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Extraction of RNA from rat uterus
BIOCHIMICA ET BIOPHYSICA ACTA
328 BBA 96338
E X T R A C T I O N OF RNA FROM RAT U T E R U S PETERANNE
B. JOEL"
AND
DWAIN
D. HAGERMAN*"
Fearing Research Laboratory at the Boston Hospital for Women and the Department o/ Biological Chemistry and o[ Obstetrics and Gynecology, Harvard Medical School, Boston, Mass. (U.S.A.) (Received J u l y Ioth, 1969)
SUMMARY
Methods for the extraction of RNA from the immature rat uterus have been critically evaluated. Primary emphasis was placed upon minimizing degradation of RNA. The procedure of choice consists of extraction with phenol and sodium dodecyl sulfate at 5°o in the presence of bentonite, followed by deoxyribonuclease treatment of the extracted material. The product contains an average of 55 % of the total RNA and > 26 % of the rapidly-labeled RNA (2o-min pulse of V5-aHluridine) of the uterus. (The recoveries are based on values obtained by hot HC104 extraction of RNA.) Almost all of the rapidly-labeled RNA in the product is of high molecular weight, with sedimentation coefficients in the range 30-50 S on sucrose gradients, which suggests that degradation of the material has been minimized. Estradiol given intraperitoneally to immature rats 2, 4, or 12 tl before sacrifice did not affect the pattern of distribution of this high-molecular-weight, rapidlylabeled RNA (io-, 2o-, or 4o-min pulse of [5-aH]uridine) on sucrose gradients. Estradiol given intraperitoneally 20 rain before sacrifice to rats ovariectomized at 28 days and used for experimentation 18 33 days after ovariectomy also did not affect the pattern of distribution of rapidly-labeled RNA (Io-min pulse of [5-3H]uridine) on sucrose gradients.
I NTRODUCTION
Several studies 1-7 have indicated that estradiol increases the synthesis of RNA as one of the hormone's early effects upon the metabolism of the rat uterus. Furthermore, some investigations s 1~ have suggested that there may be qualitative differences in the RNA synthesized in the uterus under estradiol stimulation as compared with controls. Methods previously used to extract the total uterine RNA have generally yielded a contaminated and/or degraded RNA product as judged by either the nature of the procedure or by sucrose gradients of the product. These methods are not acceptable if one is looking for qualitative differences in the new RNA. We sought to develop a procedure for the extraction of RNA from the rat uterus which would yield an undegraded and uncontaminated product coupled with a reasonable recovery of all classes of RNA. * 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 C h e m i s t r y , L a w r e n c e U n i v e r s i t y , Appleton, W i s c o n s i n ** P r e s e n t address: D e p a r t m e n t of B i o c h e m i s t r y a n d D e p a r t m e n t of O b s t e t r i c s a n d G y n e cology, U n i v e r s i t y of Colorado School of Medicine, Denver, Colorado.
Biochim. Biophys. Aeta, 195 (1969) 328-339
UTERINE RNA
329
MATERIALS AND METHODS
Immature Wistar strain rats (Charles River Breeding Co., Boston, Mass.), 22-26 days of age, were used in all of the experiments unless indicated otherwise. Estradioltreated rats received 5 #g of I7/~-estradiol in 0.5 ml of o.154 M NaC1 containing 1 % ethanol injected intraperitoneally. Control animals received the same solution without estradiol. Standard procedure/or R N A isolation Each animal received 50 t*C of [5-3H~uridine IO, 20 or 4 ° rain prior to sacrifice. The rat was decapitated; the uterus was trimmed free of adipose tissue, was removed, and was placed in a tube suspended in a --75 ° ethanol bath. Each uterus was frozen within 2 rain after the rat was decapitated. Five to eight frozen uteri were placed in a conical ground glass Potter-Elvehjem type homogenizer containing 2 ml of 0.05 M sodium acetate buffer (pH 5.2) with I m g bentonite per ml, and 2 nfl of 88 % phenol. The uteri were homogenized about 30 sec with a 5-1o ° water bath around the tube. The homogenate was combined with an additional 1.6 ml of 0.05 M sodium acetate buffer (pH 5.2) with I m g bentonite per ml, 2.0 ml of 88 % phenol, and 0. 4 ml of IO % sodium dodecyl sulfate in 0.05 M sodium acetate buffer (pH 5.2). The mixture was then blended in a Lourdes homogenizer for 3o sec at 60 V operated at room temperature. The resulting homogenate was heated for 3 rain in a bath at 52 ° (final solution temperatule of 5 o°) while being mixed with a footed stirring rod rapidly raised and lowered through the mixture. The solution was cooled in an ice bath for 2 rain and centrifuged for 6 min at 20 ooo × g (4°). The phenol layer was discarded, leaving the interphase behind, and the extraction at 5 °° was repeated with 3.2 ml of 88 % phenol. The aqueous layer was removed and set aside at 4 ° after the second extraction. 