The isolation of ribonucleic acid from plant, bacterial or animal cells

74

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95470

T H E ISOLATION OF RIBONUCLEIC ACID FROM PLANT, BACTERIAL OR ANIMAL CELLS

ROBERT

E. C L I C K * AND D A V I D P. H A C K E T T * *

Department o] Biochemistry, University of Cali]ornia, Berkeley, Cali]. (U.S.A.) ( R e c e i v e d F e b r u a r y i s t h , 1966)

SUMMARY"

I. An adaptation of the phenol procedure is described for the isolation of relatively undegraded ribonucleic acid from plant tissue, which is also useful for the isolation of ribonucleic acid from bacterial and animal cells. The novel aspects that make the isolation procedure feasible is the use of a basic buffer (pH 9.5) in the presence of versene. Ribonucleic acid was isolated from white potato tubers, the leaves, stems and roots of etiolated peas, Escherichia coli K-I2, rat fat pads, ribosomes from human reticulocytes, rabbit reticulocytes and rabbit liver. 2. The preparations, analyzed b y sucrose-gradient centrifugation, revealed that the two ribosomal RNA fractions of pea seedlings were present in equimolar concentrations and had molecular weights of 1.25" IOe and 5.7" lO6 estimated from sedimentation coefficients of 24.5 S and 16 S. 3. The molar ratios (cytidine :adenine :guanine :uridine) of the 25 S, 16 S, and 4 S RNA of pea seedlings were 22. 7:23.6:32.1:21.5, 2o.1:23.7:31.1:25.2, and 25.5 : 20.8 : 32.4 : 21.2, respectively. 4. Pea seedling leaves, which have a higher metabolic activity than stems and roots of the same tissue, also have a higher content of soluble ribonucleic acid (% of the total ribonucleic acid).

INTRODUCTION

Numerous procedures have been described for the isolation of total cellular RNA. Since many of the methods lead apparently to degradation of the RNA 1-3, or have been designed specifically either for microorganisms *-1°, plant tissue g-~3 or animal tissueg,l°, ~4-~8, comparative studies concerning RNA metabolism are difficult to evaluate. The literature concerning RNA metabolism in plant tissue is quite limited and * P r e s e n t a d d r e s s : D e p a r t m e n t of Zoology, U n i v . of W i s c o n s i n , Madison, Wisc. U.S.A. ** D e c e a s e d J a n u a r y 21, 1965.

Biochim. Biophys. Avta, ~29 (1966) 74-84

ISOLATION OF R N A

75

this may be due to the difficulty generally experienced in isolating RNA from plant tissue. The following report describes in detail an adaptation of the phenol procedure, which has been used previouslyn, zg, for the isolation of relatively undegraded RNA from plant tissue. An extension of this procedure to bacterial and animal cells is also presented.

MATERIAL AND METHODS

The following tissues were used in this study: white potato tuber (Solanum tuberosum, variety White Rose or Russet), 7-day old etiolated pea stems, leaves and roots s° (Pisum sativum, variety Alaska), Escherichia coli K-I2, rat fat pads, rabbit reticulocytes ix, rabbit liver, and ribosomes from human reticulocytes 2~. Water-saturated phenol, bentonite, sodium deoxycholate and buffer were used in the isolation of RNA. The ratio of phenol to buffer was 2 : I (v/v), but the ratio of tissue to buffer (w/v) was varied. For plant tissue, the latter ratio was 1:3; for mammalian or bacterial cells, it was important to increase this ratio to I : io in order to achieve quantitative yields of RNA and remove the protein in one extraction. The water-saturated phenol prepared b y dissolving I lb of phenol (Baker's analytical grade) in 16o ml of deionized or glass-distilled water s containing o.ooi M versene, was stored at 4 ° for use as needed. Other brands of phenol tested had to be distilled in order to obtain good yields of RNA preparations that resolved well on sucrose gradients. Addition to the phenol of a 0.5 % suspension of bentonite (5 g per 1 of buffer), prepared by the method of FRAENKEL-CONRAT, SINGER AND TSUGITA~3, Was essential to prevent RNA degradation during isolation from plant, bacterial or liver cells. On the other hand, o.I ~ bentonite, which prevented RNA degradation in the above cells, activated the nuclease present m,65,in lysates obtained from rabbit reticulocytes ~. These results support the finding that bentonite is a powerful nuclease inhibitor~, 2s-~, but suggest that it does not necessarily adsorb all cellular nucleases. Sodium deoxycholate (Difco) at a concentration of 1 % (I g per IOO ml of buffer) was used to facilitate the isolation of informational RNA. This detergent was used instead of sodium dodecyl sulfate, since the deoxycholate could be removed from the RNA preparation by precipitation of the RNA from the pH 5 buffer (see below) with ethanol ~8. The buffered solutions used in the isolation medium, o.I M sodium glycinateo.I M NaCl-o.oI M versene (pH 9-5) or o.I M sodium acetate-o.I M NaC1--o.oI M versene (pH 5.0) are modifications of those described b y GANGER AND KNIGHT11 and BROWN AND LITTI~Aze and are referred to here as the pH 9.5 and pH 5.0 buffers. The versene was used to prevent aggregation of the RNA molecules known to occur in the presence of Mg~+ (refs. 8, 29). In fact, use of Mg 2+ instead of versene in the buffers during the isolation of RNA from some plant tissue resulted in low yields of RNA and unsatisfactory sedimentation patterns (Fig. I). * S u b s t i t u t i o n of t a p water for t h e deionized water resulted in low yields of RIgA t h a t resolved poorly in sucrose-gradient centrifugation.

