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The temperature-dependent extraction of two rapidly-labeled, DNA-like RNA fractions from rat liver
649
PRELIMINARY NOTES
BBA 91162 The t e m p e r o t u r e - d e p e n d e n t extraction of two
rapidly-labeled,
DNA-like
R N A froctions from rot liver GEORGIEV AND LERMAN1 have reported that three rapidly-labeled RNA fractions remain in the gel interphase after the extraction of rat liver with aqueous phenol at low temperatures, and that these three fractions may be recovered by reextraction of the interphase at elevated temperatures (the 28-S and I8-S ribosomal RNA precursors at lO-35 ° and 45-55 °, respectively, and a DNA-like RNA at 65 75°). MORRISON AND FRAJOLA2 have reported, however, that even after extraction (of Drosophila larvae) with phenol and sodium dodecyl sulfate at 65 °, additional RNA could be recovered by a subsequent extraction of the interphase with phenol and sodium dodecyl sulfate at 95 °. In the present communication we wish to report that the residual RNA extracted (from rat liver) with phenol and sodium dodecyl sulfate at 95 ° is a rapidly-labeled, DNA-like RNA that is not extracted with aqueous phenol at 65 ° . Adult male rats were decapitated 15 rain after intraperitoneal injections of 5oo/JC of tritiated orotic acid. The livers were excised, minced with scissors, and homogenized in a Dounce tissue grinder with a mixture of 9 vol. (measured by liquid displacement) of a saline-versene solution (0. 9 % saline and o.I O/ .,o versene saturated with freshly-distilled phenol) and IO vol. of a phenol solution (freshly-distilled phenol saturated with the saline-versene solution). This and all subsequent operations were carried out at 5 ° unless otherwise specified. The homogenate from 30 g of liver was transferred to a 25o-ml centrifuge bottle and stirred for 30 rain. The aqueous phase (Fraction I), formed after a 3o-min centrifugation at I5oo ×g, was decanted and replaced by an equal volume of the saline-versene solution. This mixture was stirred and centrifuged at 15oo x g for IO ram. Once again the aqueous phase (Fraction 2) was decanted and replaced by an equal volume of the saline-versene solution. It was necessary to repeat this process 20 times in order to reduce the amount of RNA which was extractable from the interphase with cold aqueous phenol to a constant minimal level. A volume of the saline-versene solution equal to 2.5 times the original volume of liver was added to the "washed" interphase in the centrifuge bottle. This mixture was stirred for IO min at 5 °, cooled in ice water for 5 rain, and centrifuged at 15oo ×g for 5 rain. Again the aqueous phase (Fraction 21) was decanted and replaced by an equal volume of the saline-versene solution, and the mixture was stirred for IO rain at a temperature 5 ° higher than that used in the previous extraction. This five-step process was repeated at 5 ° intervals from 5 ° to 95 °. The saline-versene solution was then adjusted to 1 % with respect to sodium dodeeyl sulfate and the entire process repeated at Io ° intervals from 5 ° to 95 °. The radioactivity of each of the fractions was determined and plotted as shown in Fig. I. The three peaks in Fig. I represent radioactive, trichloroaeetic acid-precipitable materials which were extracted from the phenol interphase. On the basis of their sensitivity to ribonuclease, we concluded that the labeled materials were RNA. Biochim. Biophys. Acta, 138 (1967) 649 65I
650
I'I{ELII~IINARY NOTES EXTRACTION CONDITIONS Aq. . . . .
P. . . . . . . . . .
- . . . . c'i- SDS ~
ooi D _z
4oo~
II
,'? o
300~
x
g o
200 -
I00-
I0
20
30 FRACTION
40
50
60
NUMBER
Fig. I. The t e m p e r a t u r e - d e p e n d e n t e x t r a c t i o n of rapidly-labeled R N A from the phenol interphase. R a d i o a c t i v i t y was d e t e r m i n e d before ( 0 - 0 ) anti after ( 0 0 ) ribonuclease t r e a t m e n t . F r a c t i o n s were e x t r a c t e d as described in the text. Aliquots of o.1 ml of each fraction were precipitated onto filter p a p e r discs w i t h cold 5 ~{) trichloroacetic acid. The discs were w a s h e d by a modification of the m e t h o d of MANS AND NOVELLI~, and counted in a P a c k a r d Tri-Carb liquid scintillation s p e c t r o m e t e r . Note t h a t several e x t r a c t i o n s (Fractions 49 55) with phenol and s o d i u m dodecylsulfate (SDS) at 95 ~ were necessary to effect the complete release of the R N A (of the third peak) from the interphase.
