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The effect of deoxyribonuclease on the incorporation of precursors into nuclear ribonucleic acid in reconstituted rat-liver homogenates
BIOCHIMICA ET BIOPHYSICA ACTA
387
BBA 8138
T H E E F F E C T O F D E O X Y R I B O N U C L E A S E ON T H E I N C O R P O R A T I O N OF PRECURSORS INTO NUCLEAR RIBONUCLEIC ACID IN RECONSTITUTED RAT-LIVER HOMOGENATES J O H N H. S C H N E I D E R *
AND S H E H A D I
N. N A Y F E H * *
Department o/ Biochemistry, American University o/ Beirut, Beirut (Lebanon) (Received M a r c h 5th, I962)
SUMMARY
Isolated rat-liver nuclei were incubated with different concentrations of DNAase, and a m a x i m u m of 76 % of the "apparent D N A " {determined b y trichloroacetic acid extraction and diphenylamine reaction) could be removed in this way. The remaining 24 3/o represents a chromogen that is not DNA. After incubation, the nuclei were combined with a cytoplasmic fraction and the reconstituted homogenates were incubated with [8-1aC]adenine. Low concentrations of DNAase stimulated incorporation of adenine into nuclear RNA under a wide range of experimental conditions. Higher levels of DNAase inhibited the incorporation of adenine to a m a x i m u m of 22 % for nuclear RNA and 4 1 % for cytoplasmic RNA. When DNAase-treated nuclei were recombined with a cytoplasmic fraction, cytoplasmic RNA became associated with the nuclear fraction.
INTRODUCTION
If the genetic information carried b y DNA is transferred to nuclear RNA which is subsequently released to the cytoplasm, then the synthesis of nuclear RNA should be at least partly dependent on the presence of DNA. Evidence for such dependence has been obtained in systems from E. coli l-e, and in several cases, it has been clearly demonstrated that the amount of each base incorporated into RNA depends on the composition of the DNA present 5& A similar DNA requirement for precursor incorporation into RNA has been demonstrated in M. lysodeikticus v, L. arabinosus 8, and an enzyme system from pea embryos 9. These findings also apply to mammalian systems, since incorporation of precursors into RNA in rat liver 7,1°,n, calf-thymus nuclei 12-15 and rabbit-appendix nucleile, iv is decreased, and in some cases, completely abolished by incubation with DNAase. * P r e s e n t a d d r e s s : Biological A b s t r a c t s , 3 8 r 5 W a l n u t St., P h i l a d e l p h i a , Pa., U.S.A. (No reprints are available.) ** P r e s e n t a d d r e s s : P h a r m a c o l o g y D e p a r t m e n t , U n i v e r s i t y of N o r t h Carolina, Chapel Hill,
N. c. (U.S.A.). Biochim. Biophys. Acta, 61 (1962) 387-394
388
J.H.
SCHNEIDER, S. N. NAYFEH
In similar experiments with rat-liver homogenates in this laboratory, a stimulation of incorporation of precursors into nuclear RNA had been observed after removing part of the DNA with low concentrations of DNAase. In addition, even complete removal of DNA with DNAase resulted in only a small decrease in the incorporation of precursors into nuclear RNA. For this reason, a study of the incorporation of radioactive orotic acid and adenine into nuclear RNA as a function of DNAase concentration was undertaken. The results of this detailed study are presented in this paper. METHODS
Materials Rats were supplied by Tierzuchterei Brunger (Halle, Westfalen, Germany). DNAase (I × crystallized) and highly polymerized DNA were obtained from Worthington Biochemical Corporation (Freehold, New Jersey). E8-m4C]Adenine (specific activity, 26.2 mC/mg) was supplied by the Radiochemical Center (Amersham, England). E6-14C~Orotic acid hydrate (specific activity, 1.15 mC/mmole) was obtained from Tracerlab.
Incubation o[ homogenates with radioactive precursors and isolation o/nucleic acids Adult female rats were sacrificed and a 20 % homogenate of the excised liver was prepared in 0.25 M sucrose. Aliquots of the homogenate were transferred to 5o-ml Erlenmeyer flasks containing an equal volume of the following solution: succinate, 6 mM; pyruvate, 20 raM; glutamate, 20 mM; phosphate buffer (pH 7.4), 20 raM; magnesium chloride, 6 r a M ; niacin, I o m M ; ATP, o . 8 m M ; fructose, 12 raM; E8-1*C]adenine, 1.2#C/ml, or E6J*C]orotic acid (o.5/~C/ml); and sucrose, 0.25 M. Different concentrations of DNAase were added to each flask depending on the experiment, and the mixture was incubated with shaking in a water bath at 3 °0 for 45 rain. After incubation, the flasks were chilled in ice. Incubation mixtures were transferred to plastic tubes, rehomogenized with a motor-driven plastic pestle (also used during all subsequent resuspension and washing steps) to break up clumps formed during incubation, and then centrifuged at 6 o o × g for io min in a refrigerated centrifuge to sediment the nuclei. The nuclear pellet was resuspended in 0.25 M sucrose by homogenization, purified by underlayering with 0.34 M sucroselS, 19, and recentrifuged at 600 × g for IO rain. The supernatant was combined with the first cytoplasmic fraction. After precipitating and washing the nuclear and cytoplasmic fractions twice with ice-cold perchloric acid, the nucleic acids were extracted into hot IO % NaC1 under neutral conditions and were precipitated by the addition of 1.5 volumes of alcohol. Aliquots of these nucleic acid precipitates were taken for determination of specific activity. Since adenine is not incorporated into DNA by rat-liver homogenates (see RESULTS), it was not necessary to separate RNA from DNA in these experiments. Details of parts of this isolation procedure have been published previously 2°.
Incubation o] isolated nuclei with DNAase After preparing a 20 % rat-liver homogenate in 0.25 M sucrose at o °, 6-ml aliquots were transferred to round-bottom centrifuge tubes and centrifuged at 600 × g Biochim. Biophys. Acta, 61 (1962) 387-394
RNAASE AND INCORPORATION OF PRECURSORS INTO NUCLEAR RNA
389
for io rain at o °. The cytoplasmic fractions were then decanted and stored at o °. The nuclear pellets were resuspended in I ml of an incubation mixture containing 40 m M magnesium chloride, 2o m M phosphate buffer (pH 7) and DNAase of various concentrations depending on the experiment. After incubation of the nuclear suspension with DNAase at 3 °° for 2o min, the nuclei were sedimented by centrifugation at 6oo × g for IO min and the supernatants were discarded. The nuclei were then recombined with the corresponding cytoplasmic fractions, and 2.5-ml aliquots of these reconstituted homogenates were used for the standard incubation procedure described above.
