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The incorporation of [3H]cytidine into ribonucleic acid of liver nuclei of young and old rats
BIOCHIMICA ET BIOPHYSICA ACTA
223
BBA 95083
T H E I N C O R P O R A T I O N O F [ 3 H ] C Y T I D I N E I N T O R I B O N U C L E I C ACID O F L I V E R N U C L E I O F Y O U N G A N D OLD R A T S H A R V E Y V. SAMIS, Jr., V. J. W U L F F AND J. A. FALZONE, Jr. Masonic Medical Research Laboratory, Utica, N. Y. (U.S.A.) (Received April 2nd, 1964)
SUMMARY
The ribonucleic acid of rat-liver nuclei labeled with [~H]cytidine and harvested from liver homogenates using a phenol-NaC1 mixture may be fractionated by successive extractions at temperatures between 2o and 75 °, using a phenol-NaC1 extraction medium. Successive extractions at any one temperature produce extracts whose ribonucleic acid content decreases markedly. The analysis of the specific activity of extracted nuclear ribonucleic acid indicates two fractions: (I) a relatively low specific-activity fraction obtained at 25, 40 and 5o°; and (2) a relatively high specific-activity fraction obtained at 65 and 75 °, temperatures at which measurable amounts of deoxyribonucleic acid are also extracted. It is demonstrated that InCl~ at appropriate concentration and ionic strength, effectively precipitates all soluble ribonucleic acid as well as all deoxyribonucleic acid as the indic nucleates. The indic nucleates are solubilized in o.38 N KOH which results in the precipitation of In(OH)3 in an exchange reaction that is complete in about 2 h at room temperature. In this way the ribonucleic acid may be hydrolysed to constituent nucleotides and the unhydrolysed deoxyribonucleic acid may be recovered by perchloric acid precipitation. The results of extraction experiments with nuclei harvested from liver of young and old adult rats indicate: (i) the specific activity of ribonucleic acid extracted from "old" liver nuclei is consistently higher than that extracted from "young" nuclei; (2) the amount of ribonucleic acid extracted from "young" liver nuclei is greater than that extracted from "old" liver nuclei; and (3) the amounts of deoxyribonucleic acid extracted from "young" and "old" liver nuclei are approximately equal.
INTRODUCTION
Autoradiographic studies have revealed a greater incorporation of tritiated cytidine into RNA in liver and other tissues of old adult mice and rats than in young adults a-z. These observations stimulated a biochemical investigation of this phenomenon involving the extraction 4 of nucleic acids from "phenolic" nuclei prepared according to the method of GEORGIEV AND IV[ANT'EVA5,6 from homogenized liver of young and old adult rats previously labeled with [3H]cytidine. Pilot experiments in which "phenolic" nuclei were extracted at various temperatures yielded results which Biochim. Biophys. Acta, 91 (i964) 223-232
224
H.V.
SAMIS, J r . , v . J. WULFF, J. A. FALZONE, Jr.
were erratic and sometimes difficult to reconcile with the autoradiographic findings. This failure to obtain reproducible results stimulated the development of the techniques described in this communication. The results of the nuclear extraction experiments are also reported. GENERAL METHODS
In preparation for the extraction of nucleic acids from rat-liver nuclei adult male rats, fasted for 18 h, were given [3H]cytidine intraperitoneally and decapitated I h later. The livers were removed and minced into ice-cold 0.05 M Tris buffer (pH 7.o), made o.14 M with respect to NaC1. The liver was homogenized with three vertical strokes of a motor-driven teflon plunger in a glass grinding vessel, filtered through 8 layers of gauze and brought to a final volume to make a IO ~o homogenate. The suspension was divided into 5o-ml portions, placed in I I o - m l centrifuge tubes with 50 ml of cold water-saturated phenol 4, shaken for 25 Inin at o ° and centrifuged for 5 rain at 2000 × g at o °. The phenol and aqueous layers were withdrawn, the nuclear layer was decanted into 4o-ml conical polypropylene tubes and the nuclei were resuspended in 5 ml of Tris-NaC1 and 3 Inl of water-saturated phenol and centrifuged at o ° at 225 ° ×g. This washing procedure was repeated 5 times and each time the aqueous and phenol layers were removed and fresh buffer and phenol were added. After the 5th wash the nuclei were resuspended in 5 Inl of Tris-NaC1, centrifuged at o ° and 2250 × g, the supernatant was drawn off and the nuclear pellet was resuspended in 24 ml of Tris-NaC1. Aliquots were taken for nuclear counts (2 Inl), dry weights (2 ml) and total nuclear analysis (5 Inl). The remaining nuclei were sedimented (2250 × g, 5 rain, o°), the supernatant was decanted and a medium, consisting of 15 ml of 0.05 M Tris buffer (pH 7.o), made o.14 M with respect to NaC1 and Io Inl of water-saturated phenol, was added. This suspension was used for the extraction schedules described below. All centrifugations were carried out in an International refrigerated centrifuge, Model P R 2, using heads No. 269, 845 and 295. All solutions and washes were routinely analyzed for ultraviolet absorption, using a Cary Model i i M recording spectrophotometer, and for radioactivity using a Packard Model 314 F liquid scintillation counter. SPECIAL METHODS
Extraction Schedule
Liver nuclei from 8 adult male rats labeled with [SH~cytidine (2 l,C per g body weight, specific activity 2.0 C/inmole) were prepared according to the procedure described above. After removal of aliquots for counting, weighing and total nucleic acid extraction, the nuclear preparation was extracted in Tris-NaC1 and watersaturated phenol for 3 successive I5-min periods at each of I2 temperatures, beginning at 20 ° and proceeding to 75 ° in steps of 5 °. After each I5-nlin extraction the aqueous and phenol phases were separated by brief centrifugation (2250 ×g, 2 rain., o °) and removed and the aqueous phase was extracted three times with twice its volume of absolute ethyl ether. The ether-extracted aqueous phase was then made 68 °/o with respect to ethanol. After overnight alcoholic precipitation of tile samples at --IO ° the precipitates Biochii~. Biophys. Acla, 91 (~964) 223-23-"
AGE-RELATED CHANGES IN RAT LIVER
RNA
METABOLISM
225
were sedimented at --IO ° and 225o×g. The supernatants were removed and the precipitates were washed once with absolute ethanol and once with absolute ethyl ether. The precipitates were dissolved in 5 ml of 0.38 N K 0 H and hydrolyzed at 37.5 ° for 18 h. The hydrolysates were acidified with 0.5 N HC1Q to precipitate unhydrolyzed macromolecular material and these precipitates were sedimented at 15 ooo ×g for I rain at o °. The supernatants were decanted and neutralized with KOH, and the resulting KC104 precipitates were sedimented as before. The supernatants were decanted and aliquots, suitably diluted, were used to determine the ultraviolet absorption spectra and radioactivity. The results are presented in Table I.