Tile first of the three peaks (i) represents RNA that was extractable with aqueous phenol at 5 °. Fractions 1-2o are essentially washes of the interphase. Notice that the labeled RNA which is extractable with cold aqueous phenol is reduced to a constant minimal level by these 2o washes. The second peak (II) represents RNA that was released from the interphase with aqueous phenol at elevated temperatures (Fractions 22 39). The shoulders on Peak II at 3 °° (Fraction 26) and 55 ° (Fraction 31) probably consist of the ribosomal precursor material which GEOI',(;ILVAND LERMAN~ reported as being extractable at these temperatures. At longer labeling periods these shoulders become more prominant. With the short labeling period used in this experiment, however, most of the labeled material in Peak II was extracted at 65 ° (Fraction 33)- The third peak (III) represents RNA that was not released from the interphase with aqueous phenol at elevated temperatures nor with phenol and sodium dodecyl sulfate at any temperature below 75 °. It was, however, released with phenol and sodium dodecyl sulfate at temperatures above 75 °, and was released optimally at 95 °. Selected fractions of each of the 3 peaks shown in Fig. I were pooled and precipitated with 2.5 vol. of 95 o~, ethanol. Each of the pooled samples was reprecipitated twice with ethanol and used for nucleotide analyses a. The A + U : G + C ratio of the RNA of Peak I (Fraction I), Peak II (Fractions 32-36), and Peak I I I (Fractions 48-51) was 0.6o, o.94 and o.96, respectively. These results indicate that two rapidly-labeled RNA fractions with DNAlike base compositions exist in the phenol interphase. One type can be released with Biochi~*~. Biophys. Acla, 138 (I967) 649-651
PRELIMINARY NOTES
651
aqueous phenol at t e m p e r a t u r e s b e t w e e n 6o ° a n d 8o ° a n d p r o b a b l y corresponds to t h e D N A - l i k e R N A r e p o r t e d b y GEORG]EV AND MANTIEVA5. The o t h e r t y p e cannot be released w i t h aqueous phenol a t a n y t e m p e r a t u r e , b u t o n l y b y the use of phenol a n d s o d i u m d o d e c y l sulfate at 95 °. E x p e r i m e n t s are now in progress to characterize t h e n a t u r e a n d biological role of these two t y p e s of r a p i d l y - l a b e l e d , D N A - l i k e R N A . This work was s u p p o r t e d b y N a t i o n a l I n s t i t u t e s of H e a l t h G r a n t N B o 6615-Ol a n d A m e r i c a n Cancer S o c i e t y G r a n t I N - I 6 - G .
Departments o/ Psychiatry and Physiological Chemistry, The Ohio State University, Columbus, Ohio (U.S.A.) I 2 3 4 5
W. W. MORRISON R. H. McCLUER
G. P. GEORGIEVAND M. [. LERMAN,Biochim. Biophys. Acla, 91 (1964) 678. \'V. \¥. MORRISON AND W. J. FRAJOLA, Biochem. Biophys. Res. Commun., 17 (1964) 597. S. KATZAND D. G. COMB,J. Biol. Chem., 238 (1963) 3o65 . R. J. MANS .aND G. D. NOVELLI, Biochem. Biophys. Res. Commun., 3 (196o) 54o. G. P. GEOR~IEV AND V. L. MANTIEVA, Biochim. Biophys. Acta, 61 (1962) I53.