Determination o/ the DNA content o/ nuclei After incubating the nuclei with DNAase, 2.5-ml aliquots of the reconstituted homogenate were centrifuged at 600 × g for io rain to reisolate the nuclei and the supernatant was discarded. The nuclear pellet was precipitated and washed twice with 5 % trichloroacetic acid at o °, and centrifuged at high speed to remove most of tile trichloroacetic acid from the tissues. DNA was then extracted from the nuclear pellet with 1. 3 ml of 5 % trichloroacetic acid at 90° for 15 rain and DNA was determined on the supernatant b y the DISCHE reaction 21 using highly polymerized DNA as the standard. RESULTS
When radioactive adenine (2. 4. lO 6 counts/rain) was incubated with 5 ml of a io % rat-liver homogenate using the standard incubation procedure, the combined nuclear nucleic acid gave 411, 432, and 46o counts/rain/plate for triplicate flasks. This combined fraction was quantitatively eluted from the plates, the RNA was hydrolyzed with o.2 N N a O H at 8o ° for 15 rain, and the unhydrolyzed DNA was precipitated by adding I N HC1 at o ° to a final concentration of o.I N. When the DNA (representing the total DNA originally present in the combined nucleic acid fraction) was quantitatively plated, the resulting plates gave 12, 7 and 7 counts/rain. This experiment established that adenine is not incorporated into DNA in normal rat-liver holnogenates. This is of interest since thymidine'is incorporated by an identical system ~2. Initial experiments were directed toward determining the conditions necessary for m a x i m u m removal of DNA and highest rate of precursor incorporation into RNA. The conditions presented in the experimental section are thought to be optimal in these respects. One finding of interest is given in Fig. I which shows that at two different magnesium chloride concentrations, a 25-rain preincubation of the homogenate at 3°° before addition of the radioactive precursor resulted in an almost complete loss of ability to incorporate precursors into RNA. As a result, preincubations with DNAase to remove DNA were limited to short periods of time. Using the whole homogenate system, concentrations as high as I mg/ml of crystalline DNAase could be preincubated with the homogenate before addition of the radioactive precursor without producing a marked inhibition of orotic acid incorporation into either nuclear or cytoplasmic RNA. In fact, stimulation was noted at low DNAase concentrations (discussed later). To investigate and magnify any specific effect of DNAase on nuclear RNA, nuclei were isolated from 2o % homogenates, incubated with DNAase, and then cenBiochim. Biophyx. Acta, 61 (I962) 387 394
39 °
J. H. SCHNEIDER, S. N. NAYFEH 400
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Fig. I. Effect of p r e i n c u b a t i o n w i t h MgCI~ on t h e i n c o r p o r a t i o n of [6-~4C]orotic acid into R N A of a r a t - l i v e r h o m o g e n a t e . 2.5-ml a l i q u o t s of a 20 % r a t - l i v e r h o m o g e n a t e were p r e i n c u b a t e d w i t h 6 m M M g C 1 2 (dotted l i n e ) o r 3 m M M g C I 2 (solid line) a t 3 °° for different i n t e r v a l s of t i m e (indicated on t h e abscissa). A f t e r p r e i n c u b a t i o n , t h e h o m o g e n a t e s were i n c u b a t e d with [6-14C]orotic acid u n d e r s t a n d a r d i n c u b a t i o n c o n d i t i o n s (see text), t h e c y t o p l a s m i c a n d n u c l e a r R N A were isolated, a n d t h e specific a c t i v i t y w a s d e t e r m i n e d as c o u n t s / r a i n / r a g R N A (indicated on t h e ordinate). P o i n t s a t zero t i m e r e p r e s e n t t h e a v e r a g e of d u p l i c a t e s a m p l e s .
trifuged to remove most of the enzyme. The DNA-depleted nuclei were mixed with the cytoplasmic fraction from which they were isolated and the reconstituted homogenates were incubated with [8-1*CJadenine. It was considered desirable to incubate a reconstituted homogenate rather than isolated nuclei with adenine so that the nuclei would not be limited b y absence of reactions necessary for the conversion of adei!me to the nucleotide level required for incorporation into nuclear RNA. In addition, use of the active cytoplasmic fraction cancels any undesirable effect that DNAase might have on reactions not specifically related to precursor incorporation (such as the inhibition of nuclear ATP synthesis b y DNAase*3). The effects of preincubating isolated nuclei with DNAase are shown in the upper half of Fig. 2. It m a y be seen that the incubation of isolated nuclei with 2 mg DNAase/ ml removed about 70 % of the "apparent DNA" from the tissue, leaving behind a diphenylamine-reactive chromogen which accounted for about 30 % of the "apparent DNA". The m a x i m u m amount of "apparent D N A " removed in a number of experiments was 76 %. Both in terms of rate of reaction with diphenylamine and spectra after reaction it is clear that the remaining chromogen is not DNA. (The chromogen slowly gives a violet colour with a m a x i m u m at 534 m# rather than the pure blue colour with a 6oo-m/, m a x i m u m given b y pure DNA.) In addition, increasing the DNAase concentration from 2 mg/ml up to 5 mg/ml did not remove this chromogen, as seen in Fig. 2. It m a y be seen from Fig. 2 that incubation of isolated nuclei with low concentrations of DNAase (0.25-0.75 mg/ml) resulted in a significant increase (maximum of 21 and 44 %) in the incorporation of radioactive adenine into nuclear RNA of reconstitutBiochim. Biophys. Acta, 61 (1962) 3 8 7 - 3 9 4
RNAASE
AND INCORPORATION OF PRECURSORS INTO NUCLEAR RNA
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Fig. 2. Effect of p r e i n c u b a t i n g isolated nuclei w i t h D N A a s e on t h e s u b s e q u e n t i n c o r p o r a t i o n of [8-~4C]adenine into n u c l e a r a n d c y t o p l a s m i c R N A of a r e c o n s t i t u t e d h o m o g e n a t e . Nuclei isolated f r o m 6-ml a l i q u o t s of 2o °/o r a t - l i v e r h o m o g e n a t e s (in o.25 iV/ sucrose solution) were p r e i n c u b a t e d for 2o rain in I 2 - m l t u b e s w i t h D N A a s e ( c o n c e n t r a t i o n s i n d i c a t e d on t h e a b scissa), 4 ° m M MgCI, a n d 2o m M p h o s p h a t e b u f f e r (pH 7.o). A f t e r i n c u b a t i o n t h e nuclei were sedimented by centrifugation and recombined with the cytoplasmic fractions from which they were initially s e p a r a t e d . O n e 2.5-ml a l i q u o t of each r e c o n s t i t u t e d h o m o g e n a t e w a s i n c u b a t e d w i t h [8-14C]adenine u n d e r s t a n d a r d c o n d i t i o n s (see t e x t ) in 5o-ml E r l e n m e y e r flasks a t 3 °° for 45 m i n . C y t o p l a s m i c (solid line) a n d nuclear( d o t t e d line) R N A were isolated a n d their specific a c t i v i t i e s were d e t e r m i n e d as i n d i c a t e d b y t h e t w o e x p e r i m e n t s ( O -- ©, • -- • ) in t h e b o t t o m g r a p h . F r o m a second 2.5-ml a l i q u o t of e a c h r e c o n s t i t u t e d h o m o g e n a t e , nuclei were reisolated b y c e n t r i f u g a t i o n a t 6 o o × g . D N A w a s e x t r a c t e d f r o m t h e nuclei b y trichloroacetic acid (at 9 °o for 15 min) a n d d e t e r m i n e d b y t h e d i p h e n y l a l n i n e reaction. T h e D N A c o n c e n t r a t i o n is g i v e n in t h e t o p g r a p h . All p o i n t s a t zero c o n c e n t r a t i o n of D N A a s e r e p r e s e n t the a v e r a g e of d u p l i c a t e d e t e r m i n a t i o n s .