Biochim. Biophys. Acta, 129 (I966) 74-84

76

R. E. CLICK, D. P. HACKETT t

0.8

t

t

i

i

,~

led

E

oJ

0.2 0

0

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I

I

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5

10

15

20

25

30

35

TUBE NUMBER

Fig. I. S e d i m e n t a t i o n profiles of p o t a t o t u b e r I~NA isolated a t p H 9-5 (see t e x t ) a n d a t p H 7.3 (i m M T r i s - i m M MgCl,-o. 5 % b e n t o n i t e ) , i m l of R N A (12 a n d 16 A u n i t s of p H 9.5 a n d p H 7.3 buffers, r e s p e c t i v e l y ) w a s l a y e r e d on 3 ° m l of a linear g r a d i e n t of 2 to 20 % (w/w) a n a l y t i c a l r e a g e n t g r a d e s u c r o s e d i s s o l v e d in t h e p H 9.5 b u f f e r or i m M T r i s - i m M MgC1,-o.i M NaC1 (pH 7.3 ). T h e s u c r o s e g r a d i e n t s were p r e p a r e d a c c o r d i n g to t h e m e t h o d of BRITTEN AND :ROBERTSs°, e x c e p t t h a t a s m a l l m a g n e t i c s t i r r i n g b a r w a s u s e d in t h e m i x i n g c h a m b e r . A f t e r c e n t r i f u g a t i o n , t h e t u b e w a s p u n c t u r e d a n d t h e effluent w a s a n a l y z e d in a Gilford r e c o r d i n g s p e c t r o p h o t o m e t e r e q u i p p e d w i t h a f l o w - t h r o u g h cell (o.2-cm l i g h t p a t h w a s used; t h e a b s o r b a n c e w a s corrected to a I - c m l i g h t p a t h ) . T h e t r a c i n g s were a r b i t r a r i l y s u p e r i m p o s e d .

EXPERIMENTAL PROCEDURE

The degree of resolution, as judged from the sedimentation patterns of RNA, varied with the treatment used in preparing the source material for isolation. The best results were obtained when the tissue was frozen in liquid N,, either directly or in test tubes (for cell suspensions). Bacterial cells, which had been treated with lysozyme 31 before freezing, and mammalian cells were put in the isolation medium directly; plant cells and some bacterial suspensions were pulverized in a solid-CO, precooled mortar before they were added to the medium. The frozen tissue (plant, bacterial or animal) was then extracted with the appropriate phenol-buffer mixture at 4 ° (all subsequent steps were carried out at this temperature) in the 250 ml cup of a Servall Omni-mixer with the rheostat set at 20 (maximum setting of IOO). Higher settings resulted in the appearance of four ultraviolet-absorbing bands in the sedimentation profiles of pea-seedling RNA. The fourth peak was not characterized, but probably represents degraded RNA. The samples were blended for 15 min, then transferred to 4o-ml polyethylene tubes and centrifuged at 12 ooo ×g for 5 min to separate the phenol-buffer emulsion into two phases. The aqueous phase (upper) was removed b y suction and extracted twice with 3 volumes of cold anhydrous ether in a glass-stoppered graduated cylinder, to remove dissolved phenol. After removing most of the ether b y suction, 2 volumes of 95 % ethanol were added to precipitate the RNA. The mixture was placed at --20 ° for r5-3o min before it was centrifuged at 25 ooo × g for IO min. The RNA pellet was redissolved in the p H 9.5 buffer and precipitated 2-3 times from the buffer with 2 volumes of ethanol. The samples, dissolved in either buffer, could be stored at --20 ° for several months in the presence of 2 volumes of ethanol with no apparent degradation. However, the p H 5.0 buffer was preferred for storage. Biochim. Biophys. Acta, 129 (1966) 74-84