4 mg of bentonite were added to this aqueous layer. To the remaining phenol layer and interphase were added 2.0 ml of 0.05 M sodium acetate buffer (pH 5.2), containing 2 mg bentonite, for a third partition. The aqueous layer from this third extraction was combined with the first aqueous layer, and the combination was centrifuged (15 rain at 20 ooo ×g, 4 °) to remove most of the bentonite. RNA (and DNA) were precipitated from the resulting supernatant fluid by the addition of NaC1 to o.15 M followed by 2 vol. of ethanol and storage overnight at --20 °. This procedure for the extraction of RNA follows that of WARNER et al. 14 with some modifications. The precipitate was collected by centrifugation and was washed twice with cold 95 % ethanol. The washed precipitate was treated with o.15 mg pancreatic deoxyribonuclease (electrophoretically pure, Worthington Biochemical Corp., Freehold, N.J.) in I.O ml of o.oi M Tris buffer (pH 7.5) containing o.ooi M MgCI2 with a 5-rain incubation at o ° followed by IO rain at 25 ° (ref. 15). (A larger amount of deoxyribonuclease was required to digest the DNA than was used by DIGIROLAMOet al. is, presumably as a result of residual bentonite in the extract.) The cooled solution was made to I mg/ml with bentonite and to 1 % sodium dodecyl sulfate, and was then extracted with 0.5 vol. of 88 % phenol for IO rain at IO°. After centrifugation as before, the aqueous phase was saved and the phenol and interphase were reextracted with 0.5 ml of Tris buffer (pH 7-5) containing o.ooi M MgC12. The combined aqueous phases were centrifuged as before to remove bentonite. The RNA was then preBiochim. Biophys. Acta, 195 (1969) 328-339
330
P . B . JOEL, D. D. HAGERMAN
cipitated from the supernatant fluid with NaC1 and ethanol for 1. 5 h at --20 °. The precipitate was collected by centrifugation, dissolved in 0.5 ml of o.ooi M MgC12 and was reprecipitated with 2 M potassium acetate, 25 % ethanol and chilling as described by DIGIROLAMOet al. 1~. The reprecipitation was performed 3 times. The final RNA precipitate was washed twice with cold 95 % ethanol.
Sucrose gradients lO-5O% sucrose gradients in o.oi M sodimn acetate buffer (pH 5) containing o.I M NaC1 were used unless otherwise indicated. The initial sucrose solutions were made with o.5 mg bentonite per 1111and were centrifuged before the gradients were prepared. RNA was dissolved in o.oi M sodium acetate buffer (pH 5) containing o.i M NaC1 and o.I ml of the solution was layered on the top of 4.9-ml gradients. The gradients were centrifuged at 4 ° for 165 rain at 65 ooo rev/min in the SW65 rotor of a Model L2-65 ultracentrifuge, Beckman Instruments, Palo Alto, Calif. The gradients were fractionated into about 35 sanlples by the collection of 6-drop samples from the bottom of the tube through a siphon. The absorbance at 260 nm was determined on each fraction in mierocuvettes. When necessary, a portion was diluted with water for the absorbance reading. For determination of radioactivity, o.I ml of each fraction was mixed in a vial with I.I ml of 0.3 M "Nuclear-Chicago solubilizer" in toluene (Nuclear Chicago Corp., Des Plaines, Ill.). 15 ml of scintillator solution (4 g 2,5diphenyloxazole and o.I g 1,4-bis-E2-(5-phenyloxazolyl)~henzene per 1 of toluene) were added and the vials were counted in a Nuclear-Chicago scintillation spectrometer equipped with an external standardization device. In all of the figures, the solid lines represent the absorbance at 260 nm and the dotted lines the disint,/min in o.I ml of the fractions.
Analyses For DNA and RNA analyses, samples were extracted with 0. 5 M HC1Q for 15 rain at 7 o°. DNA was analyzed on a portion of the HCI04 extract by the procedure of BURTO~16. Calf thymus DNA (Calbioehem, Los Angeles, Calif.) was used as the standard, assuming the absorbance at 260 nm of a I mg/ml solution to be 20. RNA was analyzed on a portion of the HC104 extract by a modification of the procedure of Dische and Schwarz ~7. The concentrations in the final mixture of reagent and sample were: orcinol, 2.67 mg/ml; ferric alum, 0.59 mg/ml; HC1, 4 M. The mixtures were heated 15 rain at Ioo °, cooled and read at 660 nm. Yeast RNA (Nutritional Biochemieals Co,, Cleveland, Ohio) was used as the standard.