ed homogenates. This same effect was noted in every other experiment where low levels of DNAase were tested. As shown in Table I, the maximum stimulation of incorporation of either adenine or orotic acid into nuclear RNA ranged from 19 to 118 °/o at concentrations of DNAase ranging from 0.2 to 0.6 mg/ml under a wide range of experimental conditions. Stimulation of incorporation of precursors into cytoplasmic RNA was also observed in some experiments 24, but not as consistently as that observed for nuclear RNA. It may be seen from Fig. 2 that as the DNAase concentration was increased to high levels, the specific activity of both cytoplasmic and nuclear RNA decreased. At a level of 4-5 mg DNAase/ml, the specific activity of nuclear RNA was decreased by a maximum of 12 °/o and 22 °/o of the control value (no DNAase added), and the specific activity of cytoplasmic RNA showed a maximum decrease of 39 o, /o and 41 °"o in the same experiments. Since all the DNA is removed with much lower levels of DNAase (2-3 mg/ml), it is clear that the values just presented represent the maximum Biochim. Biophys. Acta, 61 (i962) 3 8 7 - 3 9 4
392
j. H. SCHNEIDER, S. N. NAYFEH TABLE I
STIMULATION
OF INCORPORATION RECONSTITUTED
OF
RADIOACTIVE
HOMOGENATES
PRECURSORS AFTER
INTO
TREATMENT
NUCLEAR WITH
RNA
IN WHOLE
OR
DNAASE
Three t y p e s of e x p e r i m e n t s were performed: (a) D N A a s e was added to whole h o m o g e n a t e s during i n c o r p o r a t i o n of the radioactive p r e c u r s o r ([6-14C]orotic acid or [8-14C]adenine as indicated); (b) D N A a s e was p r e i n c u b a t e d w i t h a whole h o m o g e n a t e p r i o r to the addition of the radioactive precursor; a n d (c) after i n c u b a t i o n of D N A a s e with isolated nuclei, the nuclei were sedimented b y centrifugation and were recombined w i t h a cytoplasmic fraction as in the e x p e r i m e n t s given in Fig. 2. I n all cases, g r a d u a t e d low levels of D N A a s e (less t h a n i m g / m l in the incubated solution) were tested and duplicate or triplicate control flasks (no D N A a s e added) were incubated. Only the c o n c e n t r a t i o n of D N A a s e (either non-crystalline or I × crystallized D N A a s e as indicated) t h a t gave the m a x i m u m s t i m u l a t i o n is included in this table. The percentage s t i m u l a t i o n presented was observed at the D N A a s e c o n c e n t r a t i o n indicated. Type o] expt.
Radioactive precursor
DNAase concentration ( mglml )
a c c b* a c b** b***
Orotate Adenine Adenine Orotate Orotate Adenine Orotate Orotate
o. 4 o. 4 0. 5 o.6 0. 3 o. 5 o.4 o.2
Type o] DNAase
Non-crystalline Crystalline Crystalline Non-crystalline Crystalline Crystalline Non-crystalline Crystalline
Stimulation (percentage o~control)
19 2i 25 4° 42 44 72 i 18
" P r e i n c u b a t e d w i t h D N A a s e at o ° for 3 h. ** P r e i n c u b a t e d w i t h D N A a s e a t 3 °° for 3 ° min. *** P r e i n c u b a t e d w i t h D N A a s e at 3 °° for 15 min.
decrease in precursor incorporation produced b y complete removal of DNA in this system. I t should also be mentioned that one batch of DNAase had an inhibitory effect on precursor incorporation apart from its effect in removing DNA. With this DNAase, inhibition was proportional to DNAase concentration at concentrations of 2, 3, 4 and 5 mg DNAase/ml, even though m a x i m u m removal of DNA had been achieved at the 2 mg/ml level. D a t a on the effect of this batch of DNAase on precursor incor12
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Fig. 3. Effect of p r e i n c u b a t i n g isolated nuclei with D N A a s e on the relative a m o u n t s of nuclear (-- - --) and cytoplasmic ( ) R N A . The a m o u n t s of nuclear and cytoplasmic R N A were determined f r o m d a t a obtained in the e x p e r i m e n t s described in the legend for Fig. 2.