ISOLATION OF RNA

77

Prior to preparing the samples for analysis on sucrose gradients the R N A suspension was centrifuged, the ethanol-buffer supernatant discarded and then the pellet recentrifuged for 5 min at 25 ooo ×g. The tubes were then inverted and allowed to drain for 5-1o min at --20 ° before the pellet was dissolved in buffer for layering on gradients. The trace of ethanol still present did not affect the layering or the resolution of the RNA. Thus the time-consuming step of dialyzing the RNA to remove traces of alcohol prior to layering, a method routinely employed in m a n y laboratories was eliminated. The RNA (less than 35 absorbance units in I ml) was then applied to a 2-20 % (w/w) sucrose gradient (buffered with the p H 9-5 buffer) in a 34 ml plastic centrifuge tube. All tubes were previously soaked in o.I M versene for 24 h to remove an unknown ultraviolet-absorbing material which was otherwise extracted into the gradients b y the versene present in the buffers. The tubes were centrifuged in a Spinco SW 25.1 rotor at 25 ooo rev./min at 8 ° for 16 h for plant and bacterial preparations, and for 13 h for mammalian preparations.

RESULTS Although both buffers yielded apparently undegraded RNA (Fig. 2), there were quantitative differences in the extraction (compare Tubes lO-13). When the p H 9.5

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Fig. e. Sedimentation profiles of the :RblA (33 A units) of pea seedlings (the end io mm from the top of the stem) isolated at pH 9.5 and 5.o (see text). The RNA was analyzed on pH 9.5 buffered sucrose gradients (see Fig. i). After centrifugation the tube was punctured and I-ml aliquots collected. I ml of buffer was added to each tube before the absorbance was determined on a speetrophotometer.

buffer was used on 3 g of pea seedlings, re-extraction of the phenol phase with IO ml of buffer yielded less than IO % additional ultraviolet-absorbing material. Since this was similar in sedimentation profile to t h a t of the first fraction, the second extraction was not performed routinely. In contrast, the first extraction using the p H 5.0 buffer gave only 65-80 % of the extractable RNA. Re-extraction of the phenol layer with the p H 9-5 buffer yielded the remaining extractable ultraviolet-absorbing material (Table I). With bacterial and animal cells, the yield of RNA was also higher when the isolation was done at p H 9.5 than at p H 5.0. Although the p H 9-5 extract from pea seedlings contained some DNA, as Biochim. Biopkys. Acta, 129 (1966) 74-84

78

g.E.

TABLE THE

CLICK, D. P. HACKETT

I

EFFICIENCY

OF EXTRACTING

g o] pea seedlings used

3 9 io 3 4

RNA

FROM

PEA

SEEDLINGS

AT

p H 5.0 oR 9.5

p H ~ot b*Her err~otYed [or /i~st extraction

Absorbance units* xst extraction

2nd extraction**

% isolated in the ist extraction

9.5 9-5 9.5 5.0 5 .o

32.0 87.6 91.5 26.1 32.6

2.8 11.8 io.i 9.5 19.o

92 88 9o 73 {)4

* A b s o r b a n c e d e t e r m i n e d a t 260 mtt a n d e x p r e s s e d as t o t a l a b s o r b a n c e p e r ml. * T h e second e x t r a c t i o n w a s a l w a y s m a d e w i t h t h e 9.5 buffer.

determined b y the diphenylamine reaction ~2, the p H 5.o buffer extract did not ( s ~ Fig. 2, Tubes lO-13). BROWN AND LITTNA16 have, however, reported that the p H 5.0 buffer does extract DNA from animal tissue, and we have confirmed this. The DNA accompanying the isolated RNA from pea seedlings at p H 9.5 amounted to 6-1o ~o depending on the method of estimation (Table II). Since the ratio of RNA to DNA in this tissue ~ is approx. 9:1, it appears that the DNA and RNA were extracted to nearly the same extent.