Materials Labeled V5-~H~uridine (8.o C/mmole) was purchased from Schwartz Bio-Research, Orangeburg, N.Y. Bentonite was purified according to the procedure of BROWNHILL et al. is. Sodium dodeeyl sulfate was purified according to the procedure of CRESTFIELD et al. ~9. Phenol (Mallinckrodt 0025, liquified) was taken from a newly opened bottle for each experiment or was redistilled before use. RESULTS
Comparison o] [our extraction procedures A method was sought for extraction of RNA from rat uterus which would give reasonable recovely of all classes of RNA with minimum degradation. The uteri were Biochim. Biophys. Acta, 195 (1969) 328-339
UTERINE RNA
331
labeled in vivo with a 2o-min pulse of [5-~H]uridine in order to ascertain the extent to which rapidly-labeled RNA was extracted. Rapidly-labeled RNA is the most difficult class to extract and is also the most susceptible tc degradation. Due to the small size of the immature rat uterus, it was necessary to combine several uteri for a single extraction. In order to avoid degradation of RNA in uteri between excision and homogenization, uteri were frozen at --75 ° immediately upon excision. The rat uterus has unusually potent nucleases (unpublished observations; ref. 13). Therefore no attempt was made to fractionate the cell into nuclei and cytoplasm; instead, homogenization of the uteri was carried out in the presence of phenol and bentonite, both of which inhibit ribonuclease ls,2°-~2. The large amount of connective tissue in the rat uterus makes homogenization and extraction of nucleic acids difficult. (KIRBY 23 has shown that collagen absorbs DNA under certain conditions.) The combination of homogenization first with a Potter-Elvejhem homogenizer, followed by homogenization in the Lourdes homogenizer was the most effective method found for disrupting cells and connective tissue. For the extraction of RNA four methods using phenol from the laboratories of WARNER et al. 14, KIRBY 24, DIGIROLAMO et al. 15, and ATTARDI et al. 25 were compared for their applicability to the rat uterus. The four methods differ chiefly in the temperature at which the extraction is carried out. The final temperature of the mixture in the procedure of WARNER et al. is 5 o°, whereas the procedure of KIRBY uses 20 °, and the procedures of DIGIROLAMO et al. and ATTERDI et al. extract at 4 °. The proce dure of WARNER et al. uses sodium dodecyl sulfate in conjunction with phenol to release RNA at a p H of 2. 5. The procedure of KIRBY relies on 4-aminosalicylate to release DNA as well as RNA and uses a phenol-clesol mixture, containing 8-hydroxyquinoline, as deproteinizing agent. The procedures of DIGIROLAMOet al. and ATTARDI et al. are very similar, both using sodium dodecyl sulfate and naphthalene disulfate with phenol containing 8-hydroxyquinoline. They differ in the concentration of sodium dodecyl sulfate used and in the ionic and osmotic conditions of the aqueous phase. Bentonite was added to all the extraction media except in the procedure of KIRBY.
All four procedures extracted a large portion of the uterine DNA. In the procedure of KIRBY, DNA was removed by extraction with 3 M sodium acetate. The DNA in the extraction products from the other three procedures was hydrolyzed by deoxyribonuclease. The oligodeoxyribonucleotides were then removed with 2 M potassium acetate and 25 % ethanol. The modified Darnell extraction procedure, including removal of DNA b y deoxyribonuclease, is referred to and described in ~ATERIALS AND METHODS as t h e " s t a n d a r d p r o c e d u r e " .
The yield of total RNA and rapidly-labeled RNA in the final product using each of the four methods is presented in Table I. The results given in Table I under the heading HC104 indicate values obtained when nucleic acids are extracted with hot HCIO 4 as the basis of determining approximate recoveries by the other methods. Both the recovery of total RNA and rapidly-labeled RNA were by far superior in the product extracted b y the adopted standard procedure. The iecoveries averaged 55 % for total RNA and ~ 26 % for rapidly-labeled RNA for the standard prccedure as compared to hot HC104 extraction. Analysis of the final products from the four procedures by sucrose gradient centrifugation is shown in Fig. I. Only in the product extracted by the adopted Biochim. Biophys. Acta, 195 (1969) 328-339
332
P. B. JOEL, D. D. HAGERMAN
TABLE I
COMPARISON OF FOUR METHODS FOR THE EXTRACTIONOF R N A
FROM THE
IMMATURERAT UTERUS
Descriptions of the four m e t h o d s used for extraction are given in the legend to Fig. i. The last c o l u m n (HC104) indicates the values o b t a i n e d w h e n the frozen uteri were homogenized in 0. 5 M HC104, the precipitates w a s h e d 3 times w i t h cold 0. 5 M HC104, and R N A and D N A extracted w i t h 0.5 M HCIO~ at 7 °o for 15 miD. The values for the HC104 extraction are averages of eight uteri f r o m r a t s 22 d a y s of age.
Quantity isolated per uterus ,4 *
B
C
D
HCIO~
6o-~ io 6 4 ~ 12 i I 4oo~43oo
23** 57 96o
29 73 25oo
21 79 28oo
lO9 t53 44 5 ° 0
R N A (ktg) D N A (/,g)*** Total disint./min in R N A
• The values for Method A are the m e a n s of 6-8 separate e x p e r i m e n t s i S . D , • * The 3 M p o t a s s i u m acetate used in the m e t h o d of KIRBY r e m o v e s t R N A . "** ~'rior to r e m o v a l b y deoxyribonuclease or 3 M s o d i u m acetate.