Biochim. Biophys. Acta, 61 (1962) 387-394
RNAAsE AND INCORPORATION OF PRECURSORS INTO NUCLEAR RNA
393
poration are not included in Fig. 2 since others ~7 have noted similar inhibitory effects and also attributed them to contamination of the DNAase preparation. Fig. 3 shows the amount of nuclear and cytoplasmic RNA as a function of DNAase concentration in two experiments. The amount of nuclear RNA showed a maxim u m increase of 75 and IiO °/o, and this increase was consistently observed in all similar experiments. The cytoplasmic RNA showed corresponding decreases, while the total amount of RNA remained constant, within experimental error. DISCUSSION
ALLYREY,~{IRSKYAND OSAWA12 have noted that when calf-thymus nuclei are treated with DNAase in sucrose (the conditions used in the present experiments) they lose 9 ° % of their DNA, but no more than 15 o~, of the histone is released even though these basic proteins are water-soluble and are known to be attached to DNA in the nuclei. They suggest that these chromosomal proteins are still arranged in some specific functional configuration. Retention of histone b y the nucleus after DNA removal leads to some interesting consequences, since this highly positively-charged protein must be neutralized b y the addition of polyanions such as heparin, chondroitin sulfate, polyethylene sulfonates, hyaluronic acid, RNA, or DNA before DNAase-treated nuclei will synthesize ATP or incorporate precursors into proteins or nucleic acidla, 'v. In the present experiments, when DNA-depleted nuclei are recombined with a cytoplasmic fraction, the cytoplasmic RNA can function as a polyanion which becomes associated with the nucleus. Such a phenomenon has been previously described b y SALGANIK et al. 25 who found a 2. 7- to 4.4-fold increase in the amount of nuclear RNA when DNAase-treated nuclei were exposed to a solution of RNA. This appears to be the best explanation of the direct relation between the concentration of DNAase and the increase in the amount of nuclear RNA noted in Fig. 3. It should be noted that after removal of the m a x i m u m amount of DNAase-labile DNA (at about 2.5 mg DNAase/ml), doubling the DNAase concentration did not cause further increase in the amount of nuclear RNA. The results of Fig. 3 show that cytoplasmic RNA is selectively bound to the nuclei, even though m a n y other anions are undoubtedly available in the cytoplasmic fraction. It can be calculated that as much as 38 °i, of the removed DNA has been replaced by cytoplasmic RNA. The data of Fig. 2 indicate that complete removal of DNA inhibits incorporation of adenine into nuclear RNA by a reconstituted homogenate to a m a x i m u m of 22 o~. This inhibition is very low compared to the almost complete inhibition observed in m a n y of the systems reviewed in the introduction '-~7. In addition, it would be expected that precursor incorporation into nuclear RNA would be inhibited much more than incorporation into cytoplasmic RNA. Actually, inhibition of cytoplasmic RNA reached a m a x i m u m of 4 1 % , which is almost double the 22 Uo m a x i m u m inhibition observed for nuclear RNA. Two explanations m a y be offered for this unexpected finding: (a) The cytoplasmic RNA which becomes associated with the DNA-depleted nuclei acts in the same way as exogenous polyanions which are known to restore the ability of DNAasetreated nuclei to incorporate precursors into RNA ~3,17. This would explain the failure to observe larger inhibition of precursor incorporation into nuclear RNA. (b) RNA of the "post-microsomal" and supernatant fractions is known to be the most active Biochim. Biophy,,..4cta, 6r (I962) 3 8 7 - 3 9 4
394
j . H . SCHNEIDER, S. N. NAYFEH
in incorporating precursors into RNA *n. These very active fractions (rather than the microsomal or mitochondrial fractions which contain relatively inactive RNA 2~) may be selectively associated with the nucleus, and may continue to be active after the association occurs. This would result in an increased specific activity of the nuclear RNA and a decreased specific activity of the cytoplasmic RNA. The two explanations just presented could be elaborated to explain most of the changes observed in Fig. 2, and it is clear that they must be considered when interpreting the data. However, they cannot be considered completely adequate until more is known about the effect of DNAase in stimulating precursor incorporation into nuclear RNA and until the fractions of cytoplasmic RNA that become associated with nuclear RNA after DNAase treatment have been investigated. ACKNOWLEDGEMENTS
This work was supported in part by grants from the Anna Fuller Fund and the National Science Foundation (G-9995 and G-5178), and was performed in partial fulfillment of a M.S. degree in Biochemistry by one of the authors (S.N.N.). REFERENCES 1 * 3 4 5
7 s 9 10 11 12 18 54 in is 17 18 19 20 ~1 22 23 24 26
2s
A. STEVENS, Biochem. Biophys. Research Communs., 3 (196o) 92. A. STEVENS, Federation Proc., 20 (I96I) 363 . j . HURWITZ, A. ]3RESLER AND R. DIRINGER, Biochem. Biophys. Research Communs., 3 (196o) 15. j . j . FURTH, J. HURWITZ AND M. GOLDMANN, Federation Proc., 20 (I96I) 363. j . j . FURTH, J. HURWITZ AND M. CTOLDMANN, Biochem. Biophys. Research Communs., 4 (I96I) 362. J. J. FURTH, J. HURWITZ AND M. GOLDMANN, Biochem. Biophys. Research Communs., 4 (I96I) 431 . S. B. WEiss, Federation Proc., 20 (1961) 362. S. OCHOA, D. P. BURMA, H. KROGER AND J. D. WEILL, Federation Proc., 20 (1961) 362. R. C. HUANG, N. MAHESHWARI AND J. BONNER, Biochem. Biophys. Research Communs., 3 (196o) 689. S. ]3. WEISS AND L. GLADSTONE, J. Am. Chem. Soc., 81 (1959) 4118. S. ]3. WEISS, Proc. Natl. Acad. Sci. U.S., 46 (196o) lO2O. V. G. ALLER]~Y, A. E. MIRSKY AND S. OSAWA, J. Gen. Physiol., 4 ° (1956) 451. V. G. ALLFREY AND A. E. MIRSKY, Proc. Natl. Acad. Sci. U.S., 44 (1958) 981. j. S. KRAEOW AND H. O. KAMMEN, Federation Proc., 19 (196o) 307 . B. B. ]3ISWA$ AND R. ABRAMS, Federation Proc., 20 (1961) 362. M. SEKIGUCHI AND A. SIBATANI, Biochim. Biophys. Acta, 28 (1958) 455. M. SEKIGUCHI AND A. SIBATANI, Biochim. Biophys. Acta, 34 (1959) 444. G. H. HOGEBOOM, W. C. SCHNEIDER AND i . J. STRIEBICH, J. Biol. Chem., 196 (1952) i i i . G. H. HOGEBOOM, W. C. SCHNEIDER AND M. J. STRIEBICH, Cancer Research, 13 (1953) 617. j . H. SCHNEIDER AND V. R. POTTER, J. Biol. Chem., 233 (1958 ) 154. Z. DISCHE, in E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Academic Press, Inc., New York, 1955, p. 285. j . H. SCHNEIDER, R. CASSIR AND V. CHORDIKIAN, Biochim. Biophys. Acta, 42 (196o) 225. V. G. ALLFREY AND A. E. MIRSKY, Proc. Natl. Acad. Sci. U.S., 43 (1957) 589. S. N. NAYFEH, M. S. THESIS, University of Beirut, 1961". R. I. SALGANIK, T. M. MOROZOVAAND I. I. KIKNADZE, Proc. Acad. Sci. U.S.S.R. (Biochemistry Section), 124-129 (1959) 287. (Translation from the American Institute of Biological Sciences, Washington, D. C.) j. H. SCHNEIDER, Biochim. Biophys. Acta, 47 (1961) lO 7.
* Microfilm copies m a y be obtained from Jaffet Library, American University of Beirut, Beirut (Lebanon).