TABLE I ABSORBANCIES AND SPECIFIC ACTIVITIES OF R N A EXTRACTED FROM RAT-LIVER NUCLEI LABELED WITH [3H]cYTIDINE A b s o r b a n c y w a s m e a s u r e d a t ~m~x" Specific a c t i v i t y is e x p r e s s e d i n c o u n t s × lO 5 p e r m i n p e r u n i t of a b s o r b a n c y p e r ml. Absorbancy Extraction temp. (°C)
Specific activity Extraction No. z
Group
0.075 o.o6o
1.32 1.44
I
o.o75 o.145 0.340
-o.13o o.125
o.2i o.75 o.70
45 50
o.31o o.355
o.125 0.085
1.61 2.98
55 60 65
0.290 o.285 0.245
o.o9o 0.095 0.075
4.86 5.74 5.28
7° 75
o.13o o.o5o
o.o45 o.o35
2.52 0.65
Extraction NO. x
Extraction No. 3
20 25
0.560 o.o9o
3° 35 4°
2 3
4 5
I t was found that the hydrolysates from the last extraction period at each temperature contained less ultraviolet absorbing material than did the hydrolysates from the first extraction period at the same temperature and the first period at the next higher temperature (Table I). The specific activities of the R N A extracted at the various temperatures were grouped as shown in Table I. A second experiment using the temperature schedule suggested b y the 5 groups in Table I was undertaken. Liver nuclei from eight adult male rats labeled and prepared as described above were subjected to four I5-min extractions at 25 °, 4 o°, 50 °, 65 ° and 75 °. At 65 ° a fifth I5-min extraction was obtained. The extracts were treated as described and the results are presented in Table II. At each temperature the absorbancies of the successive extracts decrease, with the exception of 4o°--4, and the first extracts exhibit greater absorbancies than the last extract of the next lower temperature, with the exception of 75°--1. These results prompted us to adopt a i-h extraction at each of the temperatures listed in Table II. Biochim.
Biophys.
Acta,
91 (1964) 223-232
H.V. SAMIS, Jr., v. J. WULFF, J. A. FALZONE, jr.
226
TABLE
II
ABSORBANCIES AND SPECIFIC ACTIVITIES OF R N A EXTRACTED FROI~I RAT-LIVER NUCLEI LABELED ~VITFI [3H]cYTIDINE A b s o r b a n c y w a s m e a s u r e d a t '~max' S p e c i f i c a c t i v i t y is e x p r e s s e d i n c o u n t s >,~ lO 5 p e r m i n p e r u n i t of a b s o r b a n c y p e r m l . T h e f i g u r e s i n t h e c o l u m n h e a d i n g s i n d i c a t e t h e e x t r a c t i o n n u m b e r s . l"xtraction
,4 bsorbancy
temp.
r
:
3
25 4° 50 65 75
0.290 -0.705 o.815 o.o95
o. I i O o.145 0 . 2 7 ,5 o.275 o.o6o
0.050 o.i1o o.15o o.2o0 o.o5o
S p e c i / i c activity 4
5
0.040 o.17o 0.095 o.155 o.o4o
o. i 3 o
•
2
2.12 i.i0 6.57 4-43
2.30 0.73 1.96 6.1o 2.33
3
o,88 0,63 1,54 6,35 1,5S
4
].oo 0.43 2.Ol 5.76 o.95
5
5.44
The precipitation o/ R N A with InCl 3 During the experiments described above the alcoholic supernatants from the initial precipitation of the aqueous extracts of liver nuclei were found to contain sufficient radioactive material to give counts above background. Consequently, the effectiveness of the alcoholic precipitation was investigated. 2 ml of a 50//o solution of the sodium salt of yeast R N A (Schwarz) was diluted to a final volume of 3o ml with different concentrations of ethanol and precipitated overnight at IO °. The supernatants were decanted and their ultraviolet absorption spectra determined. The percentage of the total R N A precipitated was found to v a r y as a function of the ethanol concentration (Fig. I). The ethanol precipitate was then redissolved in 2 ml of Tris-NaC1 and reprecipitated overnight at IO ° with the same concentrations of ethanol. The percentage of the total RNA precipitated was not, in this case, a function of the ethanol concentration. The lack of complete precipitation with ethanol necessitated a more effective precipitation method. [nC1a is a precipitant for R N A and DNA at high H+ concen12
. 10
9 8 7
6
\ \' \ \
\
First precipltGtJon
5 <
4
\
3
2
2
1
Second
preopltGtlOn
0 6tO
710
8to
9ro
o/o Ethanol F i g . i. A g r a p h o f t h e p e r c e n t a g e R N A ( y e a s t ) n o t p r e c i p i t a t e d ( i . e . , r e m a i n i n g i n s o l u t i o n ) p l o t t e d a s a f u n c t i o n of e t h a n o l c o n c e n t r a t i o n . T h e c u r v e l a b e l e d " s e c o n d p r e c i p i t a t i o n " w a s obtained using the precipitated RNA from the "first precipitation". t3iochim.
Biophys..Ida,
91 (I9641 2 2 3 - 2 3 2
AGE-RELATED CHANGES IN RAT LIVER ] { N A
METABOLISM
227
trations and low concentrations of NaC1 (ref. 7). In order to determine if InC13 would be a more effective precipitant than ethanol, 2 ml of a 5 ~o solution of the sodium salt of yeast RNA in Tris-NaC1 was made to a final volume of 30 ml with different concentrations of InC13. Concentrations of InC13 above 3 mM precipitated essentially all of the RNA from this solution (Fig. 2). Measurement of the p H of each of these solutions (Fig. 2) indicated thai I n 3+ served to adjust the p H of the solutions to a sufficiently acid state to permit tile precipitation of the nucleic acids present, thus eliminating the necessity of adjusting the pI~. It was also determined that, under these conditions, all DNA present was precipitated. 100-
90-
so- "i 70 -
°", \
\
6O
5 i
•~. 50
[X
'
+o 30'
10
oi~.,, ,]
......................................... concn, lnCL3 (mivl)
Fig. 2. A g r a p h s h o w i n g t h e relation b e t w e e n t h e p e r c e n t a g e R N A (yeast) p r e c i p i t a t e d p l o t t e d as a f u n c t i o n of t h e c o n c e n t r a t i o n of InCt 3 (solid line). T h e d a s h e d line s h o w s t h e p H of t h e InC18-RNA solution in relation to t h e c o n c e n t r a t i o n of InCl.s.