TABLE II RATIO OF D N A TO R N A

IN THE FRACTION ISOLATED AT p H 9.5 FROM PEA SEEDLINGS

R N A w a s d e t e r m i n e d b y t h e o r i c i n o l m e t h o d 83. D N A w a s d e t e r m i n e d b y t h e d i p h e n y l a m i n e m e t h o d 32.

Expt. No.

Total #g R N A per g [resh weight

Total ttg D N A per g /resh weight

% DNA

I

I5OO

2 3

12oo 15oo

89 95 lO6

4*

--

--

IO

5*"

--

--

8

6

8 7

E s t i m a t e d from t h e i n c r e a s e d a b s o r b a n c e in t h e 4-S r e g i o n a f t e r d e o x y r i b o n u c l e a s e t r e a t m e n t (see Fig. 3). ** E s t i m a t e d f r o m t h e a b s o r b a n c e in t h e I7-S r e g i o n a f t e r r i b o n u c l e a s e t r e a t m e n t (see Fig. 4).

Even though Fig. 2 suggests that the contaminating DNA is in the 16-17 S region (Tubes IO-I3), it was further characterized b y two other methods. Treatment of an aliquot of the RNA with deoxyribonuclease (EC 3.1.4.5) resulted in a decrease in the relative area (Fig. 3) of the 16-17 S peak, an increase in the 4 S peak, and no change in the 25 S peak of a sucrose density-gradient pattern. This effect is demonstrated better b y summing the area underneath these peaks (Fig. 3). A similar aliquot treated with pancreatic ribonuclease (EC 2.7.7.17) revealed a ribonuclease-resistant, Biochim. Biophys. Acta, I29 (t966) 74-84

ISOLATION OF R N A

79

+ Deoxyrlbonucleose

- Oeoxyribonucleose 2.(

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34.6

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20

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Fig. 3. S e d i m e n t a t i o n profile of 25 A u n i t s a l i q u o t s of p e a s e e d l i n g R N A isolated at p H 9-5O n e a l i q u o t w a s a n a l y z e d directly. T h e o t h e r a l i q u o t w a s d i g e s t e d w i t h 5 / ~ g m d e o x y r i b o n u c l e a s e ( W o r t h i n g t o n , 2 × crystallized) p e r m l a t r o o m t e m p e r a t u r e for 2o rain in o.oi M s o d i u m a c e t a t e 1 m M MgClz ( p H 6.0) a n d 1.5/~gm d e x t r a n s u l f a t e p e r m l to p r e v e n t r i b o n u c l e a s e a c t i v i t y . P r i o r to d i g e s t i o n t h e R N A w a s d i s s o l v e d a n d p r e c i p i t a t e d 2 - 3 t i m e s f r o m o. i M s o d i u m a c e t a t e o . i M N a C I - I m M MgC12 (pH 5.0) w i t h e t h a n o l . A f t e r digestion, t h e s a m p l e w a s p r e c i p i t a t e d b y a d d i n g NaC1 to o. 1 M a n d 2 v o l u m e s of e t h a n o l . T h e pellet w a s t h e n d i s s o l v e d a n d p r e c i p i t a t e d t w i c e f r o m t h e p H 9.5 b u f f e r w i t h e t h a n o l to a t t e m p t to r e m o v e t h e Mg ~+ before p r e p a r i n g t h e s a m p l e for layering. T h e s a m p l e s were a n a l y z e d as d e s c r i b e d in t h e legend for Fig. 2.

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Fig. 4. S e d i m e n t a t i o n profile of 33 A u n i t s a l i q u o t s of p e a seedling R N A isolated a t p H 9.5 before a n d a f t e r t r e a t m e n t w i t h i / 2 g of p a n c r e a t i c r i b o n u c l e a s e p e r ml. T h e s a m p l e s were a n a l y z e d as d e s c r i b e d in t h e legend for Fig. 2, e x c e p t t h a t t h e r i b o n u c l e a s e - t r e a t e d a l i q u o t w a s n o t d i l u t e d w i t h b u f f e r p r i o r to t h e m e a s u r e m e n t of t h e a b s o r b a n c e . To c o m p a r e t h e curves, t h e u n t r e a t e d c o n t r o l h a s to be m u l t i p l i e d b y t h e d i l u t i o n factor 2. Fig. 5. S e d i m e n t a t i o n profile of 2o A u n i t s of r a b b i t r e t i c u l o c y t e (plus l y m p h o c y t e ) R N A isolated a t p H 9.5 before a n d a f t e r r i b o n u c l e a s e t r e a t m e n t . T h e s a m p l e s were a n a l y z e d as described in t h e legend for Fig. 4.