Biochim. Biophys. Acta, 61 (1962) 387-394
387
BBA 8138
T H E E F F E C T O F D E O X Y R I B O N U C L E A S E ON T H E I N C O R P O R A T I O N OF PRECURSORS INTO NUCLEAR RIBONUCLEIC ACID IN RECONSTITUTED RAT-LIVER HOMOGENATES J O H N H. S C H N E I D E R *
AND S H E H A D I
N. N A Y F E H * *
Department o/ Biochemistry, American University o/ Beirut, Beirut (Lebanon) (Received M a r c h 5th, I962)
SUMMARY
Isolated rat-liver nuclei were incubated with different concentrations of DNAase, and a m a x i m u m of 76 % of the "apparent D N A " {determined b y trichloroacetic acid extraction and diphenylamine reaction) could be removed in this way. The remaining 24 3/o represents a chromogen that is not DNA. After incubation, the nuclei were combined with a cytoplasmic fraction and the reconstituted homogenates were incubated with [8-1aC]adenine. Low concentrations of DNAase stimulated incorporation of adenine into nuclear RNA under a wide range of experimental conditions. Higher levels of DNAase inhibited the incorporation of adenine to a m a x i m u m of 22 % for nuclear RNA and 4 1 % for cytoplasmic RNA. When DNAase-treated nuclei were recombined with a cytoplasmic fraction, cytoplasmic RNA became associated with the nuclear fraction.
INTRODUCTION
If the genetic information carried b y DNA is transferred to nuclear RNA which is subsequently released to the cytoplasm, then the synthesis of nuclear RNA should be at least partly dependent on the presence of DNA. Evidence for such dependence has been obtained in systems from E. coli l-e, and in several cases, it has been clearly demonstrated that the amount of each base incorporated into RNA depends on the composition of the DNA present 5& A similar DNA requirement for precursor incorporation into RNA has been demonstrated in M. lysodeikticus v, L. arabinosus 8, and an enzyme system from pea embryos 9. These findings also apply to mammalian systems, since incorporation of precursors into RNA in rat liver 7,1°,n, calf-thymus nuclei 12-15 and rabbit-appendix nucleile, iv is decreased, and in some cases, completely abolished by incubation with DNAase. * P r e s e n t a d d r e s s : Biological A b s t r a c t s , 3 8 r 5 W a l n u t St., P h i l a d e l p h i a , Pa., U.S.A. (No reprints are available.) ** P r e s e n t a d d r e s s : P h a r m a c o l o g y D e p a r t m e n t , U n i v e r s i t y of N o r t h Carolina, Chapel Hill,
N. c. (U.S.A.). Biochim. Biophys. Acta, 61 (1962) 387-394
388
J.H.
SCHNEIDER, S. N. NAYFEH
In similar experiments with rat-liver homogenates in this laboratory, a stimulation of incorporation of precursors into nuclear RNA had been observed after removing part of the DNA with low concentrations of DNAase. In addition, even complete removal of DNA with DNAase resulted in only a small decrease in the incorporation of precursors into nuclear RNA. For this reason, a study of the incorporation of radioactive orotic acid and adenine into nuclear RNA as a function of DNAase concentration was undertaken. The results of this detailed study are presented in this paper. METHODS
Materials Rats were supplied by Tierzuchterei Brunger (Halle, Westfalen, Germany). DNAase (I × crystallized) and highly polymerized DNA were obtained from Worthington Biochemical Corporation (Freehold, New Jersey). E8-m4C]Adenine (specific activity, 26.2 mC/mg) was supplied by the Radiochemical Center (Amersham, England). E6-14C~Orotic acid hydrate (specific activity, 1.15 mC/mmole) was obtained from Tracerlab.
Incubation o[ homogenates with radioactive precursors and isolation o/nucleic acids Adult female rats were sacrificed and a 20 % homogenate of the excised liver was prepared in 0.25 M sucrose. Aliquots of the homogenate were transferred to 5o-ml Erlenmeyer flasks containing an equal volume of the following solution: succinate, 6 mM; pyruvate, 20 raM; glutamate, 20 mM; phosphate buffer (pH 7.4), 20 raM; magnesium chloride, 6 r a M ; niacin, I o m M ; ATP, o . 8 m M ; fructose, 12 raM; E8-1*C]adenine, 1.2#C/ml, or E6J*C]orotic acid (o.5/~C/ml); and sucrose, 0.25 M. Different concentrations of DNAase were added to each flask depending on the experiment, and the mixture was incubated with shaking in a water bath at 3 °0 for 45 rain. After incubation, the flasks were chilled in ice. Incubation mixtures were transferred to plastic tubes, rehomogenized with a motor-driven plastic pestle (also used during all subsequent resuspension and washing steps) to break up clumps formed during incubation, and then centrifuged at 6 o o × g for io min in a refrigerated centrifuge to sediment the nuclei. The nuclear pellet was resuspended in 0.25 M sucrose by homogenization, purified by underlayering with 0.34 M sucroselS, 19, and recentrifuged at 600 × g for IO rain. The supernatant was combined with the first cytoplasmic fraction. After precipitating and washing the nuclear and cytoplasmic fractions twice with ice-cold perchloric acid, the nucleic acids were extracted into hot IO % NaC1 under neutral conditions and were precipitated by the addition of 1.5 volumes of alcohol. Aliquots of these nucleic acid precipitates were taken for determination of specific activity. Since adenine is not incorporated into DNA by rat-liver homogenates (see RESULTS), it was not necessary to separate RNA from DNA in these experiments. Details of parts of this isolation procedure have been published previously 2°.
Incubation o] isolated nuclei with DNAase After preparing a 20 % rat-liver homogenate in 0.25 M sucrose at o °, 6-ml aliquots were transferred to round-bottom centrifuge tubes and centrifuged at 600 × g Biochim. Biophys. Acta, 61 (1962) 387-394
RNAASE AND INCORPORATION OF PRECURSORS INTO NUCLEAR RNA
389
for io rain at o °. The cytoplasmic fractions were then decanted and stored at o °. The nuclear pellets were resuspended in I ml of an incubation mixture containing 40 m M magnesium chloride, 2o m M phosphate buffer (pH 7) and DNAase of various concentrations depending on the experiment. After incubation of the nuclear suspension with DNAase at 3 °° for 2o min, the nuclei were sedimented by centrifugation at 6oo × g for IO min and the supernatants were discarded. The nuclei were then recombined with the corresponding cytoplasmic fractions, and 2.5-ml aliquots of these reconstituted homogenates were used for the standard incubation procedure described above.