~.5
t
2B,
-! 5oc
500 1 3O0
5~ ? 0 0 05 Ec oo
Z 100 z
300 i I0
2I0
i
110
3I0
210
3t0
D
1.5
E
~
-
~
"~ 300100"~ T~
-j ~-
(?.5
I0
2O
10
30 ER#,CTION
20
30
NUMBER
Fig. i. Sucrose g r a d i e n t analysis of the R N A extracted f r o m r a t u t e r u s b y four methods. The m e t h o d s used for e x t r a c t i o n and purification of R N A were as follows. A. WARNER el al. a4 as modified and described in detail in MATERIALS AND METHODS. ]3. KIRBY, Method 2 f r o m ref. 24. C. DIGIROLAMO et al. 1~, the conditions used for the D N A h y d r o l y s i s varied f r o m the published procedure in t h a t the deoxyribonuclease c o n c e n t r a t i o n w a s 25/~g/ml and the i n c u b a t i o n was for 5 miD at 25 °. D. ATTARDI et al. 25. I n all of the m e t h o d s , i m m a t u r e r a t s each received 5 ° #C of [5-DH~uridine 20 miD prior to sacrifice. Frozen uteri were homogenized in the combined a q u e o u s and phenol p h a s e s described for each m e t h o d . I n A, B and C, seven uteri were extracted w i t h an initial a q u e o u s plus phenol v o l u m e of 8.0 ml; in D, five uteri in an initial v o l u m e of 8.0 ml. I n A, C and D the sucrose gradients were p r e p a r e d as described in MATERIALS AND METHODS; in B the purified R N A was dissolved in o.i M s o d i u m acetate buffer (pH 5.2) and the gradients were p r e p a r e d in o.i M s o d i u m acetate buffer (pH 5.2). standard mentation
procedure
did the major
p o r t i o n of t h e l a b e l e d R N A
coefficients equal to or greater than
sediment
with sedi-
28 S.
EMect o/variations in the standard procedure upon the extraction o/ R N A Several conditions determine
whether
of t h e s t a n d a r d
procedure
were varied systematically
these conditions were critical and whether
Biochim. Biophys. Acta, 195 (1969) 328-339
to
the procedure might
UTERINE R N A
333
°I I' ,
/'\
I!
l
"\.
2.A /
• o
$
t~500 900
;st ~ Io
20
30
E
9oo
z iz
300
~o 20
2o
3o
20
Io
30
z
~I
~ t0
20
30 F~AC'ClO~
I0
3°0 2O
30
NUMIER
Fig. 2. Sucrose gradient analysis of the effect of variations in the standard procedure for extraction of RNA from rat uterus. Immature rats received 5o/~C of F5-3H]uridine 20 mid prior to sacrifice. In Expt. I each group contained six uteri; in Expt. II, five uteri. RNA was extracted according to the standard procedure (see MATERIALS AND METHODS) with individual variations as indicated below. In each group the final RNA product was dissolved in o.15 ml, and o. I ml was layered on the sucrose gradient. Expt. L A. Control; no variation. B. 1 % sodium dodecyl sulfate was present in the 2 ml of 0.05 M sodium acetate buffer added for the third extraction at 5o°. C. The final temperature of the extraction mixture at the end of the 3-mid heating was 6o ° rather than 5 o°. D. The uteri were cut into 2-ram slices and placed in o.154 M NaC1 at 4 ° rather than being frozen. Expt. II. A. Control; no variation. B. The initial homogenization of the uteri in the glass conical homogenizer was carried out in 2 ml of 0.05 M sodium acetate buffer (pN 5.2) with i mg bentonite per ml (no phenol present). Phenol was added prior to the subsequent homogenization in the Lourdes homogenizer.