Received F e b r u a r y 27th, 1967
Biochim. Biophys. Acta, 138 (1967) 649-65 1
BBA 91165 Synthesis of mitochondriol proteins. The synthesis of cytochrome c in vitro E a r l i e r claims concerning t h e d e m o n s t r a t i o n of the synthesis of c y t o c h r o m e c in isolated r a t liver 1 a n d calf h e a r t m i t o c h o n d r i a 2 were l a t e r w i t h d r a w n a. In fact, several workers have now e s t a b l i s h e d t h a t isolated m i t o c h o n d r i a are unable to synthesize their soluble p r o t e i n s 4-G. O n l y the insoluble fraction ~,8, m a i n l y the struct u r a l p r o t e i n ~,8,9, is s y n t h e s i z e d in vitro. Therefore it was assumed, a n d also i n d i r e c t l y shown, t h a t m o s t of t h e m i t o c h o n d r i a l enzymes are s y n t h e s i z e d at the microsomes 9-11. Direct evidence i n d i c a t i n g t h a t the microsomes represent the site of synthesis of c y t o c h r o m e c has been o b t a i n e d r e c e n t l y b y CAMPBELL AND CADAVID12. However, t h e m e c h a n i s m b y which these proteins enter the m i t o c h o n d r i a was a m a t t e r of speculation. In a previous p a p e r we d e m o n s t r a t e d the t r a n s f e r of labelled proteins from microsomes into m i t o c h o n d r i a in vitro 6. This p a p e r describes the labelling of c y t o c h r o m e c in m i t o c h o n d r i a which h a v e been i n c u b a t e d with labelled microsomes. A special c h r o m a t o g r a p h i c t e c h n i q u e was d e v e l o p e d for the purification of v e r y small a m o u n t s of c y t o c h r o m e c. The c o m b i n a t i o n of an a n i o n - e x c h a n g e ( T E A E cellulose) a n d a c a t i o n - e x c h a n g e column (CM-cellulose) in tandem (i.e. so t h a t t h e effluent from the first enters t h e second) allows t h e one-step exclusion of 95 % of t h e p h o s p h a t e - s o l u b l e m i t o c h o n d r i a l protein. The a n i o n - e x c h a n g e colunm retains 83 o0, whereas 12 % is e x c l u d e d from t h e c a t i o n - e x c h a n g e c o l u m n b y elution w i t h T r i s HC1 (pH 7-5). C y t o c h r o m e c a p p e a r s at t h e t o p of t h e CM-cellulose c o l u m n as a n a r r o w red b a n d . The elution of the l a t t e r column with a linear g r a d i e n t results in an almost pure fraction of c y t o c h r o m e c (Fig. I). R e c h r o m a t o g r a p h y of t h e c y t o chrome c on CM-cellulose d i d not change t h e specific a c t i v i t y .
Biochim. Biophys. Acta, 138 (1967) 651-65¢
PRELIMINARY NOTES
BBA 91162 The t e m p e r o t u r e - d e p e n d e n t extraction of two
rapidly-labeled,
DNA-like
R N A froctions from rot liver GEORGIEV AND LERMAN1 have reported that three rapidly-labeled RNA fractions remain in the gel interphase after the extraction of rat liver with aqueous phenol at low temperatures, and that these three fractions may be recovered by reextraction of the interphase at elevated temperatures (the 28-S and I8-S ribosomal RNA precursors at lO-35 ° and 45-55 °, respectively, and a DNA-like RNA at 65 75°). MORRISON AND FRAJOLA2 have reported, however, that even after extraction (of Drosophila larvae) with phenol and sodium dodecyl sulfate at 65 °, additional RNA could be recovered by a subsequent extraction of the interphase with phenol and sodium dodecyl sulfate at 95 °. In the present communication we wish to report that the residual RNA extracted (from rat liver) with phenol and sodium dodecyl sulfate at 95 ° is a rapidly-labeled, DNA-like RNA that is not extracted with aqueous phenol at 65 ° . Adult male rats were decapitated 15 rain after intraperitoneal injections of 5oo/JC of tritiated orotic acid. The livers were excised, minced with scissors, and homogenized in a Dounce tissue grinder with a mixture of 9 vol. (measured by liquid displacement) of a saline-versene solution (0. 9 % saline and o.I O/ .,o versene saturated with freshly-distilled phenol) and IO vol. of a phenol solution (freshly-distilled phenol saturated with the saline-versene solution). This and all subsequent operations were carried out at 5 ° unless otherwise specified. The homogenate from 30 g of liver was transferred to a 25o-ml centrifuge bottle and stirred for 30 rain. The aqueous phase (Fraction I), formed after a 3o-min centrifugation at I5oo ×g, was decanted and replaced by an equal volume of the saline-versene solution. This mixture was stirred and centrifuged at 15oo x g for IO ram. Once again the aqueous phase (Fraction 2) was decanted and replaced by an equal volume of the saline-versene solution. It was necessary to repeat this process 20 times in order to reduce the amount of RNA which was extractable from the interphase with cold aqueous phenol to a constant minimal level. A volume of the saline-versene solution equal to 2.5 times the original volume of liver was added to the "washed" interphase in the centrifuge bottle. This mixture was stirred for IO min at 5 °, cooled in ice water for 5 rain, and centrifuged at 15oo ×g for 5 rain. Again the aqueous phase (Fraction 21) was decanted and replaced by an equal volume of the saline-versene solution, and the mixture was stirred for IO rain at a temperature 5 ° higher than that used in the previous extraction. This five-step process was repeated at 5 ° intervals from 5 ° to 95 °. The saline-versene solution was then adjusted to 1 % with respect to sodium dodeeyl sulfate and the entire process repeated at Io ° intervals from 5 ° to 95 °. The radioactivity of each of the fractions was determined and plotted as shown in Fig. I. The three peaks in Fig. I represent radioactive, trichloroaeetic acid-precipitable materials which were extracted from the phenol interphase. On the basis of their sensitivity to ribonuclease, we concluded that the labeled materials were RNA. Biochim. Biophys. Acta, 138 (1967) 649 65I
650
I'I{ELII~IINARY NOTES EXTRACTION CONDITIONS Aq. . . . .