Prior to hydrolysing the RNA in the form of the indic nucleate it was necessary to redissolve the precipitate and remove the In 3+ from the solution. In basic solution In 3+ forms insoluble In(OH)3. To determine if this property of In 3+ could be used to free the RNA and precipitate In 3+, 5 ml of 0.38 N K O H was added to indic RNA and the tubes were shaken until the precipitate was dissolved and the solutions were clear. The solutions were allowed to stand at room temperature for periods of time ranging up to 2 h after the addition of the base. After selected time periods, one sample was centrifuged for IO rain at 2500 ×g, and o °, one aliquot of the supernatant was diluted and measured spectrophotometrically, and a second aliquot was acidified with HC1 to precipitate any indic RNA. The precipitate was sedimented as Biochim. Biophys. Acta, 91 (1964) 223-232
228
H.V.
SAMIS,
Jr., v. j. WULFF, J. A. FALZONE, Jr.
before and an aliquot of this supernatant was diluted for measurement of its ultraviolet absorption spectrum. The acidified supernatant contained that nucleic acid fraction not complexed with In 3+. In this way, it was determined that complete removal of the In 3+ could be achieved in 2 h under these conditions (Fig. 3).
Total
RNA
2.11
_ _ 1----jj--
/;
/ r<
/
/ ,//
>~ u
g
/
x~ m <
/
Indium nucleate-KOH exchange r'eoctlon
"
~o / t
15 Elapsed
30 tffne
60 ( rain ) after
90 addition
12Q of
0.38N
KOH
Fig. 3. A g r a p h (solid line) s h o w i n g t h e t i m e course of t h e i n d i u m n u c l e a t e - K O H e x c h a n g e reaction. T h e c u r v e defines t h e rate a t w h i c h R N A is released f r o m t h e i n 3+ b y v i r t u e of t h e f o r m a t i o n of insoluble I n ( O H ) v T h e d a s h e d line defines' t h e t o t a l R N A (indic R N A plus soluble RNA) present.-
After the removal of the In s+ as insoluble In(OH)3 the basic supernatant containing RNA was placed in a thermostat at 37.5 ° for 18 h. After hydrolysis the samples were acidified with I-IC104, neutralized with KOI-I as before, dried over P205 in a vacuum dessicator and the dried samples were dissolved in o.5 ml of water. The remaining KC104 was removed by repeatedly freezing the samples in acetone-dry ice, thawing at o ° and filtering through Millipore filters of o.2 # porosity by means of a stainless steel Swinny hypodermic adapter (Millipore). The samples were then applied to strips of Whatman No. 541 filter paper which were subsequently placed in a Spinco model R Durrum type electrophoresis cell containing formate buffer at p H 3.5 at I o.o2. The samples were separated at a constant potential of 49 ° V. The resulting electrophoretograms, examined under an ultraviolet source giving a peak transmission at approximately 26o m#, showed four spots corresponding to standard spots of known commercial AMP, CMP, a M P and UMP.
A n extraction procedure/or nuclear R N A and D N A . Liver nuclei from 5 male adult rats labeled with I pC of EaH]cytidine per g body weight were prepared as described and were extracted successively at 25, Biochim. B i o p h y s . . q c l a ,
91 (I964) 223-232
AGE-RELATED CHANGES IN RAT LIVER R N A
229
METABOLISM
4 o, 50, 65 and 75L Extractions were for I h and two successive I-h extractions were carried out at 65 ° (65°--1 and 65°--2). The resulting aqueous solutions were extracted five times with ether, after which 2 ml of a 0.2 M aqueous solution of InC13 was added and, after IO rain, the precipitate was sedimented in the cold at IOOO×g for IO min. The supernatant was removed and the precipitate was washed once with ethanol-ether (I :3, v/v) and 4 times with absolute ether. The indic nucleates were solubilized and the In 8+ was precipitated as In(OH)3 b y the addition of 5 ml of 0.38 N K O H and the In(OH)3 was sedimented at IOOOXg for I o m i n in the cold. The supernatant was placed in a thermostat at 37.5 ° for I8 h. The unhydrolysed material was precipitated with I-IC1Q, the precipitate was sedimented and the supernatants were neutralized with K O H and the resulting KC104 sedimented. The ultraviolet absorption and specific activity of the neutral supernatants were determined and are presented in Fig. 4. The unhydrolysed macromolecular material, precipitated with
Absorbancy
of DNA
!!;!ii 1.5 ~
3
~..
r',-
0
E e<
+6
~ iiil;
1.0 >, @
Ea m >
×
•- -
c-
~ 0
..Q
iiiiti
Specific activity o f RNA
0.5
i~!~!: ~,
25
40 50 65 (i) Extraction. t e m p e r a t u r e
65(2)
75
Fig. 4. A b a r g r a p h s h o w i n g t h e specific a c t i v i t y of R N A (shaded bars) a n d t h e a b s o r b a n c y of D N A (open bars) e x t r a c t e d p e r lO 9 liver nuclei in relation to t h e e x t r a c t i n g t e m p e r a t u r e .
HC104 was redissolved, ultraviolet absorption spectra were determined, and aliquots were analyzed for radioactivity. These samples were found to contain very little radioactivity, to give a negative orcinol test s for RNA, and positive cysteine 9 and tryptophan 1° tests for DNA. I t was also found that these samples yielded no HC104Biochim. Biophys. Acta, 91 (1964) 223-232
230
H.V.
SAMIS, J r . , v. j. WULFF,
j.
A. FALZONE, Jr.
soluble material after 3 successive I8-h periods in o.38 N KOt{ at 37.5 °. The high specific activity RNA was extracted only at or above 65 °. The extraction o/ liver nuclei/rorn young and old adult rats Liver nuclei from 14 young (12o days) and 9 old (822 days) adult rats labeled with I/zC of [~H]cytidine per g body weight were prepared, extracted and analyzed as described. The results of the experiments with liver nuclei from young and old adult rats show similarities and differences. DNA, identified b y positive cysteine and tryptophan and negative orcinol tests, is extracted in measurable amounts at 65 ° and higher from both "young" and "old" nuclei (Fig. 5) and the total amounts extracted are similar (Fig. 5, extreme right). I~NA is extracted at all temperatures employed but the quantity and specific activity vary with temperature in a characteristic fashion (Figs. 5 and 6). Most of the nuclear RNA is extracted at 5 ° and 65 ° and the amount obtained from liver nuclei of young rats exceeds that from liver
2.5-
T
l
E K
~ l.5ui
g m
\/
RNA
DNA
I
6 E
~<
6*6 c oc~
1.6-
4
L_
o £)
<
< 0.5
25
40
/1
65(1) 65(2) Extraction t e m p e r a t u r e 50
75
TOTAL '25 "75
Fig. 5- The a b s o r b a n c y of R N A (shaded bars) and D N A (open bars) extracted from lo 9 liver nuclei at t e m p e r a t u r e s from 25 to 75 '~ is plotted, left to right, using the ordinate on the left. The two columns on the e x t r e m e right indicate the s u m of the absorbaneies of the extracted nucleic acids a n d are plotted using the ordinate on the right. The height of the left side of each b a r represents the average value for liver nuclei of y o u n g rats and the height of the r i g h t side of each b a r represents the average value for liver nuclei of old rats. The vertical lines t h r o u g h the average values indicate the range of the data.