Expt. I A RNA per uterus (/,g) Total disint./min in RNA per uterus Recovery of disint./min from gradient (%) be of to in
Expt. I I B
C
D
A
B
55
5°
56
5°
53
54
12 500
12 800
13 200
6850
12 4oo
18 ooo
64
79
37
80
75
87
i m p r o v e d (Fig. 2). T h e f o u r a l t e r a t i o n s , o m i s s i o n of f r e e z i n g of t h e u t e r i , o m i s s i o n p h e n o l f r o m t h e f i r s t h o m o g e n i z a t i o n , a d d i t i o n of m o r e s o d i u m d o d e c y l s u l f a t e t h e t h i r d e x t r a c t i o n a t 5 °0 , a n d i n c r e a s e of t h e f i n a l t e m p e r a t u r e of t h e m i x t u r e t h e e x t r a c t i o n s t e p s t o 60 °, h a d l i t t l e o r n o e f f e c t o n t h e t o t a l q u a n t i t y of R N A
e x t r a c t e d (Fig. 2). W h e n t h e u t e r i w e r e k e p t in s a l i n e r a t h e r t h a n b e i n g f r o z e n b e f o r e h o m o g e n i z a t i o n , c o n s i d e r a b l y less of t h e r a p i d l y - l a b e l e d R N A w a s e x t r a c t e d (Fig. 2, E x p t . I D). S i n c e t h e m a i o r p o r t i o n of t h e r a p i d l y - l a b e l e d R N A s e d i m e n t e d f a s t e r t h a n t h e 28-S R N A , t h e d e c r e a s e d a m o u n t of r a p i d l y - l a b e l e d R N A in t h e p r o d u c t is p r o b a b l y
Biochim. Biophys. Acta, 195 (1969) 328-330
334
P . B . JOEL, D. D. HAGERMAN
not due to degradation while the uteri were in saline. Freezing and thawing may facilitate release of rapidly-labeled RNA, or the uteri may be disrupted more effectively when they are frozen at the initiation of the homogenization. When the phenol was omitted from the first homogenization step, the total amount of rapidly-labeled RNA extracted increased (Fig. 2, Expt. II B). This finding may reflect the fact that the uteri are more easily homogenized when phenol is absent and may therefore be more thoroughly disrupted. A greater portion of the rapidly-labeled RNA extracted was of a size less than the 28-S RNA. Either the increased amount of rapidly-labeled RNA which was extracted actually has a lower molecular weight or, more likely, some degradation occurred when phenol was absent from the homogenization medium. Adding sodium dodecyl sulfate to the third extraction at 5 °° did not affect the amount of rapidly-labeled RNA extracted (Fig. 2, Expt. I C). The recovery of labeled RNA from the sucrose gradient was, however, greater with the RNA isolated in the presence of extra sodium dodecyl sulfate than with the control RNA. The lower recovery of labeled RNA flom the sucrose gradient in the control plus the shape of the curve of the control labeled RNA suggests that some material may have sedimented to the bottom of the tube (v.i.). Increasing the final temperature of the mixture to 6o ° in the extraction steps did not affect the amount of rapidly-labeled RNA extracted (Fig. 2, Expt. I B). The recovery of the RNA extracted at 6o ° (both labeled and unlabeled) from the sucrose gradient was very poor. The shape of the curves suggest that RNA had sedimented to the bottom of the tube. WAGNER et al. 26 have reported that extraction of RNA by the procedure of WARNER et al. at high temperatures (65 °) may result in noncovalently bonded aggregates of the two species of rRNA. The shape of tile absorbance curve with a large amount of absorbance at positions greater than 28 S for the material extracted at 6o ° suggests that a similar type of aggregation may have occurred here.
E//ect o/ varying conditions in the sucrose gradient analysis upon the high-molecularweight labeled R N A The possibility exists that the high-molecular-weight rapidly-labeled RNA (about 30-50 S) seen in the sucrose gradients represents aggregates of RNA. WAGNER et al. 26 reported aggregation of rRNA when the RNA was extracted at temperatures higher than 55 ° or when the RNA in the sodimn dodecyl sulfate buffer solution was more concentrated than 0. 5 mg/ml. According to their results this phenomenon would not be expected to occur to any great extent at the 5°° extraction temperature and RNA concentration of 0.2 mg/ml used in the present experiments. Although there is an occasional shoulder or trailing on the heavy side of the 28-S absorbance peak, the curves generally suggest little or no significant aggregation of rRNA. Under appropriate conditions rapidly-labeled RNA will associate with rRNA 27. This association apparently requires high concentrations of NaC1 (0.6 M) or the presence of Mg 2+ (o.oi M). The complex between rapidly-labeled RNA and rRNA disscciates only partially in o.I M NaC1 but separates completely in o.oi M Tris buffer (pH 7.0). The complex does not form at pH > 9-5 since the - N H - C O groups are dissociated and unable to form hydrogen bonds. Biochim. t~iophys. ,4cta, 195 (1969) 328-339
UTERINE RNA
335
40 ~A t ~ O 0
+ZB
500
~0
0c 2 I
~00 + I0
O~ 3r0
i 20
~00
t .... !
\..~
~00
~00
trio 210
° •'/
z
\
,~00i
\"~
zo *00
i i 10 ~10 ~0 F/ACTIONNUMBER
Fig. 3. Effect of variations in the sucrose gradient media on the s e d i m e n t a t i o n p a t t e r n of rapidlylabeled uterine RNA. I m m a t u r e r a t s received 5 o,uC of ~5-3HJuridine 20 rain (A and B) or 4 ° min (C) prior to sacrifice. Uterine RNA was extracted b y the s t a n d a r d procedure, i o - 5 o % sucrose gradients were p r e p a r e d in the media indicated below. The purified R N A was dissolved in the same m e d i u m for layering on the gradient. These R N A p r e p a r a t i o n s were also centrifuged on sucrose gradients prepared in o.oi M s o d i u m acetate buffer (pH 5.2) containing o.i M NaC1 and gave p a t t e r n s of d i s t r i b u t i o n of labeled R N A essentially the same as t h a t s h o w n in Fig. IA. A. 0.02 M g l y c i n e - N a O H buffer (pH 9-7) containing o.i M NaC1. B. 0.02 M glycine N a O H buffer (pH 9.7). C. 0.005 M Tris buffer (pH 7.2) containing o.oot M EDTA.