Determination o/ the DNA content o/ nuclei After incubating the nuclei with DNAase, 2.5-ml aliquots of the reconstituted homogenate were centrifuged at 600 × g for io rain to reisolate the nuclei and the supernatant was discarded. The nuclear pellet was precipitated and washed twice with 5 % trichloroacetic acid at o °, and centrifuged at high speed to remove most of tile trichloroacetic acid from the tissues. DNA was then extracted from the nuclear pellet with 1. 3 ml of 5 % trichloroacetic acid at 90° for 15 rain and DNA was determined on the supernatant b y the DISCHE reaction 21 using highly polymerized DNA as the standard. RESULTS
When radioactive adenine (2. 4. lO 6 counts/rain) was incubated with 5 ml of a io % rat-liver homogenate using the standard incubation procedure, the combined nuclear nucleic acid gave 411, 432, and 46o counts/rain/plate for triplicate flasks. This combined fraction was quantitatively eluted from the plates, the RNA was hydrolyzed with o.2 N N a O H at 8o ° for 15 rain, and the unhydrolyzed DNA was precipitated by adding I N HC1 at o ° to a final concentration of o.I N. When the DNA (representing the total DNA originally present in the combined nucleic acid fraction) was quantitatively plated, the resulting plates gave 12, 7 and 7 counts/rain. This experiment established that adenine is not incorporated into DNA in normal rat-liver holnogenates. This is of interest since thymidine'is incorporated by an identical system ~2. Initial experiments were directed toward determining the conditions necessary for m a x i m u m removal of DNA and highest rate of precursor incorporation into RNA. The conditions presented in the experimental section are thought to be optimal in these respects. One finding of interest is given in Fig. I which shows that at two different magnesium chloride concentrations, a 25-rain preincubation of the homogenate at 3°° before addition of the radioactive precursor resulted in an almost complete loss of ability to incorporate precursors into RNA. As a result, preincubations with DNAase to remove DNA were limited to short periods of time. Using the whole homogenate system, concentrations as high as I mg/ml of crystalline DNAase could be preincubated with the homogenate before addition of the radioactive precursor without producing a marked inhibition of orotic acid incorporation into either nuclear or cytoplasmic RNA. In fact, stimulation was noted at low DNAase concentrations (discussed later). To investigate and magnify any specific effect of DNAase on nuclear RNA, nuclei were isolated from 2o % homogenates, incubated with DNAase, and then cenBiochim. Biophyx. Acta, 61 (I962) 387 394
39 °
J. H. SCHNEIDER, S. N. NAYFEH 400
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i
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30
35
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7" ///
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lb
15
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Fig. I. Effect of p r e i n c u b a t i o n w i t h MgCI~ on t h e i n c o r p o r a t i o n of [6-~4C]orotic acid into R N A of a r a t - l i v e r h o m o g e n a t e . 2.5-ml a l i q u o t s of a 20 % r a t - l i v e r h o m o g e n a t e were p r e i n c u b a t e d w i t h 6 m M M g C 1 2 (dotted l i n e ) o r 3 m M M g C I 2 (solid line) a t 3 °° for different i n t e r v a l s of t i m e (indicated on t h e abscissa). A f t e r p r e i n c u b a t i o n , t h e h o m o g e n a t e s were i n c u b a t e d with [6-14C]orotic acid u n d e r s t a n d a r d i n c u b a t i o n c o n d i t i o n s (see text), t h e c y t o p l a s m i c a n d n u c l e a r R N A were isolated, a n d t h e specific a c t i v i t y w a s d e t e r m i n e d as c o u n t s / r a i n / r a g R N A (indicated on t h e ordinate). P o i n t s a t zero t i m e r e p r e s e n t t h e a v e r a g e of d u p l i c a t e s a m p l e s .
trifuged to remove most of the enzyme. The DNA-depleted nuclei were mixed with the cytoplasmic fraction from which they were isolated and the reconstituted homogenates were incubated with [8-1*CJadenine. It was considered desirable to incubate a reconstituted homogenate rather than isolated nuclei with adenine so that the nuclei would not be limited b y absence of reactions necessary for the conversion of adei!me to the nucleotide level required for incorporation into nuclear RNA. In addition, use of the active cytoplasmic fraction cancels any undesirable effect that DNAase might have on reactions not specifically related to precursor incorporation (such as the inhibition of nuclear ATP synthesis b y DNAase*3). The effects of preincubating isolated nuclei with DNAase are shown in the upper half of Fig. 2. It m a y be seen that the incubation of isolated nuclei with 2 mg DNAase/ ml removed about 70 % of the "apparent DNA" from the tissue, leaving behind a diphenylamine-reactive chromogen which accounted for about 30 % of the "apparent DNA". The m a x i m u m amount of "apparent D N A " removed in a number of experiments was 76 %. Both in terms of rate of reaction with diphenylamine and spectra after reaction it is clear that the remaining chromogen is not DNA. (The chromogen slowly gives a violet colour with a m a x i m u m at 534 m# rather than the pure blue colour with a 6oo-m/, m a x i m u m given b y pure DNA.) In addition, increasing the DNAase concentration from 2 mg/ml up to 5 mg/ml did not remove this chromogen, as seen in Fig. 2. It m a y be seen from Fig. 2 that incubation of isolated nuclei with low concentrations of DNAase (0.25-0.75 mg/ml) resulted in a significant increase (maximum of 21 and 44 %) in the incorporation of radioactive adenine into nuclear RNA of reconstitutBiochim. Biophys. Acta, 61 (1962) 3 8 7 - 3 9 4
RNAASE
AND INCORPORATION OF PRECURSORS INTO NUCLEAR RNA
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Fig. 2. Effect of p r e i n c u b a t i n g isolated nuclei w i t h D N A a s e on t h e s u b s e q u e n t i n c o r p o r a t i o n of [8-~4C]adenine into n u c l e a r a n d c y t o p l a s m i c R N A of a r e c o n s t i t u t e d h o m o g e n a t e . Nuclei isolated f r o m 6-ml a l i q u o t s of 2o °/o r a t - l i v e r h o m o g e n a t e s (in o.25 iV/ sucrose solution) were p r e i n c u b a t e d for 2o rain in I 2 - m l t u b e s w i t h D N A a s e ( c o n c e n t r a t i o n s i n d i c a t e d on t h e a b scissa), 4 ° m M MgCI, a n d 2o m M p h o s p h a t e b u f f e r (pH 7.o). A f t e r i n c u b a t i o n t h e nuclei were sedimented by centrifugation and recombined with the cytoplasmic fractions from which they were initially s e p a r a t e d . O n e 2.5-ml a l i q u o t of each r e c o n s t i t u t e d h o m o g e n a t e w a s i n c u b a t e d w i t h [8-14C]adenine u n d e r s t a n d a r d c o n d i t i o n s (see t e x t ) in 5o-ml E r l e n m e y e r flasks a t 3 °° for 45 m i n . C y t o p l a s m i c (solid line) a n d nuclear( d o t t e d line) R N A were isolated a n d their specific a c t i v i t i e s were d e t e r m i n e d as i n d i c a t e d b y t h e t w o e x p e r i m e n t s ( O -- ©, • -- • ) in t h e b o t t o m g r a p h . F r o m a second 2.5-ml a l i q u o t of e a c h r e c o n s t i t u t e d h o m o g e n a t e , nuclei were reisolated b y c e n t r i f u g a t i o n a t 6 o o × g . D N A w a s e x t r a c t e d f r o m t h e nuclei b y trichloroacetic acid (at 9 °o for 15 min) a n d d e t e r m i n e d b y t h e d i p h e n y l a l n i n e reaction. T h e D N A c o n c e n t r a t i o n is g i v e n in t h e t o p g r a p h . All p o i n t s a t zero c o n c e n t r a t i o n of D N A a s e r e p r e s e n t the a v e r a g e of d u p l i c a t e d e t e r m i n a t i o n s .