nuclei of old rats (Fig. 5). The specific activity (Fig. 6) of the extracted RNA is relatively low at 25, 4 ° and 5 °° but increases markedly at 65 and 75 ° and the specific activity of the RNA from "old" nuclei (Fig. 6, shaded bars) is consistently higher than the specific activity of RNA extracted from "young" liver nuclei (Fig. 6, open bars). The radioactivity of all extracted DNA is much lower than that of RNA (Table I I I ) and all other solutions, except those known to contain RNA or its hydrolysis products, exhibited little or no radioactivity. Biochim. Biophys. ,:tcta, 91 (1904) 223 :z32
AGE-RELATED
CHANGES IN
RAT LIVER ~RNA
METABOLISM
231
3
r~ n"~ ~,
o oJ
2
~b u .-
oE
Q_o
1
Young iiii
25
40
50
65 (1)
65 (i
75
Extraction temperature F i g . 6. T h e specific a c t i v i t y (counts/min per unit of absorbancy at 2 6 0 m / , p e r m l ) of the extracted R N A from liver nuclei of y o u n g (open bars) and old (shaded bars) rats is s h o w n for the extraction temperatures 2.5-75 ° . The top of each bar represents the average value and the range is s h o w n b y the vertical line. TABLE
III
AVERAGE SPECIFIC ACTIVITY OF R N A AND D N A EXTRACTED AT 65 AND 75 ° FROM LIVER NUCLEI OF YOUNG AND OLD ADULT RATS
Speci/ic activity (counts/rain per unit o/absorbancy per ml)
"Young" nuclei "Old" nuclei
RNA
DNA
191 73 ° 335 3 ° 0
192 233
DISCUSSION
The possibilityof fractional extraction of R N A from rat-liver nuclei by the arbitrary extraction schedule used by us (based in part on the work of GEORGIEVAND ~¢[ANT'EVA~,~) is indicated by the two criteria used in the present study: amount and specific activity. The decrease in absorbancy with successive extractions at any one temperature suggests the exhaustion of that part of the nuclear R N A which is soluble under one set of conditions. This procedure would seem to permit a rather fine degree of fractionation of nuclear R N A but the functional significance of such an accomplishment is, at this time, obscure. The analysis of specific activity clearly indicates two Biochim. Biophys. Acta, 91 (1964) 2 2 3 - 2 3 2
232
H.V.
SAMIS, Jr., v. j. WULFF, J. A. FALZONE, jr.
different nuclear RNA fractions: an RNA fraction of relatively low specific activity (obtained at 25, 4 ° and 5 o°) and a relatively high specific activity fraction (obtained at 65 and 75°). These findings are in agreement with those of GEORGIEV AND MANT'ERAS,6 It is of considerable interest that the nuclear RNA of high specific activity is extracted only at those temperatures at which DNA is also extracted (Fig. 4). This was dramatically emphasized in our laboratory when a nuclear suspension being extracted at 5 °0 was inadvertently exposed to a temperature "shock" of 65 ° for 5 rain and was found to contain high specific activity RNA and a measurable amount of DNA. The experimental demonstration of fractional extraction of nuclear RNA was facilitated b y the use of the InC1a precipitation technique. Although we suspected some loss of nuclear RNA by virtue of incomplete precipitation with ethanol, we were surprised at the magnitude of the loss at 68 o,/o (Fig. i). It is of considerable interest that yeast RNA, which had just been precipitated b y ethanol, was virtually completely recovered after a second precipitation with ethanol. The implication of this observation will be explored. I t was gratifying to find that InC1 a completely precipitated all RNA from solution (Fig. 2). The results of the extraction of liver nuclei from young and old adult rats confirm and extend the earlier observations *-a which suggested that liver-cell nuclei in old mice and rats incorporate more [aH]cytidine than do liver nuclei of young adults. This enhanced incorporation in old animals is manifested as an increased specific activity of the extracted nuclear RNA. The differences in specific activity between "old" and "young" nuclear RNA are particularly striking in the t)5 ° extracts (Fig. 6). This is the temperature at which measurable amounts of DNA are first extracted, and where the specific activities of RNA from both old and young rat-liver nuclei are appreciably higher than those obtained at lower temperatures. This suggests that the R N A extracted at 65 ° and higher temperatures m a y contain a DNAassociated RNA of high specific activity, as proposed previously by GEORGIEV AND iVfANT'EVAS,6. It has been suggested 2 that the enhanced incorporation of [aH]cytidine into nuclear IRNA of liver of old rats m a y signify a compensatory increase in RNA synthesis produced by the occurrence of errors in "messenger" RNA. ACKNOWLEDGEMENTS
We wish to acknowledge the excellent technical assistance of M. P1EKIELNIAK, G. SFEIR, J . PELLERICO, D. SAMIS, J . L. LISZCZYNSKYJ, J . FAHY AND J . LOCHNEF.. This investigation was supported by grants NB o27o7 and H D ooo56 from the National Institutes of Health, Public Health Service, Department of Health, Education and Welfare. I(EFERENCES I V. J. WULVV, H. QUASTLER AND F. G. SHERMAN, Arch. Biochem. Biophys., 95 (1961) 548. V. J. \¥ULFF, H. QUASTLER AND F. G. SHERMAN, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1373. 3 V. J. WULFF, t-I. QUASTLER AND F. G. SHERMAN, J. Gerontol., 19 (t964) 294. 4 K. S. KIRBY, Biochem. J., 63 (1956 ) 405 • 5 G. P. GEORGIEV AND V. L. MANT'EVA, Biokhimiya, 27 (1962) 949. 6 G. P. GEORGIEV AND V. g. MANT'EVA, Biochim. Biophys. Acta, 61 (1962) 153 7 V~;. O. ALDRIGE, Nature, 187 (196o) 323 . 8 \V. MEJBAUM, Z. Physiol. Chem., 258 (1939) 117. 9 p. K. STUMPF, J. Biol. Chem., 169 (1947) 367 • 10 S. S. COHEN, J. Biol. Chem., 156 (1944) 691.