To determine whether association of rapidly-labeled RNA and rRNA was responsible for the rapidly-labeled RNA sedimenting between 3o and 5° S, RNA isolated by the standard procedure was centrifuged through sucrose gradients prepared in glycine buffer at pH 9.7 with or without o.I M NaC1 (Figs. 3A and 3 B) and in o.oo 5 M Tris buffer (pH 7.2) containing o.ooi M EDTA (Fig. 3C) to remove any residual Mg2÷. Increasing the pH to 9.7 did not decrease the portion of the rapidlylabeled RNA sedimenting between 3o and 5o S. The heavy side of the 28-S RNA absorbance curve was somewhat steeper at pH 9.7 and the rapidly-labeled RNA was slightly more concentrated toward 3o S. Therefore association of rapidly-labeled RNA with rRNA may contribute slightly but not to any great extent to the patterns seen in o.oi M sodium acetate buffer (pH 5) containing o.I M NaC1. In the gradients prepared at low ionic strengths in the presence of EDTA there was a shift of the peak of rapidly-labeled RNA toward 24-3o S. The shift in these latter gradients may reflect the fact that lowering the ionic strength reduces the sedimentation coefficients of a single stranded RNA.
E//ect o/the administration o~ estradiol and varying pulse times on the sucrose gradient sedimentation characteristics o/ rapidly-labeled uterine R N A The adopted standard procedure for extraction of uterine RNA was used to investigate whether the rapidly-labeled RNA made under estradiol-stimulation might contain a greater predominance of precursor rRNA of 45 and 32 S (refs. 28-30) than in the control. Estradiol-I7/~ was administered to immature rats 2 h prior to sacrifice. The F5-aHluridine (5o/~C) was injected IO, 20 or 40 rain prior to sacrifice. RNA was extracted by the standard procedure and analyzed on sucrose gradients. The labeled RNA from the control uteri gave the same pattern of distribution on sucrose gradients regardless whether the pulse time had been IO, 20 or 4 ° rain. The labeled RNA from the estradiol-treated animals gave the same pattern of distribution as the control RNA at each pulse time. No specific increase in radioactivity in the region of 45 and 32 S could be distinguished. Since all of these patterns were similar to that shown in Fig. IA, they are not included. In additional experiments estradiol-I7/~ was administered to immature rats at 4 or 12 h prior to sacrifice and [5-3HJuridine injected 20 rain prior to sacrifice. Biochim. Biophys. Acts, I95 (1969) 328-339
336
P.B. JOEL, D. D. HAGERMAN
Again the uterine rapidly-labeled RNA from estradiol-treated rats gave the same pattern of distribution on sucrose gradients as that from the controls. In these experiments on the effects of estradiol, an average of 6 8 ± 1 9 ~g (S.E., 9 groups) of RNA per uterus were isolated. This represents an average recovery of 62 % compared to RNA levels determined by hot HC104 extraction (Table I). (The group receiving estradiol 12 h prior to sacrifice is not included in the average since JERVELL et al. 31 have shown that there is a 30 O//oincrease in total uterine RNA by 12 h after administration of estradiol.) In the experiment in which estradiol was administered 2 h prior to sacrifice and I5-3Hluridine injected 20 rain prior to sacrifice, the specific activity of the isolated RNA was 85 % greater than that of the control. The same percent increase (89 %) was found when the total RNA was extracted by hot HC10 4 in a comparable experiment. Thus although the recovery of RNA is not quantitative, the increase in the amount of I5-aHluridine incorporated into RNA due to estradiol administration is evident to the same extent in the fraction of RNA isolated as in the total RNA. Although the uteri after 4 h of estradiol treatment were distended and obviously stimulated by the estradiol, the specific activity of the RNA was 30 % lower than the control level. The same decrease occurred when RNA was extracted by hot HC10 4. This finding is not interpreted as a decrease in RNA synthesis but rather as a consequence of a large increase in the size of the uridine nucleotide pool which is caused by estradio131. The E5-3Hluridine incorporated into F5-3HJUMP is diluted to a much greater extent by unlabeled UMP in the case of the estradiol-treated uteri than in the control. Effects of estradiol on the turnover rate 32 and specific activity of uterine nucleoside triphosphates 31,3a and possible effects on transport processes make changes in the specific activity of RNA uninterpretable without concomitant measurement of the specific activity of the immediate precursor (UTP in this case). E[lect o/estradiol on the sucrose density sedimentation characteristics oI rapidly-labeled uterine R N A [rom ovariectomized rats Rats were ovariectomized at 28 days of age and were used for experimentation 18-33 days after ovariectomy (I9O-25o-g rats). Ovariectomized rats received IO/~g of estradiol in 0.2 ml propanediol intraperitoneally 20 nfin prior to sacrifice and 50 /,C of E5-~Hjuridine in 0.2 ml of o.154 M NaC1 intraperitoneally io rain prior to sacrifice as described by HAMILTON et al. 4. RNA was extracted from the total uterus by the standard procedure and examined on a sucrose gradient. RNA isolated from uteri of estradiol-treated rats had the same pattern of isotope and mass distribution on the sucrose gradient as did the RNA from control animals. Both patterns resembled the pattern of RNA from immature rats. Four such experiments were carried out and the average total disint./min in RNA per uterus extracted from the estradioltreated rats (396O:klO5 o (S.E.)) in these four experiments was not significantly different from that of the controls (478o~:27oo). An average of 48:ki6 #g RNA per uterus were isolated.