P. . . . . . . . . .
- . . . . c'i- SDS ~
ooi D _z
4oo~
II
,'? o
300~
x
g o
200 -
I00-
I0
20
30 FRACTION
40
50
60
NUMBER
Fig. I. The t e m p e r a t u r e - d e p e n d e n t e x t r a c t i o n of rapidly-labeled R N A from the phenol interphase. R a d i o a c t i v i t y was d e t e r m i n e d before ( 0 - 0 ) anti after ( 0 0 ) ribonuclease t r e a t m e n t . F r a c t i o n s were e x t r a c t e d as described in the text. Aliquots of o.1 ml of each fraction were precipitated onto filter p a p e r discs w i t h cold 5 ~{) trichloroacetic acid. The discs were w a s h e d by a modification of the m e t h o d of MANS AND NOVELLI~, and counted in a P a c k a r d Tri-Carb liquid scintillation s p e c t r o m e t e r . Note t h a t several e x t r a c t i o n s (Fractions 49 55) with phenol and s o d i u m dodecylsulfate (SDS) at 95 ~ were necessary to effect the complete release of the R N A (of the third peak) from the interphase.
Tile first of the three peaks (i) represents RNA that was extractable with aqueous phenol at 5 °. Fractions 1-2o are essentially washes of the interphase. Notice that the labeled RNA which is extractable with cold aqueous phenol is reduced to a constant minimal level by these 2o washes. The second peak (II) represents RNA that was released from the interphase with aqueous phenol at elevated temperatures (Fractions 22 39). The shoulders on Peak II at 3 °° (Fraction 26) and 55 ° (Fraction 31) probably consist of the ribosomal precursor material which GEOI',(;ILVAND LERMAN~ reported as being extractable at these temperatures. At longer labeling periods these shoulders become more prominant. With the short labeling period used in this experiment, however, most of the labeled material in Peak II was extracted at 65 ° (Fraction 33)- The third peak (III) represents RNA that was not released from the interphase with aqueous phenol at elevated temperatures nor with phenol and sodium dodecyl sulfate at any temperature below 75 °. It was, however, released with phenol and sodium dodecyl sulfate at temperatures above 75 °, and was released optimally at 95 °. Selected fractions of each of the 3 peaks shown in Fig. I were pooled and precipitated with 2.5 vol. of 95 o~, ethanol. Each of the pooled samples was reprecipitated twice with ethanol and used for nucleotide analyses a. The A + U : G + C ratio of the RNA of Peak I (Fraction I), Peak II (Fractions 32-36), and Peak I I I (Fractions 48-51) was 0.6o, o.94 and o.96, respectively. These results indicate that two rapidly-labeled RNA fractions with DNAlike base compositions exist in the phenol interphase. One type can be released with Biochi~*~. Biophys. Acla, 138 (I967) 649-651
PRELIMINARY NOTES
651
aqueous phenol at t e m p e r a t u r e s b e t w e e n 6o ° a n d 8o ° a n d p r o b a b l y corresponds to t h e D N A - l i k e R N A r e p o r t e d b y GEORG]EV AND MANTIEVA5. The o t h e r t y p e cannot be released w i t h aqueous phenol a t a n y t e m p e r a t u r e , b u t o n l y b y the use of phenol a n d s o d i u m d o d e c y l sulfate at 95 °. E x p e r i m e n t s are now in progress to characterize t h e n a t u r e a n d biological role of these two t y p e s of r a p i d l y - l a b e l e d , D N A - l i k e R N A . This work was s u p p o r t e d b y N a t i o n a l I n s t i t u t e s of H e a l t h G r a n t N B o 6615-Ol a n d A m e r i c a n Cancer S o c i e t y G r a n t I N - I 6 - G .