Biochim. Biophys. Acta, 91 (I964) 223 232
223
BBA 95083
T H E I N C O R P O R A T I O N O F [ 3 H ] C Y T I D I N E I N T O R I B O N U C L E I C ACID O F L I V E R N U C L E I O F Y O U N G A N D OLD R A T S H A R V E Y V. SAMIS, Jr., V. J. W U L F F AND J. A. FALZONE, Jr. Masonic Medical Research Laboratory, Utica, N. Y. (U.S.A.) (Received April 2nd, 1964)
SUMMARY
The ribonucleic acid of rat-liver nuclei labeled with [~H]cytidine and harvested from liver homogenates using a phenol-NaC1 mixture may be fractionated by successive extractions at temperatures between 2o and 75 °, using a phenol-NaC1 extraction medium. Successive extractions at any one temperature produce extracts whose ribonucleic acid content decreases markedly. The analysis of the specific activity of extracted nuclear ribonucleic acid indicates two fractions: (I) a relatively low specific-activity fraction obtained at 25, 40 and 5o°; and (2) a relatively high specific-activity fraction obtained at 65 and 75 °, temperatures at which measurable amounts of deoxyribonucleic acid are also extracted. It is demonstrated that InCl~ at appropriate concentration and ionic strength, effectively precipitates all soluble ribonucleic acid as well as all deoxyribonucleic acid as the indic nucleates. The indic nucleates are solubilized in o.38 N KOH which results in the precipitation of In(OH)3 in an exchange reaction that is complete in about 2 h at room temperature. In this way the ribonucleic acid may be hydrolysed to constituent nucleotides and the unhydrolysed deoxyribonucleic acid may be recovered by perchloric acid precipitation. The results of extraction experiments with nuclei harvested from liver of young and old adult rats indicate: (i) the specific activity of ribonucleic acid extracted from "old" liver nuclei is consistently higher than that extracted from "young" nuclei; (2) the amount of ribonucleic acid extracted from "young" liver nuclei is greater than that extracted from "old" liver nuclei; and (3) the amounts of deoxyribonucleic acid extracted from "young" and "old" liver nuclei are approximately equal.
INTRODUCTION
Autoradiographic studies have revealed a greater incorporation of tritiated cytidine into RNA in liver and other tissues of old adult mice and rats than in young adults a-z. These observations stimulated a biochemical investigation of this phenomenon involving the extraction 4 of nucleic acids from "phenolic" nuclei prepared according to the method of GEORGIEV AND IV[ANT'EVA5,6 from homogenized liver of young and old adult rats previously labeled with [3H]cytidine. Pilot experiments in which "phenolic" nuclei were extracted at various temperatures yielded results which Biochim. Biophys. Acta, 91 (i964) 223-232
224
H.V.
SAMIS, J r . , v . J. WULFF, J. A. FALZONE, Jr.
were erratic and sometimes difficult to reconcile with the autoradiographic findings. This failure to obtain reproducible results stimulated the development of the techniques described in this communication. The results of the nuclear extraction experiments are also reported. GENERAL METHODS
In preparation for the extraction of nucleic acids from rat-liver nuclei adult male rats, fasted for 18 h, were given [3H]cytidine intraperitoneally and decapitated I h later. The livers were removed and minced into ice-cold 0.05 M Tris buffer (pH 7.o), made o.14 M with respect to NaC1. The liver was homogenized with three vertical strokes of a motor-driven teflon plunger in a glass grinding vessel, filtered through 8 layers of gauze and brought to a final volume to make a IO ~o homogenate. The suspension was divided into 5o-ml portions, placed in I I o - m l centrifuge tubes with 50 ml of cold water-saturated phenol 4, shaken for 25 Inin at o ° and centrifuged for 5 rain at 2000 × g at o °. The phenol and aqueous layers were withdrawn, the nuclear layer was decanted into 4o-ml conical polypropylene tubes and the nuclei were resuspended in 5 ml of Tris-NaC1 and 3 Inl of water-saturated phenol and centrifuged at o ° at 225 ° ×g. This washing procedure was repeated 5 times and each time the aqueous and phenol layers were removed and fresh buffer and phenol were added. After the 5th wash the nuclei were resuspended in 5 Inl of Tris-NaC1, centrifuged at o ° and 2250 × g, the supernatant was drawn off and the nuclear pellet was resuspended in 24 ml of Tris-NaC1. Aliquots were taken for nuclear counts (2 Inl), dry weights (2 ml) and total nuclear analysis (5 Inl). The remaining nuclei were sedimented (2250 × g, 5 rain, o°), the supernatant was decanted and a medium, consisting of 15 ml of 0.05 M Tris buffer (pH 7.o), made o.14 M with respect to NaC1 and Io Inl of water-saturated phenol, was added. This suspension was used for the extraction schedules described below. All centrifugations were carried out in an International refrigerated centrifuge, Model P R 2, using heads No. 269, 845 and 295. All solutions and washes were routinely analyzed for ultraviolet absorption, using a Cary Model i i M recording spectrophotometer, and for radioactivity using a Packard Model 314 F liquid scintillation counter. SPECIAL METHODS
Extraction Schedule
Liver nuclei from 8 adult male rats labeled with [SH~cytidine (2 l,C per g body weight, specific activity 2.0 C/inmole) were prepared according to the procedure described above. After removal of aliquots for counting, weighing and total nucleic acid extraction, the nuclear preparation was extracted in Tris-NaC1 and watersaturated phenol for 3 successive I5-min periods at each of I2 temperatures, beginning at 20 ° and proceeding to 75 ° in steps of 5 °. After each I5-nlin extraction the aqueous and phenol phases were separated by brief centrifugation (2250 ×g, 2 rain., o °) and removed and the aqueous phase was extracted three times with twice its volume of absolute ethyl ether. The ether-extracted aqueous phase was then made 68 °/o with respect to ethanol. After overnight alcoholic precipitation of tile samples at --IO ° the precipitates Biochii~. Biophys. Acla, 91 (~964) 223-23-"
AGE-RELATED CHANGES IN RAT LIVER
RNA
METABOLISM
225
were sedimented at --IO ° and 225o×g. The supernatants were removed and the precipitates were washed once with absolute ethanol and once with absolute ethyl ether. The precipitates were dissolved in 5 ml of 0.38 N K 0 H and hydrolyzed at 37.5 ° for 18 h. The hydrolysates were acidified with 0.5 N HC1Q to precipitate unhydrolyzed macromolecular material and these precipitates were sedimented at 15 ooo ×g for I rain at o °. The supernatants were decanted and neutralized with KOH, and the resulting KC104 precipitates were sedimented as before. The supernatants were decanted and aliquots, suitably diluted, were used to determine the ultraviolet absorption spectra and radioactivity. The results are presented in Table I.
