h e a b s o r b a n c e scale are i n d i c a t e d as if 6 A 2So m# u n i t s of R N A was s t r a t i f i e d o n t h e g r a d i e n t . T r i c h l o r o a c e t i c acidp r e c i p i t a b l e r a d i o a c t i v i t y is i n d i c a t e d b y t h e o p e n circles.
depleted rats is a decrease of the radioactivity in the 18 S- 4 S region and corresponds to the differences of their stimulatory activity in vitro; we have therefore considered these as unstable messenger RNA's*. In order to clarify this result we have performed experiments using actinomycin D. Table I compares, for protein-depleted and control rats, the ratios between * T h e p o s s i b i l i t y t h a t t h e differences o b s e r v e d in t h e p a t t e r n of r a d i o a c t i v i t y a l o n g t h e g r a d i e n t could be p r o d u c e d b y d i f f e r e n t n u c l e a s e a c t i v i t i e s i n t h e two sets of r a t s w a s r u l e d o u t b y t h e following e x p e r i m e n t : D e p l e t e d a n d c o n t r o l r a t s were pulse-labelled w i t h [14C]orotic acid. T o t a l liver h o m o g e n a t e s f r o m d e p l e t e d r a t s were t h e n m i x e d w i t h cold h o m o g e n a t e s f r o m c o n t r o l a n i m a l s a n d vice versa. R N A w a s e x t r a c t e d a n d r a d i o a c t i v i t y w a s m e a s u r e d i n a s u c r o s e g r a d i e n t . T h e differences in t h e p a t t e r n of r a d i o a c t i v i t y f o u n d were as s h o w n in Fig. 2 (A a n d B), a n d are t h e r e f o r e real differences. Biochim. Biophys. Acta, 114 (1966) 195-198
197
SHORT COMMUNICATIONS TABLE I
EFFECT OF ACTINOMYCIN D ON STIMULATORY ACTIVITY OF NUCLEAR ~RNA FROM CONTROL A N D PROTEIN-DEPLETeD RATS R N A was p r e p a r e d f r o m r a t s injected i n t r a p e r i t o n e a l l y w i t h a c t i n o m y c i n D (i.5 m g per kg b o d y wt.) 13 h before sacrifice a n d s e p a r a t e d into 6 fractions, as described in Fig. i. The n u m b e r s are r a t i o s b e t w e e n s t i m u l a t i o n of R N A e x t r a c t e d f r o m liver nuclei of a c t i n o m y c i n D - t r e a t e d r a t s a n d n o n - t r e a t e d rats.
Fractions
Aproteic r a t s Pair-fed r a t s
>28S
28S
<28S >I8S
I8S
4 S
0.40 0.35
0.38 0.35
0.40 0.40
0.35 0.20
0.30 o.15
0.55 0.25
the total stimulation of each fraction from rats treated with actinomycin D and the stimulation of the corresponding fraction from untreated animals. The results show significant decreases in stimulation b y all fractions. In both groups of animals stimulation decreases most in Fraction 5, which corresponds to an RNA with a sedimentation rate between 18 and 4 S. Moreover, there is less decrease in stimulatory activity in depleted than in control rats. These results confirm the presence in this gradient region of unstable messenger RNA's. These fractions of messenger RNA's are relatively less in protein- depleted rats. In protein-depleted rats, we found an increase in the stimulatory activity in heavy regions of the gradient, but there is no corresponding increase in pulse- labelling. These results are interpreted as reflecting the presence, in heavy regions, of species of messenger RNA's which are more stable; in protein-depleted animals, there is an increase of these messenger RNA's. The presence of these more stable messengers could be accounted for b y two hypotheses: (I) We are really dealing with a large messenger molecule or (2) the apparently large "messenger" molecule is actually an aggregate of messenger RNA strongly bound to some other type of molecule. Since, in the latter case, these messenger RNA's are affected b y nutritional conditions, they must either preexist in vivo, or their large size is an artifact due to physicochemical differences between the RNA's, in vivo, in protein-depleted and control rats. Of the two hypotheses the first seems to be substantiated b y the results of SIDRANSKY, STAEHELIN AND VERNEYTMwho called attention to a shift from lighter to heavier polysomes in rats on a threonine-free diet. This work was partially carried out under the Association Euratom-C.N.R.C.N.E.N., Contract No. o12-61-12 BIAI. We gratefully acknowledge the skilled technical assistance of Mr. E. BUSIELLO. Actinomycin D was a gift from Merck, Sharpe and Dohme.