Departments o/ Psychiatry and Physiological Chemistry, The Ohio State University, Columbus, Ohio (U.S.A.) I 2 3 4 5
W. W. MORRISON R. H. McCLUER
G. P. GEORGIEVAND M. [. LERMAN,Biochim. Biophys. Acla, 91 (1964) 678. \'V. \¥. MORRISON AND W. J. FRAJOLA, Biochem. Biophys. Res. Commun., 17 (1964) 597. S. KATZAND D. G. COMB,J. Biol. Chem., 238 (1963) 3o65 . R. J. MANS .aND G. D. NOVELLI, Biochem. Biophys. Res. Commun., 3 (196o) 54o. G. P. GEOR~IEV AND V. L. MANTIEVA, Biochim. Biophys. Acta, 61 (1962) I53.
Received F e b r u a r y 27th, 1967
Biochim. Biophys. Acta, 138 (1967) 649-65 1
BBA 91165 Synthesis of mitochondriol proteins. The synthesis of cytochrome c in vitro E a r l i e r claims concerning t h e d e m o n s t r a t i o n of the synthesis of c y t o c h r o m e c in isolated r a t liver 1 a n d calf h e a r t m i t o c h o n d r i a 2 were l a t e r w i t h d r a w n a. In fact, several workers have now e s t a b l i s h e d t h a t isolated m i t o c h o n d r i a are unable to synthesize their soluble p r o t e i n s 4-G. O n l y the insoluble fraction ~,8, m a i n l y the struct u r a l p r o t e i n ~,8,9, is s y n t h e s i z e d in vitro. Therefore it was assumed, a n d also i n d i r e c t l y shown, t h a t m o s t of t h e m i t o c h o n d r i a l enzymes are s y n t h e s i z e d at the microsomes 9-11. Direct evidence i n d i c a t i n g t h a t the microsomes represent the site of synthesis of c y t o c h r o m e c has been o b t a i n e d r e c e n t l y b y CAMPBELL AND CADAVID12. However, t h e m e c h a n i s m b y which these proteins enter the m i t o c h o n d r i a was a m a t t e r of speculation. In a previous p a p e r we d e m o n s t r a t e d the t r a n s f e r of labelled proteins from microsomes into m i t o c h o n d r i a in vitro 6. This p a p e r describes the labelling of c y t o c h r o m e c in m i t o c h o n d r i a which h a v e been i n c u b a t e d with labelled microsomes. A special c h r o m a t o g r a p h i c t e c h n i q u e was d e v e l o p e d for the purification of v e r y small a m o u n t s of c y t o c h r o m e c. The c o m b i n a t i o n of an a n i o n - e x c h a n g e ( T E A E cellulose) a n d a c a t i o n - e x c h a n g e column (CM-cellulose) in tandem (i.e. so t h a t t h e effluent from the first enters t h e second) allows t h e one-step exclusion of 95 % of t h e p h o s p h a t e - s o l u b l e m i t o c h o n d r i a l protein. The a n i o n - e x c h a n g e colunm retains 83 o0, whereas 12 % is e x c l u d e d from t h e c a t i o n - e x c h a n g e c o l u m n b y elution w i t h T r i s HC1 (pH 7-5). C y t o c h r o m e c a p p e a r s at t h e t o p of t h e CM-cellulose c o l u m n as a n a r r o w red b a n d . The elution of the l a t t e r column with a linear g r a d i e n t results in an almost pure fraction of c y t o c h r o m e c (Fig. I). R e c h r o m a t o g r a p h y of t h e c y t o chrome c on CM-cellulose d i d not change t h e specific a c t i v i t y .
Biochim. Biophys. Acta, 138 (1967) 651-65¢