TABLE I ABSORBANCIES AND SPECIFIC ACTIVITIES OF R N A EXTRACTED FROM RAT-LIVER NUCLEI LABELED WITH [3H]cYTIDINE A b s o r b a n c y w a s m e a s u r e d a t ~m~x" Specific a c t i v i t y is e x p r e s s e d i n c o u n t s × lO 5 p e r m i n p e r u n i t of a b s o r b a n c y p e r ml. Absorbancy Extraction temp. (°C)
Specific activity Extraction No. z
Group
0.075 o.o6o
1.32 1.44
I
o.o75 o.145 0.340
-o.13o o.125
o.2i o.75 o.70
45 50
o.31o o.355
o.125 0.085
1.61 2.98
55 60 65
0.290 o.285 0.245
o.o9o 0.095 0.075
4.86 5.74 5.28
7° 75
o.13o o.o5o
o.o45 o.o35
2.52 0.65
Extraction NO. x
Extraction No. 3
20 25
0.560 o.o9o
3° 35 4°
2 3
4 5
I t was found that the hydrolysates from the last extraction period at each temperature contained less ultraviolet absorbing material than did the hydrolysates from the first extraction period at the same temperature and the first period at the next higher temperature (Table I). The specific activities of the R N A extracted at the various temperatures were grouped as shown in Table I. A second experiment using the temperature schedule suggested b y the 5 groups in Table I was undertaken. Liver nuclei from eight adult male rats labeled and prepared as described above were subjected to four I5-min extractions at 25 °, 4 o°, 50 °, 65 ° and 75 °. At 65 ° a fifth I5-min extraction was obtained. The extracts were treated as described and the results are presented in Table II. At each temperature the absorbancies of the successive extracts decrease, with the exception of 4o°--4, and the first extracts exhibit greater absorbancies than the last extract of the next lower temperature, with the exception of 75°--1. These results prompted us to adopt a i-h extraction at each of the temperatures listed in Table II. Biochim.
Biophys.
Acta,
91 (1964) 223-232
H.V. SAMIS, Jr., v. J. WULFF, J. A. FALZONE, jr.
226
TABLE
II
ABSORBANCIES AND SPECIFIC ACTIVITIES OF R N A EXTRACTED FROI~I RAT-LIVER NUCLEI LABELED ~VITFI [3H]cYTIDINE A b s o r b a n c y w a s m e a s u r e d a t '~max' S p e c i f i c a c t i v i t y is e x p r e s s e d i n c o u n t s >,~ lO 5 p e r m i n p e r u n i t of a b s o r b a n c y p e r m l . T h e f i g u r e s i n t h e c o l u m n h e a d i n g s i n d i c a t e t h e e x t r a c t i o n n u m b e r s . l"xtraction
,4 bsorbancy
temp.
r
:
3
25 4° 50 65 75
0.290 -0.705 o.815 o.o95
o. I i O o.145 0 . 2 7 ,5 o.275 o.o6o
0.050 o.i1o o.15o o.2o0 o.o5o
S p e c i / i c activity 4
5
0.040 o.17o 0.095 o.155 o.o4o
o. i 3 o
•
2
2.12 i.i0 6.57 4-43
2.30 0.73 1.96 6.1o 2.33
3
o,88 0,63 1,54 6,35 1,5S
4
].oo 0.43 2.Ol 5.76 o.95
5
5.44
The precipitation o/ R N A with InCl 3 During the experiments described above the alcoholic supernatants from the initial precipitation of the aqueous extracts of liver nuclei were found to contain sufficient radioactive material to give counts above background. Consequently, the effectiveness of the alcoholic precipitation was investigated. 2 ml of a 50//o solution of the sodium salt of yeast R N A (Schwarz) was diluted to a final volume of 3o ml with different concentrations of ethanol and precipitated overnight at IO °. The supernatants were decanted and their ultraviolet absorption spectra determined. The percentage of the total R N A precipitated was found to v a r y as a function of the ethanol concentration (Fig. I). The ethanol precipitate was then redissolved in 2 ml of Tris-NaC1 and reprecipitated overnight at IO ° with the same concentrations of ethanol. The percentage of the total RNA precipitated was not, in this case, a function of the ethanol concentration. The lack of complete precipitation with ethanol necessitated a more effective precipitation method. [nC1a is a precipitant for R N A and DNA at high H+ concen12
. 10
9 8 7
6
\ \' \ \
\
First precipltGtJon
5 <
4
\
3
2
2
1
Second
preopltGtlOn
0 6tO
710
8to
9ro
o/o Ethanol F i g . i. A g r a p h o f t h e p e r c e n t a g e R N A ( y e a s t ) n o t p r e c i p i t a t e d ( i . e . , r e m a i n i n g i n s o l u t i o n ) p l o t t e d a s a f u n c t i o n of e t h a n o l c o n c e n t r a t i o n . T h e c u r v e l a b e l e d " s e c o n d p r e c i p i t a t i o n " w a s obtained using the precipitated RNA from the "first precipitation". t3iochim.
Biophys..Ida,
91 (I9641 2 2 3 - 2 3 2
AGE-RELATED CHANGES IN RAT LIVER ] { N A
METABOLISM
227
trations and low concentrations of NaC1 (ref. 7). In order to determine if InC13 would be a more effective precipitant than ethanol, 2 ml of a 5 ~o solution of the sodium salt of yeast RNA in Tris-NaC1 was made to a final volume of 30 ml with different concentrations of InC13. Concentrations of InC13 above 3 mM precipitated essentially all of the RNA from this solution (Fig. 2). Measurement of the p H of each of these solutions (Fig. 2) indicated thai I n 3+ served to adjust the p H of the solutions to a sufficiently acid state to permit tile precipitation of the nucleic acids present, thus eliminating the necessity of adjusting the pI~. It was also determined that, under these conditions, all DNA present was precipitated. 100-
90-
so- "i 70 -
°", \
\
6O
5 i
•~. 50
[X
'
+o 30'
10
oi~.,, ,]
......................................... concn, lnCL3 (mivl)
Fig. 2. A g r a p h s h o w i n g t h e relation b e t w e e n t h e p e r c e n t a g e R N A (yeast) p r e c i p i t a t e d p l o t t e d as a f u n c t i o n of t h e c o n c e n t r a t i o n of InCt 3 (solid line). T h e d a s h e d line s h o w s t h e p H of t h e InC18-RNA solution in relation to t h e c o n c e n t r a t i o n of InCl.s.