Biochim. Biophys. Acta, 129 (1966) 74-84

80

R . E . CLICK, D. P. HACKETT

ultraviolet-absorbing band in the 16-17 S area (Fig. 4), which corresponds to the deoxyribonuclease-sensitive portion of the sedimentation profile. Treatment of the RNA preparations with both deoxyribonuclease and ribonuclease resulted in the conversion of all the high molecular weight ultraviolet-absorbing material into products that sedimented in the 4 S area. The percent DNA contamination in the RNA preparations (Table II), calculated from these experiments, agrees with the values obtained b y direct determinations. This suggests that the major portion of the DNA in the RNA preparations sediments primarily in the 16-17 S region in sucrose gradients. RNA preparations from bacteria and animal tissue were similarly contaminated with DNA. As shown in Fig. 5 for rabbit reticulocytes (plus lymphocytes), the 5 DNA contamination from the lymphocytes in the RNA preparations sedimented in the 18-2o S region in sucrose gradients. While the p H 9-5 buffer could be used on all tissues studied, the p H 5.0 buffer could not be used effectively to extract RNA from some plant tissue. For example, RNA isolated from potato tubers, using the p H 5.0 buffer, was granular in appearance and resolved poorly in sucrose gradients. The protein content of pea seedling RNA preparations was about 3 % as determined b y the method of LOWRY eg al. 85, and was independent of the p H of the buffer used for the isolation. Re-extraction of the aqueous phase with phenol did not decrease this apparent contamination. To assess the extent to which the method outlined above m a y lead to RNA degradation, TMV-RNA was exposed to the entire p H 9.5 procedure, exploiting the finding of GmRERSe,s~ t h a t one break in the RNA molecule of TMV destroys its infectivity. A sample of purified TMV-RNA, prepared by the method of GIERER AND SCHRAMM 38, w a s divided into 4 equal parts and treated in the following manner. One part was mixed with frozen pea segments, which were then pulverized and treated as described above to isolate the RNA. The second part was added to the isolation medium after the pea stems had been ground in the Omni-mixer. The mixture was then carried through the remainder of the isolation procedure. The third part was added to a solution of I I °/o sucrose in p H 9.5 buffer and stored at 4 ° overnight. The 4th part was frozen and kept as a control. Each of these samples was then tested for infectivity on tobacco plants 39. Although the total extraction procedure (Table III) resulted in a 39 % loss of infectivity, this loss of activity could not be attributed to the pulverizing or Omni-mixer grinding of the tissue. Overnight storage in sucrose

TABLE

III

EFFECTS

OF

THE

ISOLATION

PROCEDURE

(pH 9.5) O N

Treatment o/ RNA

TMV-RNA

No. of lesions"

Control 3o7 Total isolation procedure 186 Total isolation procedure except grinding in the Omni-mixer 2o2 Stored in 9-5 buffer- I I ~o sucrose overnight at 4° 161 *

IO leaves assayed per experiment.

Biochim. Biophys. Acta, 129 (1966) 74-84

% loss of infectivity

-39 34 48

ISOLATION OF RNA

81

at p H 9.5 resulted in a 5o ~o loss of infectivity as expected, since RNA is labile to alkali. These results suggest t h a t over 60 ~o of the added TMV-RNA is essentially unaffected when isolated under these conditions, and therefore if the cellular RNA behaves as did the added RNA, it must be accordingly undegraded. Another method to detect breaks in RNA molecules has been described b y ~SPIRINL After heating at 9 °0 for io min, samples are subjected to centrifugation to detect any change in molecule size. We have found t h a t the heated samples of pea stem RNA give well resolved patterns similar to those obtained before heating suggesting that the three classes of RNA molecules suffer only minor damage, if any, during the isolation procedure.