DISCUSSION Of the four procedures for the extraction of RNA initially tested with rat uterus, the procedure developed in the laboratory of WARNER et al. 14 clearly gave the Biochim. Biophys. Mcta, 195 (1969) 328-339
UTERINE RNA
337
highest recovery both of total RNA and of rapidly-labeled RNA. Most likely it is the higher temperature (5°0 ) of extraction used in this procedure which is responsible for the higher extraction efficiency. However, unlike H e L a cells from which little if any DNA is extracted by the procedure of SCHERRER AND DARNELL34, a considerable portion of the uterine DNA is extracted and must be removed from the extract. Therefore, the final procedure found most suitable for extraction of RNA from rat uterus combines the extraction technique developed in the laboratory of WARNER et al. 14 with the removal of DNA from the product by procedures used in the laboratory of DIGIROLAMO et al. 15. The modifications introduced (freezing the tissue after excision, omitting fractionation of the cells, using phenol in the homogenization medium and keeping bentonite present whenever feasible) were for the purpose of minimizing degradation of RNA. The rapidly-labeled RNA extracted from uteri b y the procedure developed was predominantly of high molecular weight (i.e., with sedimentation coefficients in the range of 30-50 S). The fact that the sedimentation coefficients of the rapidly-labeled RNA changed very little when the sucrose gradients were carried out at p H 9.7 (at similar ionic strength) suggests that the high-molecular-weight rapidly-labeled RNA is not the result of aggregation of individual RNA molecules by hydrogen bonding. The possibility of residual DNA or protein causing aggregation of smaller molecules of rapidly-labeled RNA to give the material sedimenting between 30 and 50 S cannot be ruled out but seems unlikely for several reasons: the DNA digestion is extensive; the extracted RNA is treated a second time with phenol after the deoxyribonuclease treatment for removal of protein; and the general pattern of the labeled RNA on sucrose gradients is very reproducible and similar to that seen with other tissues when care is taken to avoid RNA degradation 25,34,s5. The existence of rapidly-labeled rRNA precursors of 32 and 45 S is well substantiated 2s-a°. CHAUDHURIAND LIEBERMAN~6 have reported the labeling in rat liver of 45-, 32- and I8-S RNA within 8 rain after intravenous injection of F14C~orotate. Rapidly-labeled RNA of 30-80 S differing from rRNA in base ratios has been observed in other tissues 14,25,3v-39. The primary disadvantage of the standard procedure is the fact that recovery of total RNA averaged 55 % while that of the rapidly-labeled RNA was probably lower. Comparison of the amount of radioactive label in the RNA extracted by the standard procedure with the amount of label extracted by hot HC1Q suggests an average recovery of only 26 % for rapidly-labeled RNA. However, it is quite likely that the hot HC104 extract contains some short (incomplete) labeled oligonucleotides and possibly some contaminating E5-3H~uridine and acid-soluble [5-3Hluridine nucleotides. In contrast, the RNA isolated by the standard procedure contains very little labeled material with a sedimentation rate less than 4 S. Therefore the average recovery of RNA ~ 4 S with the standard procedure is probably significantly higher than 26 %. The large amount of connective tissue in the uterus makes it difficult to homogenize the tissue completely and to extract RNA by any method which does not degrade the RNA. This is presumably the major factor in the low recoveries. The major advantage of the standard procedure is the fact that the rapidlylabeled RNA isolated is of high molecular weight indicating that RNA degradation has been minimized. In addition, DNA and protein are specifically removed from the product. For studies of possible qualitative changes in the nature of RNA produced under hormonal stimulation, it is essential to avoid degradation of RNA and to reBiochim. Biophys. dcta, I95 (I969) 328-339
338
P.B.