ed homogenates. This same effect was noted in every other experiment where low levels of DNAase were tested. As shown in Table I, the maximum stimulation of incorporation of either adenine or orotic acid into nuclear RNA ranged from 19 to 118 °/o at concentrations of DNAase ranging from 0.2 to 0.6 mg/ml under a wide range of experimental conditions. Stimulation of incorporation of precursors into cytoplasmic RNA was also observed in some experiments 24, but not as consistently as that observed for nuclear RNA. It may be seen from Fig. 2 that as the DNAase concentration was increased to high levels, the specific activity of both cytoplasmic and nuclear RNA decreased. At a level of 4-5 mg DNAase/ml, the specific activity of nuclear RNA was decreased by a maximum of 12 °/o and 22 °/o of the control value (no DNAase added), and the specific activity of cytoplasmic RNA showed a maximum decrease of 39 o, /o and 41 °"o in the same experiments. Since all the DNA is removed with much lower levels of DNAase (2-3 mg/ml), it is clear that the values just presented represent the maximum Biochim. Biophys. Acta, 61 (i962) 3 8 7 - 3 9 4
392
j. H. SCHNEIDER, S. N. NAYFEH TABLE I
STIMULATION
OF INCORPORATION RECONSTITUTED
OF
RADIOACTIVE
HOMOGENATES
PRECURSORS AFTER
INTO
TREATMENT
NUCLEAR WITH
RNA
IN WHOLE
OR
DNAASE
Three t y p e s of e x p e r i m e n t s were performed: (a) D N A a s e was added to whole h o m o g e n a t e s during i n c o r p o r a t i o n of the radioactive p r e c u r s o r ([6-14C]orotic acid or [8-14C]adenine as indicated); (b) D N A a s e was p r e i n c u b a t e d w i t h a whole h o m o g e n a t e p r i o r to the addition of the radioactive precursor; a n d (c) after i n c u b a t i o n of D N A a s e with isolated nuclei, the nuclei were sedimented b y centrifugation and were recombined w i t h a cytoplasmic fraction as in the e x p e r i m e n t s given in Fig. 2. I n all cases, g r a d u a t e d low levels of D N A a s e (less t h a n i m g / m l in the incubated solution) were tested and duplicate or triplicate control flasks (no D N A a s e added) were incubated. Only the c o n c e n t r a t i o n of D N A a s e (either non-crystalline or I × crystallized D N A a s e as indicated) t h a t gave the m a x i m u m s t i m u l a t i o n is included in this table. The percentage s t i m u l a t i o n presented was observed at the D N A a s e c o n c e n t r a t i o n indicated. Type o] expt.
Radioactive precursor
DNAase concentration ( mglml )
a c c b* a c b** b***
Orotate Adenine Adenine Orotate Orotate Adenine Orotate Orotate
o. 4 o. 4 0. 5 o.6 0. 3 o. 5 o.4 o.2
Type o] DNAase
Non-crystalline Crystalline Crystalline Non-crystalline Crystalline Crystalline Non-crystalline Crystalline
Stimulation (percentage o~control)
19 2i 25 4° 42 44 72 i 18
" P r e i n c u b a t e d w i t h D N A a s e at o ° for 3 h. ** P r e i n c u b a t e d w i t h D N A a s e a t 3 °° for 3 ° min. *** P r e i n c u b a t e d w i t h D N A a s e at 3 °° for 15 min.
decrease in precursor incorporation produced b y complete removal of DNA in this system. I t should also be mentioned that one batch of DNAase had an inhibitory effect on precursor incorporation apart from its effect in removing DNA. With this DNAase, inhibition was proportional to DNAase concentration at concentrations of 2, 3, 4 and 5 mg DNAase/ml, even though m a x i m u m removal of DNA had been achieved at the 2 mg/ml level. D a t a on the effect of this batch of DNAase on precursor incor12
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Fig. 3. Effect of p r e i n c u b a t i n g isolated nuclei with D N A a s e on the relative a m o u n t s of nuclear (-- - --) and cytoplasmic ( ) R N A . The a m o u n t s of nuclear and cytoplasmic R N A were determined f r o m d a t a obtained in the e x p e r i m e n t s described in the legend for Fig. 2.