CHARACTERIZATION

OF

RNA

FROM PLANT TISSUE

The sedimentation coefficients of the three RNA fractions of pea stems were determined at room temperature using a model E ultracentrifuge equipped with ultraviolet-absorption optics *°. The s20,w values obtained in o.I M NaC1 were 24-25 S, 16-17 S and 3.7-4 S, which are typical of RNA from other sourcesST,41,42 but different from values reported for this tissue b y both SPIRIN9 and TS'O AND SQUIRES~. The significance of this difference will be more extensively treated in a subsequent report. The molecular weight of the ribosomal RNA was estimated to be 5.8" lO 6 and 1.2. IOe using equations cited b y KURLAND42, and 5.6" lO 5 and 1.3.io e when compared to E. coti b y the method described b y SPIRIN 9. Thus, the 25-S RNA molecule is 2.0-2.3 times larger t h a n the I6-S molecule. The concentration of RNA was determined in two ways from the ultraviolet absorbance in each of the three fractions obtained b y sucrose gradient centrifugation. Assuming no DNA contamination when the isolation was carried out at p H 5.0, the areas under the curves (Fig. 2) give a direct indication of the relative amounts of the components. The second method assumes t h a t the DNA contaminant of the p H 9-5 extraction migrates in the 16-17 S region, and t h a t after deoxyribonuclease treatment, it is possible to calculate the relative amounts of the three types of RNA. As shown in Table IV, the two methods give values that are in good agreement. Moreover, the amount (A units) of the 25-S RNA was 2.1 times more than the I6-S material, a value very close to the ratio of the calculated molecular weights. Assuming

TABLE

IV

THE RELATIVE AMOUNTS OF 25-S , I 6 - S AND 4-S R N A ISOLATED FROM PEA STEMS AT p H 5.0 AND 9"5

% of total" RNA isolated Fraction

25 S x6 S 4S

pH 9.5""

pH 5.0

57.9 27.2 I4.9

26.8

57 .8 x5.4

* Total represents the sum of the areas under the 25, i6 and 4 S areas only. ** Corrected by comparing the area of the fractions before and after deoxyribonuclease treatment. Biochim. Biophys. Aaa, I29 (I966) 74-84

82

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CLICK, D. P. HACKETT

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0.8

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0.6 0.4 0.2 0

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0.75

0.9

0.50

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0.3

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20

30

40

lO

TUBE NUMBER

20

30

0

10

20

30

FRACTION NUMBER

Fig. 6. S e d i m e n t a t i o n p r o f i l e of 12. 5 A u n i t s of p e a r o o t a n d i o A u n i t s of p e a leaf R N A i s o l a t e d a t p H 9.5. T h e s a m p l e s w e r e a n a l y z e d on p H 9.5 b u f f e r e d g r a d i e n t s d e s c r i b e d i n Fig. i . Fig. 7. S e d i m e n t a t i o n profiles of 6 A u n i t s of r a t f a t - p a d 12. 5 A u n i t s of R N A f r o m s t a t i o n a r y - p h a s e E. coli, a n d c u l o c y t e r i b o s o m e s . All t h e R N A w a s i s o l a t e d a t p H 9.5 d i e n t s d e s c r i b e d in Fig. i. I n t h e case of t h e f a t cell R N A i n s t e a d of t h e o.2-cm cell.

R N A , 13 A u n i t s of r a b b i t - l i v e r R N A , 16 A u n i t s of R N A f r o m h u m a n r e t i a n d a n a l y z e d on p H 9.5 b u f f e r e d gra t h e o.5-c m f l o w - t h r o u g h cell w a s u s e d

that the areas under the curves give an estimate of the relative amounts of the various kinds of RNA in a cell, this suggests that the two ribosomal RNA molecules exist in pea stems in equimolar amounts. The molecular weight (2.8. IOe and 1. 4. io e) and concentration of ribosomal subunits in pea stem aa ribosomes intimate that the concentration of subunits is equimolar. If it is assumed that the RNA exists in the individual subunits as continuous polynucleotide chains, the calculated molecular weight of the RNA (40 % of the ribosomal mass ~) is 1.12. IOe and 5.6" lO 5, values that are similar to those found experimentally. These findings, with the previously discussed equimolar concenTABLE V NUCLEOTIDE

COMPOSITION

OF

THE 25-8 , I6-S AND 4-8 R N A OF PEA STEMS

T h e R N A p o o l e d f r o m s u c r o s e g r a d i e n t s w a s d i g e s t e d i n o.2 m l of 0. 3 M N a O H for 16 h a t 37 °. The mononucleotides, w i t h o u t neutralization, were then separated b y paper electrophoresis (CLICK,i n p r e p a r a t i o n ) b y a m o d i f i c a t i o n of t h e p r o c e d u r e of MARKHAM AND SMITH 46. I n d i v i d u a l m o n o n u c l e o t i d e s w e r e l o c a t e d w i t h a n u l t r a v i o l e t l a m p , e l u t e d w i t h o. 1 M HC1 a n d t h e i r conc e n t r a t i o n d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y 4 L S t a n d a r d d e v i a t i o n r a n g e d f r o m o.2o t o o.5o.