ln/,'rnational Laboratory o/ Genetics amt l¢iophysics, Naples (Italy) and IslilHIo Nazionale della Nutrizione, /~',,;;t,: (II,dy)
A. DI GIROLAMO M. DI GIROLAMO S. GAETANI M.A. SPADONI Biochim. Biophys. Acta, 114 (1966) 195-198
198
SHORT COMMUNICATIONS
1 F. GRos, H. HIATT, W. GILBER'r, C. C. KURLAND, }{. W. I{ISEBROUGH AND J. D. WATSON, Nature, 19o (1961) 581. 2 M. W. NIREMBERG AND J, H. MATTItAEI, JProe. Natl. Acad. Sci. U.S., 47 (1961) 1588. 3 A. T]SSII~RES AND J. W. HOPKINS, Proc. Natl. Acad. Sci. U.S., 47 (1961) 2oi 5. 4 H. H. HIATT, .]. ]~lol. Biol., 5 ( 1 9 6 2 ) 217. 5 A. DI GIROLAMO, E. C. HENSHAW AND H. H. HIATT, J. Mo!. Biol., 8 (1964) 479. 6 H. H. HIATT, F. GROS AND I~. JACOB, Biochim. Biophys. Acta, 72 (1963) 15. 7 G. ATTARDI, S. NAONO, J. t{OUVIERE, F. JACOB AND F. GROS, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 363 . 8 B. S. GUTTMAN AND A. NOVlCK, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 373. 9 M. HAYASHI, S. SPIEGELMAN, N. FRANKLIN AND S. E. LURIA, Proc. Natl. Acad. Sci. U.S., 49 (I963) 729. Io W. E. KNox, W. H. AUERBACK AND E. C. LIN, Physiol. Rev., 36 (1956) 164. I I J. C. WATERLOW, Federation -Proc., 18 (1959) 1143. I2 H. C. PITOT, VV. IR. POTTER AND H. P. MORRlS, Cancer Res., 21 (1961) i o o i . 13 J. B. ALLISON, R. W. WANNEMACHER, W. L. BANKS, W. H. WUNNER AND R. A. COMEZBRENES, J. Nutrition, 78 (1962) 333. 14 J. N. WILLIAMS, J. Nutrition, 82 (1964) 51 . 15 S. GAETANI, A. M. I)AOLUCCI, M. A. SPADON1 AND G. TOMASS1, J. Nutrition, 84 (1964) 173. I6 H. G. ALBAUM AND W. W. UMBREI% J. Biol. Chem., 167 (I947) 369 . 17 A. B. PARDEE, K. PAIGEN AND L. PRESTIDGE, Biochim. Biophys. Acta, 23 (1957) 162. 18 O. H. LOWRY, INT.J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J . Biol. Chem., 193 (1951) 265 . I9 H. SIDRANSKY, T. STAEHELIN AND E. VERNEY, Science, 146 (1964) 766.
Received April 2oth, 1965 Revised manuscript received August 2nd, 1965
BBA 93106
Patterns of mRNA synthesis during rat-liver carcinogenesis induced by
diethylnitrosamine and thioacetamide Changes in the m R N A patterns on counter-current distribution were observed during isolation of the m R N A - D N A complex from rat livers when rats were fed a diet containing 4'-fluoro-4-dimethylaminoazobenzene 1. The changes were reversible during the first month by removing the carcinogen and refeeding the control diet. After this time the patterns on counter-current distribution became irreversible and a complete change in the pattern had appeared by the time the liver tumour arose. Upon feeding the weakly carcinogenic dye 4-aminoazobenzene ~ the counter-current distribution patterns remained reversible up to 4 months. Since the incorporation of orotic acid had greatly decreased prior and subsequent to tumour induction we have examined 2 different carcinogens to determine how general the change is during the carcinogenic process. We have chosen diethylnitrosamine 3 and thioacetamide 4 since, while these are both hepatocarcinogens, it seemed possible that each m a y have different biochemical reactions within the hepatic cells and hence induce different biochemical lesions. Adult Wistar rats (3 months old) of both sexes were maintained on a 20 % protein diet with normal supplements. In one group a solution of 0.006 % (w/v) of diethylnitrosamine in darkened drinking bottles was supplied in place of drinking water (c/. ref. 3). The diet of the second group was supplemented with thioacetamide (320 mg per kg diet) and a third group received the 20 % protein control diet. Biochim. Biophys. dcta, 114 (I966) 198-2oo