Prior to hydrolysing the RNA in the form of the indic nucleate it was necessary to redissolve the precipitate and remove the In 3+ from the solution. In basic solution In 3+ forms insoluble In(OH)3. To determine if this property of In 3+ could be used to free the RNA and precipitate In 3+, 5 ml of 0.38 N K O H was added to indic RNA and the tubes were shaken until the precipitate was dissolved and the solutions were clear. The solutions were allowed to stand at room temperature for periods of time ranging up to 2 h after the addition of the base. After selected time periods, one sample was centrifuged for IO rain at 2500 ×g, and o °, one aliquot of the supernatant was diluted and measured spectrophotometrically, and a second aliquot was acidified with HC1 to precipitate any indic RNA. The precipitate was sedimented as Biochim. Biophys. Acta, 91 (1964) 223-232
228
H.V.
SAMIS,
Jr., v. j. WULFF, J. A. FALZONE, Jr.
before and an aliquot of this supernatant was diluted for measurement of its ultraviolet absorption spectrum. The acidified supernatant contained that nucleic acid fraction not complexed with In 3+. In this way, it was determined that complete removal of the In 3+ could be achieved in 2 h under these conditions (Fig. 3).
Total
RNA
2.11
_ _ 1----jj--
/;
/ r<
/
/ ,//
>~ u
g
/
x~ m <
/
Indium nucleate-KOH exchange r'eoctlon
"
~o / t
15 Elapsed
30 tffne
60 ( rain ) after
90 addition
12Q of
0.38N
KOH
Fig. 3. A g r a p h (solid line) s h o w i n g t h e t i m e course of t h e i n d i u m n u c l e a t e - K O H e x c h a n g e reaction. T h e c u r v e defines t h e rate a t w h i c h R N A is released f r o m t h e i n 3+ b y v i r t u e of t h e f o r m a t i o n of insoluble I n ( O H ) v T h e d a s h e d line defines' t h e t o t a l R N A (indic R N A plus soluble RNA) present.-
After the removal of the In s+ as insoluble In(OH)3 the basic supernatant containing RNA was placed in a thermostat at 37.5 ° for 18 h. After hydrolysis the samples were acidified with I-IC104, neutralized with KOI-I as before, dried over P205 in a vacuum dessicator and the dried samples were dissolved in o.5 ml of water. The remaining KC104 was removed by repeatedly freezing the samples in acetone-dry ice, thawing at o ° and filtering through Millipore filters of o.2 # porosity by means of a stainless steel Swinny hypodermic adapter (Millipore). The samples were then applied to strips of Whatman No. 541 filter paper which were subsequently placed in a Spinco model R Durrum type electrophoresis cell containing formate buffer at p H 3.5 at I o.o2. The samples were separated at a constant potential of 49 ° V. The resulting electrophoretograms, examined under an ultraviolet source giving a peak transmission at approximately 26o m#, showed four spots corresponding to standard spots of known commercial AMP, CMP, a M P and UMP.
A n extraction procedure/or nuclear R N A and D N A . Liver nuclei from 5 male adult rats labeled with I pC of EaH]cytidine per g body weight were prepared as described and were extracted successively at 25, Biochim. B i o p h y s . . q c l a ,
91 (I964) 223-232
AGE-RELATED CHANGES IN RAT LIVER R N A
229
METABOLISM
4 o, 50, 65 and 75L Extractions were for I h and two successive I-h extractions were carried out at 65 ° (65°--1 and 65°--2). The resulting aqueous solutions were extracted five times with ether, after which 2 ml of a 0.2 M aqueous solution of InC13 was added and, after IO rain, the precipitate was sedimented in the cold at IOOO×g for IO min. The supernatant was removed and the precipitate was washed once with ethanol-ether (I :3, v/v) and 4 times with absolute ether. The indic nucleates were solubilized and the In 8+ was precipitated as In(OH)3 b y the addition of 5 ml of 0.38 N K O H and the In(OH)3 was sedimented at IOOOXg for I o m i n in the cold. The supernatant was placed in a thermostat at 37.5 ° for I8 h. The unhydrolysed material was precipitated with I-IC1Q, the precipitate was sedimented and the supernatants were neutralized with K O H and the resulting KC104 sedimented. The ultraviolet absorption and specific activity of the neutral supernatants were determined and are presented in Fig. 4. The unhydrolysed macromolecular material, precipitated with
Absorbancy
of DNA
!!;!ii 1.5 ~
3
~..
r',-
0
E e<
+6
~ iiil;
1.0 >, @
Ea m >
×
•- -
c-
~ 0
..Q
iiiiti
Specific activity o f RNA
0.5
i~!~!: ~,
25
40 50 65 (i) Extraction. t e m p e r a t u r e
65(2)
75
Fig. 4. A b a r g r a p h s h o w i n g t h e specific a c t i v i t y of R N A (shaded bars) a n d t h e a b s o r b a n c y of D N A (open bars) e x t r a c t e d p e r lO 9 liver nuclei in relation to t h e e x t r a c t i n g t e m p e r a t u r e .
HC104 was redissolved, ultraviolet absorption spectra were determined, and aliquots were analyzed for radioactivity. These samples were found to contain very little radioactivity, to give a negative orcinol test s for RNA, and positive cysteine 9 and tryptophan 1° tests for DNA. I t was also found that these samples yielded no HC104Biochim. Biophys. Acta, 91 (1964) 223-232
230
H.V.
SAMIS, J r . , v. j. WULFF,
j.
A. FALZONE, Jr.
soluble material after 3 successive I8-h periods in o.38 N KOt{ at 37.5 °. The high specific activity RNA was extracted only at or above 65 °. The extraction o/ liver nuclei/rorn young and old adult rats Liver nuclei from 14 young (12o days) and 9 old (822 days) adult rats labeled with I/zC of [~H]cytidine per g body weight were prepared, extracted and analyzed as described. The results of the experiments with liver nuclei from young and old adult rats show similarities and differences. DNA, identified b y positive cysteine and tryptophan and negative orcinol tests, is extracted in measurable amounts at 65 ° and higher from both "young" and "old" nuclei (Fig. 5) and the total amounts extracted are similar (Fig. 5, extreme right). I~NA is extracted at all temperatures employed but the quantity and specific activity vary with temperature in a characteristic fashion (Figs. 5 and 6). Most of the nuclear RNA is extracted at 5 ° and 65 ° and the amount obtained from liver nuclei of young rats exceeds that from liver
2.5-
T
l
E K
~ l.5ui
g m
\/
RNA
DNA
I
6 E
~<
6*6 c oc~
1.6-
4
L_
o £)
<
< 0.5
25
40
/1
65(1) 65(2) Extraction t e m p e r a t u r e 50
75
TOTAL '25 "75
Fig. 5- The a b s o r b a n c y of R N A (shaded bars) and D N A (open bars) extracted from lo 9 liver nuclei at t e m p e r a t u r e s from 25 to 75 '~ is plotted, left to right, using the ordinate on the left. The two columns on the e x t r e m e right indicate the s u m of the absorbaneies of the extracted nucleic acids a n d are plotted using the ordinate on the right. The height of the left side of each b a r represents the average value for liver nuclei of y o u n g rats and the height of the r i g h t side of each b a r represents the average value for liver nuclei of old rats. The vertical lines t h r o u g h the average values indicate the range of the data.