Fraction

25 S 16 S 4 S

Moles per i o o moles of nudeotides CMP

AMP

GMP

UMP

22.7 2o.1 25.5

23.6 23. 7 20.8

32.1 31.1 32. 4

21. 5 25.2 21.2

Biochim. Biophys. Acta, 129 (1966) 74-84

ISOLATION OF R N A

83

trations of the ribosomal RNA's, suggest that the 58-S and 39-S ribosomal subunits contain only 25-S and I6-S RNA respactively. The proportion of the total RNA present as sRNA in pea stems and E. coli was found to be similar .5. The sRNA content in young leaves from a given plant (Fig. 6) was higher than that in the stem and root. Since young leaves have a higher metabolic rate than stems, this content correlates with the rate. By contrast, E. coli cells in stationary phase contain much more sRNA than logarithmically growing cells45. The significance of the apparent contradiction is unclear at the moment. The base composition of the pea stem RNA, isolated from the individual peaks of IO gradients similar to Fig. 2, is summarized in Table V. The composition found is similar to that reported for other plant RNA preparationsl3, 4s-5°, and agrees well with that for total pea seedling ribosomal RNASS,5". The values obtained for sRNA, however, differed significantly from both those reported by LOENING51 and HUANG AND BONNER ~ .

RNA OF OTHER CELLS This method (the pH 9.5 buffer) m a y also be used for the isolation of RNA from animal and bacterial cells. Fig. 7 shows the absorbance density profiles of RNA isolated from rabbit liver, E. coli, human-reticulocyte ribosomes and rat fat cells ~. With the exception of the reticulocyte ribosome preparations, these materials contained DNA in the 16-2o S areas. The pH 9-5 buffer extraction method has been utilized b y us with equal success for the isolation of RNA from rat liver, Bacillus subtitis, chick fibroblasts, and chick embryo 5e.

ACKNOWLEDGEMENTS

This investigation was supported b y National Science Foundation Grant GB-I75I. One of us (R.E.C.) was a Predoctoral Fellow of the Public Health Service. We wish to express our appreciation to Dr. D. MAY for carrying out the experiments on viral infectivity, Drs. F. PUTNEY and H. K. SCHACHMANfor determining the sedimentation coefficients of the RNA, to Dr. P. A. MARKS for the generous supply of reticulocyte cells, and to Drs. C. A. DEKKER AND A. BENDICH for help in preparing this manuscript.

REFERENCES M. I. H. CHIPCHASE AND M. L. BIRNSTIEL, Proc. Natl. Acad. Sci. U.S., 49 (1963) 692. B. H. SELLS, Biochim. Biopkys. Acta, 8o (1964) 23o. J- D. WILSON, Proc. Natl. Acad. Sci. U.S., 50 (1963) 93. K. SHORTMAN AND H. FUKUHARA, Biochim. Biophys. Acta, 76 (1963) 5Ol. G. BRAWERMAN, Biochim. Biophys. Acta, 76 (1963) 322. F. GROS, H. HIATT, W. GILBERT, C. G. KURLAND, R. W. RISEBROUGH AND J. D. WATSON, Nature, 19° (1961) 581. 7 N. NOMURA, K. OKAMOTO AND K. ASANO, J. Mol. Biol., 4 (1962) 376. 8 W. M. SXANLEY, Jr. AND R. M. BOCK, Biochemistry, 4 (1965) I3O2. i 2 3 4 5 6

Biochim. Biophys. Acta, 129 (1966) 74-84

84 9 Io II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43 44 45 46 47 48 49 5° 51 52 53 54 55 56