Biochim. Biophys. Acta, 61 (1962) 387-394
RNAAsE AND INCORPORATION OF PRECURSORS INTO NUCLEAR RNA
393
poration are not included in Fig. 2 since others ~7 have noted similar inhibitory effects and also attributed them to contamination of the DNAase preparation. Fig. 3 shows the amount of nuclear and cytoplasmic RNA as a function of DNAase concentration in two experiments. The amount of nuclear RNA showed a maxim u m increase of 75 and IiO °/o, and this increase was consistently observed in all similar experiments. The cytoplasmic RNA showed corresponding decreases, while the total amount of RNA remained constant, within experimental error. DISCUSSION
ALLYREY,~{IRSKYAND OSAWA12 have noted that when calf-thymus nuclei are treated with DNAase in sucrose (the conditions used in the present experiments) they lose 9 ° % of their DNA, but no more than 15 o~, of the histone is released even though these basic proteins are water-soluble and are known to be attached to DNA in the nuclei. They suggest that these chromosomal proteins are still arranged in some specific functional configuration. Retention of histone b y the nucleus after DNA removal leads to some interesting consequences, since this highly positively-charged protein must be neutralized b y the addition of polyanions such as heparin, chondroitin sulfate, polyethylene sulfonates, hyaluronic acid, RNA, or DNA before DNAase-treated nuclei will synthesize ATP or incorporate precursors into proteins or nucleic acidla, 'v. In the present experiments, when DNA-depleted nuclei are recombined with a cytoplasmic fraction, the cytoplasmic RNA can function as a polyanion which becomes associated with the nucleus. Such a phenomenon has been previously described b y SALGANIK et al. 25 who found a 2. 7- to 4.4-fold increase in the amount of nuclear RNA when DNAase-treated nuclei were exposed to a solution of RNA. This appears to be the best explanation of the direct relation between the concentration of DNAase and the increase in the amount of nuclear RNA noted in Fig. 3. It should be noted that after removal of the m a x i m u m amount of DNAase-labile DNA (at about 2.5 mg DNAase/ml), doubling the DNAase concentration did not cause further increase in the amount of nuclear RNA. The results of Fig. 3 show that cytoplasmic RNA is selectively bound to the nuclei, even though m a n y other anions are undoubtedly available in the cytoplasmic fraction. It can be calculated that as much as 38 °i, of the removed DNA has been replaced by cytoplasmic RNA. The data of Fig. 2 indicate that complete removal of DNA inhibits incorporation of adenine into nuclear RNA by a reconstituted homogenate to a m a x i m u m of 22 o~. This inhibition is very low compared to the almost complete inhibition observed in m a n y of the systems reviewed in the introduction '-~7. In addition, it would be expected that precursor incorporation into nuclear RNA would be inhibited much more than incorporation into cytoplasmic RNA. Actually, inhibition of cytoplasmic RNA reached a m a x i m u m of 4 1 % , which is almost double the 22 Uo m a x i m u m inhibition observed for nuclear RNA. Two explanations m a y be offered for this unexpected finding: (a) The cytoplasmic RNA which becomes associated with the DNA-depleted nuclei acts in the same way as exogenous polyanions which are known to restore the ability of DNAasetreated nuclei to incorporate precursors into RNA ~3,17. This would explain the failure to observe larger inhibition of precursor incorporation into nuclear RNA. (b) RNA of the "post-microsomal" and supernatant fractions is known to be the most active Biochim. Biophy,,..4cta, 6r (I962) 3 8 7 - 3 9 4
394
j . H . SCHNEIDER, S. N. NAYFEH
in incorporating precursors into RNA *n. These very active fractions (rather than the microsomal or mitochondrial fractions which contain relatively inactive RNA 2~) may be selectively associated with the nucleus, and may continue to be active after the association occurs. This would result in an increased specific activity of the nuclear RNA and a decreased specific activity of the cytoplasmic RNA. The two explanations just presented could be elaborated to explain most of the changes observed in Fig. 2, and it is clear that they must be considered when interpreting the data. However, they cannot be considered completely adequate until more is known about the effect of DNAase in stimulating precursor incorporation into nuclear RNA and until the fractions of cytoplasmic RNA that become associated with nuclear RNA after DNAase treatment have been investigated. ACKNOWLEDGEMENTS
This work was supported in part by grants from the Anna Fuller Fund and the National Science Foundation (G-9995 and G-5178), and was performed in partial fulfillment of a M.S. degree in Biochemistry by one of the authors (S.N.N.). REFERENCES 1 * 3 4 5
7 s 9 10 11 12 18 54 in is 17 18 19 20 ~1 22 23 24 26
2s
A. STEVENS, Biochem. Biophys. Research Communs., 3 (196o) 92. A. STEVENS, Federation Proc., 20 (I96I) 363 . j . HURWITZ, A. ]3RESLER AND R. DIRINGER, Biochem. Biophys. Research Communs., 3 (196o) 15. j . j . FURTH, J. HURWITZ AND M. GOLDMANN, Federation Proc., 20 (I96I) 363. j . j . FURTH, J. HURWITZ AND M. CTOLDMANN, Biochem. Biophys. Research Communs., 4 (I96I) 362. J. J. FURTH, J. HURWITZ AND M. GOLDMANN, Biochem. Biophys. Research Communs., 4 (I96I) 431 . S. B. WEiss, Federation Proc., 20 (1961) 362. S. OCHOA, D. P. BURMA, H. KROGER AND J. D. WEILL, Federation Proc., 20 (1961) 362. R. C. HUANG, N. MAHESHWARI AND J. BONNER, Biochem. Biophys. Research Communs., 3 (196o) 689. S. ]3. WEISS AND L. GLADSTONE, J. Am. Chem. Soc., 81 (1959) 4118. S. ]3. WEISS, Proc. Natl. Acad. Sci. U.S., 46 (196o) lO2O. V. G. ALLER]~Y, A. E. MIRSKY AND S. OSAWA, J. Gen. Physiol., 4 ° (1956) 451. V. G. ALLFREY AND A. E. MIRSKY, Proc. Natl. Acad. Sci. U.S., 44 (1958) 981. j. S. KRAEOW AND H. O. KAMMEN, Federation Proc., 19 (196o) 307 . B. B. ]3ISWA$ AND R. ABRAMS, Federation Proc., 20 (1961) 362. M. SEKIGUCHI AND A. SIBATANI, Biochim. Biophys. Acta, 28 (1958) 455. M. SEKIGUCHI AND A. SIBATANI, Biochim. Biophys. Acta, 34 (1959) 444. G. H. HOGEBOOM, W. C. SCHNEIDER AND i . J. STRIEBICH, J. Biol. Chem., 196 (1952) i i i . G. H. HOGEBOOM, W. C. SCHNEIDER AND M. J. STRIEBICH, Cancer Research, 13 (1953) 617. j . H. SCHNEIDER AND V. R. POTTER, J. Biol. Chem., 233 (1958 ) 154. Z. DISCHE, in E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Academic Press, Inc., New York, 1955, p. 285. j . H. SCHNEIDER, R. CASSIR AND V. CHORDIKIAN, Biochim. Biophys. Acta, 42 (196o) 225. V. G. ALLFREY AND A. E. MIRSKY, Proc. Natl. Acad. Sci. U.S., 43 (1957) 589. S. N. NAYFEH, M. S. THESIS, University of Beirut, 1961". R. I. SALGANIK, T. M. MOROZOVAAND I. I. KIKNADZE, Proc. Acad. Sci. U.S.S.R. (Biochemistry Section), 124-129 (1959) 287. (Translation from the American Institute of Biological Sciences, Washington, D. C.) j. H. SCHNEIDER, Biochim. Biophys. Acta, 47 (1961) lO 7.
* Microfilm copies m a y be obtained from Jaffet Library, American University of Beirut, Beirut (Lebanon).
Biochim. Biophys. Acta, 61 (1962) 387-394