nuclei of old rats (Fig. 5). The specific activity (Fig. 6) of the extracted RNA is relatively low at 25, 4 ° and 5 °° but increases markedly at 65 and 75 ° and the specific activity of the RNA from "old" nuclei (Fig. 6, shaded bars) is consistently higher than the specific activity of RNA extracted from "young" liver nuclei (Fig. 6, open bars). The radioactivity of all extracted DNA is much lower than that of RNA (Table I I I ) and all other solutions, except those known to contain RNA or its hydrolysis products, exhibited little or no radioactivity. Biochim. Biophys. ,:tcta, 91 (1904) 223 :z32
AGE-RELATED
CHANGES IN
RAT LIVER ~RNA
METABOLISM
231
3
r~ n"~ ~,
o oJ
2
~b u .-
oE
Q_o
1
Young iiii
25
40
50
65 (1)
65 (i
75
Extraction temperature F i g . 6. T h e specific a c t i v i t y (counts/min per unit of absorbancy at 2 6 0 m / , p e r m l ) of the extracted R N A from liver nuclei of y o u n g (open bars) and old (shaded bars) rats is s h o w n for the extraction temperatures 2.5-75 ° . The top of each bar represents the average value and the range is s h o w n b y the vertical line. TABLE
III
AVERAGE SPECIFIC ACTIVITY OF R N A AND D N A EXTRACTED AT 65 AND 75 ° FROM LIVER NUCLEI OF YOUNG AND OLD ADULT RATS
Speci/ic activity (counts/rain per unit o/absorbancy per ml)
"Young" nuclei "Old" nuclei
RNA
DNA
191 73 ° 335 3 ° 0
192 233
DISCUSSION
The possibilityof fractional extraction of R N A from rat-liver nuclei by the arbitrary extraction schedule used by us (based in part on the work of GEORGIEVAND ~¢[ANT'EVA~,~) is indicated by the two criteria used in the present study: amount and specific activity. The decrease in absorbancy with successive extractions at any one temperature suggests the exhaustion of that part of the nuclear R N A which is soluble under one set of conditions. This procedure would seem to permit a rather fine degree of fractionation of nuclear R N A but the functional significance of such an accomplishment is, at this time, obscure. The analysis of specific activity clearly indicates two Biochim. Biophys. Acta, 91 (1964) 2 2 3 - 2 3 2
232
H.V.
SAMIS, Jr., v. j. WULFF, J. A. FALZONE, jr.
different nuclear RNA fractions: an RNA fraction of relatively low specific activity (obtained at 25, 4 ° and 5 o°) and a relatively high specific activity fraction (obtained at 65 and 75°). These findings are in agreement with those of GEORGIEV AND MANT'ERAS,6 It is of considerable interest that the nuclear RNA of high specific activity is extracted only at those temperatures at which DNA is also extracted (Fig. 4). This was dramatically emphasized in our laboratory when a nuclear suspension being extracted at 5 °0 was inadvertently exposed to a temperature "shock" of 65 ° for 5 rain and was found to contain high specific activity RNA and a measurable amount of DNA. The experimental demonstration of fractional extraction of nuclear RNA was facilitated b y the use of the InC1a precipitation technique. Although we suspected some loss of nuclear RNA by virtue of incomplete precipitation with ethanol, we were surprised at the magnitude of the loss at 68 o,/o (Fig. i). It is of considerable interest that yeast RNA, which had just been precipitated b y ethanol, was virtually completely recovered after a second precipitation with ethanol. The implication of this observation will be explored. I t was gratifying to find that InC1 a completely precipitated all RNA from solution (Fig. 2). The results of the extraction of liver nuclei from young and old adult rats confirm and extend the earlier observations *-a which suggested that liver-cell nuclei in old mice and rats incorporate more [aH]cytidine than do liver nuclei of young adults. This enhanced incorporation in old animals is manifested as an increased specific activity of the extracted nuclear RNA. The differences in specific activity between "old" and "young" nuclear RNA are particularly striking in the t)5 ° extracts (Fig. 6). This is the temperature at which measurable amounts of DNA are first extracted, and where the specific activities of RNA from both old and young rat-liver nuclei are appreciably higher than those obtained at lower temperatures. This suggests that the R N A extracted at 65 ° and higher temperatures m a y contain a DNAassociated RNA of high specific activity, as proposed previously by GEORGIEV AND iVfANT'EVAS,6. It has been suggested 2 that the enhanced incorporation of [aH]cytidine into nuclear IRNA of liver of old rats m a y signify a compensatory increase in RNA synthesis produced by the occurrence of errors in "messenger" RNA. ACKNOWLEDGEMENTS
We wish to acknowledge the excellent technical assistance of M. P1EKIELNIAK, G. SFEIR, J . PELLERICO, D. SAMIS, J . L. LISZCZYNSKYJ, J . FAHY AND J . LOCHNEF.. This investigation was supported by grants NB o27o7 and H D ooo56 from the National Institutes of Health, Public Health Service, Department of Health, Education and Welfare. I(EFERENCES I V. J. WULVV, H. QUASTLER AND F. G. SHERMAN, Arch. Biochem. Biophys., 95 (1961) 548. V. J. \¥ULFF, H. QUASTLER AND F. G. SHERMAN, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1373. 3 V. J. WULFF, t-I. QUASTLER AND F. G. SHERMAN, J. Gerontol., 19 (t964) 294. 4 K. S. KIRBY, Biochem. J., 63 (1956 ) 405 • 5 G. P. GEORGIEV AND V. L. MANT'EVA, Biokhimiya, 27 (1962) 949. 6 G. P. GEORGIEV AND V. g. MANT'EVA, Biochim. Biophys. Acta, 61 (1962) 153 7 V~;. O. ALDRIGE, Nature, 187 (196o) 323 . 8 \V. MEJBAUM, Z. Physiol. Chem., 258 (1939) 117. 9 p. K. STUMPF, J. Biol. Chem., 169 (1947) 367 • 10 S. S. COHEN, J. Biol. Chem., 156 (1944) 691.
Biochim. Biophys. Acta, 91 (I964) 223 232