R. E. CLICK, D. P. HACKETT A. S. SPIEIN, Biohhimiya, 26 (1961) 511. R. K. RALPH AND A. R. BELLAMY, Biochim. Biophys. Acta, 87 (1964) 9H. L. S•NGER AND C. A. KNIGHT, Biochem. Biophys. Res. Commun., 13 (1963) 455. J. H. CHERRY, H. CHROBOCZEK, W . J. G. CARPENTER AND A. RICHMOND, Plant Physiol., 4 ° (1965) 582. J. INGLE, J. L. KEY AND R. E. HOLM, J. Mol. Biol., I I (1965) 73 o. K. SCHERRER AND J. E. DARNELL, Biochem. Biophys. Res. Commun., 7 (1962) 486. R. A. C o x AND H. R. V. ARNSTEIN, Biochem. J., 89 (1963) 574. D. D. BROWN AND E. LITTNA, J. Mol. Biol., 8 (1964) 669. L. MONTAGNIER AND A. D. BELLAMY, Biochim. Biophys. Acta, 8o (I964) 157. K. S. KIRBY, Biochem. J., 96 (1965) 266. W . 13ENJAMIN AND A. GELLHORN, J. Lipid Res., 7 (1966) 285. R. CLELAND, Plant Physiol., 38 (1963) 78. H. 13ORSOOK, C. L. DEASY, A. J. HAAGEN-SMIT, G. KEIGHLEY AND P. H. LOWRY, J. Biol. Chem., 196 (1952 ) 669. A. S. WEISBERGER, S. ARMENTROUT AND S. WOLFE, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 86. H. FRAENKEL-CONRAT, ]3. SINGER AND A. TSUGITA, Virology, 14 (1961) 54" W . R. FARKAS, M. SINGER AND P. A. MARKS, Federation Proc., 23 (1964) 220. T. J. 13ROWNHILL, A. S. JONES AMD i . STACEY, Biochem. J., 73 (1959) 434. J. HUPPERT AND J. PELMONT, Arch. Biochem. Biophys., 98 (1962) 214. U. Z. LITTAUER AND M. SELA, Biochim. Biophys. Acta, 61 (1962) 6o9. D. M. 13LOW AND A. RICH, J. Am. Chem. Soc., 82 (196o) 3566. M. 13. SPORN AND W . DINGMAN, Biochim. Biophys. Acta, 68 (1963) 389. R. J. BRITTEN AND R. 13. ROBERTS, Science, 131 (196o) 32. U. Z. LITTAUER AND H. EISENBERG, Biochim. Biophys. Acta, 32 (1959) 32o. K. 13URTON, Biochem. J., 62 (1956) 315 . R. 13. HURLBERT, H. SCHMITZ, A. F. 13RUMMAND V. R. POTTER, J. Biol. Chem., 2o9 (1954) 23R. F. LYNDON, J. Exptl. Bot., 14 (1963) 419 . O. H. LOWRY, i'q. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . A. GIERER, Nature, 179 (I957) I297. A. GIERER, Z. Natur/orsch., 13b (1958) 788. A. GIERER AND G. SCHRAMM, Nature, 177 (1956) 702. H. FRAENKEL-CONRAT, The Viruses, I (1959) 429. K. LAMARS, F. PUTNEY, I. Z. STEINBERG AND H. K. SCHACHMAN,Arch. Biochem. Biophys., lO3 (1963) 379. 13. J. HALL AND P. DOTY, J. Mol. Biol., i (1959) i i i . C. G. KURLAND, ]. Mol. Biol., 2 (196o) 83. P' O. P. TS'O AND R. SQUIRES, Federation Proc., 18 (1959) 341. P. O. P. T s ' o , J. 13ONNER AND J. VINOGRAD, Biochim. Biophys. Acta, 3o (1958) 57 o. N. V. KJELDGAARD AND C. G. KURLAND, dr. Mol. Biol., 6 (1963) 341. R. MARKHAM AND J. D. SMITH, Nature, 168 (1951) 4o5. G. H. 13EAVEN, E. R. HOLIDAY AND E. A. JOHNSON, in E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Vol. I, A c a d e m i c Press, N e w York, 1955, p. 493. C. J. POLLARD, Bioehem. Biophys. Res. Commun., 17 (i964) 171. R. E. CLICK AND D. P. HACKETT, J . Mol. Biol., 17 (1966) 279. J. W. LYTTLETON, Biochem. J., 74 (196o) 82. U. E. LOENING, Biochem. J., 94 (1965) I I P . J. M. WALLACE AND P. O. P. T s ' o , Biochem. Biophys. Res. Commun., 5 (196I) 125. R. C. HUANG AND J. BONNER, Proc. Natl. Acad. Sci. U.S., 54 (1965) 96o. M. RODBELL, J. Biol. Chem., 239 (1964) 375. R. E. CLICK AND D. P. HACKETT, u n p u b l i s h e d findings. W. R. ROBERTS, Biochim. Biophys. Acta, lO8 (1965) 474.

Biochim. Biophys. Acta, 129 (1966) 74-84