J O E L , D. D. HAGERMAN
move contaminants which may be bound to RNA and influence its behavior. (The work of LEADERAND BERRY4° and JACKSONAND SELLS41 suggests that the differences in countercurrent distribution profiles of rapidly-labeled liver RNA from hormonetreated and control rats observed by KIDSON AND KIRBY8 may have been introduced during preparation of the RNA.) Although recovery of RNA by the standard procedure is not quantitative, the relative specific activities of uterine RNA from control and estradiol-treated rats have been the same whether isolated by the standard procedure or hot HC1Q. Hence the standard procedure does seem to isolate a similar sample of RNA from uteri of control and estradiol-treated ra~s. Although the standard procedure would not be applicable in studies requiring rigorous quantitative recovery of RNA, it would be applicable in studies of possible qualitative changes in RNA due to estradiol. There has been speculation 42 that estradiol may specifically increase the synthesis of rRNA as one of the earliest effects on the uterus. Although no specific increases in RNA with the sedimentation values of precursor rRNA were found 2o rain, or 2, 4, or 12 h after estradiol administration, the limited resolving power of tile sucrose gradient technique makes further study of this question necessary. In 1965 GORSKI AND NELSON4a performed experiments similar to some reported here. The rapidly-labeled RNA which they isolated, however, sedimented predominantly between 5 and 2o S in contrast to the 3o-5o-S rapidly-labeled RNA iso-. lated by the procedure in this paper. HAMILTON"and co-workers4-v reported that the rate of incorporation of i5-3H] uridine into rapidly-labeled RNA in the ovariectomized rat uterus was accelerated 5-6-fold within 20 nlin after estradiol was injected intraperitoneally. Judging from tile DNA concentrations and weights reported for the uteri of their ovariectomized rats 5, HAMILTON and co-workers used rats ovariectomized after they had reached maturity. In the similar experiments reported in the present paper, the rats used were ovariectomized while immature (28 days). Whether the difference in age at which the rats were ovariectomized can account for the failure to repeat the observation of HAMILTONand co-workers remains to be investigated. ACKNOWLEDGMENTS
We thank Mrs. Sandra Cressman for capable technical assistance. This investigation was supported in part by National Institutes of Health Research Grant No. AM-II846 and its predecessors, National Institute of Arthritis and Metabolic Diseases. REFERENCES i 2 3 4 .5 6 7 8 9 IO
J. GORSKI AND J. NICOLETTE, Arch. Biochem. Biophys., i o 3 (1963) 284. H . U I ANn G. C. [VIUELLER, Pros. Natl. Acad. Sci. U.S., 5 ° (1963) 256, T. H. HAMILTON, Pros. Natl. Acad. Sci. U.S., 51 (1964) 83. T. H, HAMILTON, C. C. W'IDNELL AND J. R. TATA, Biochim. Biophys. Acre, lO8 (1965) 165. T. H. HAMILTON, C. C. \VIDNELL AND J. R. TATA, J. Biol. Chen~., 243 (1968) 408. A. R. MEANS AND T. I-I. HAMILTON, Pros. Natl. Acad. Sci. U.S., 56 (1966) 686. A. R. MEANS AND T. H . HAMILTON, Pros. Nail. Acad. Sci. U.S., 56 (1966) 1594. C. KIDSON AND K. S. KIRBY, Nature, 203 (1964) 599. K. g . BARKER AND J. C. WARREN, Pros. Natl. Acad. Sci. U.S., 56 ( i 9 6 6 ) 1298. M. L. GOLDBERG AND W . A. ATCHLEY, Pros. Natl. Acad. Sci. U.S., 55 (1966) 989 .
Biochim. Biophys. Acta. 195 (1969) 328 339
UTERINE R N A
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i i B. W'. O'MALLEY AND W. L. McGuIRE, Proc. Natl. Acad. Sci. U.S., 60 (1968) 1527. 12 W. E. HAHN, 1~. B. CHURCH, A. GORBMAN AND L. WILMOT, GeE. Comp. Endocrinol., i o (1968) 438 . 13 D. L. GREENMAN AND F. T. KENNEY, Arch. Biochem. Biophys., lO 7 (1964) I. 14 J. R. WARNER, R. SOEIRO, H. C. BIRNBOIM, M. GIRARD AND J. E. DARNELL, J. Mol. Biol., 19 (1966) 34915 A. DIGIROLAMO, E, C. HENSHAW AND H. H. HIATT, .[. Mol. Biol., 8 (1964) 479. 16 K. BURTON, Biochem. J., 62 (1956 ) 315 . 17 Z. DISCHE, The Nucleic Acids, Vol. i, A c a d e m i c Press, N e w Y ork, 1955, p. 3Ol. i8 T. J. BROWNHILL, A. S. JONES AND M. STACEY, Biochem. J., 73 (1959) 434. 19 A. M. CRESTFIELD, K. C. SMITtI ANn V. ~V. ALLEN, .]. Biol. Chem., 216 (1955) 185. 2o [7. Z. LITTAUER AND H. EISENBERG, Biochim. Biophys. Acta, 32 (1959) 320. 21 H. FRAENKEL-CONRAT, B. SINGER AND A. TSUGITA, Virology, 14 (1961) 54. 22 U. Z. LITTAUER AND M. SELA, Biochim. Biophys. Acta, 61 (1962) 609. 23 K. S. KIRBY, Biochem. J., 66 (1957) 495. 24 K. S. KIRlCY, Biochem. J., 96 (1965) 266. 25 G. ATTARDI, H. PARNAS, M-I. H. HWANG AND ]3. ATTARDI, J. Mol. Biol., 20 (1966) 145. 26 E. K. WAGNER, L. KATZ AND S. PENMAN, Biochem. Biophys. Res. Commun., 28 (1967) 152. 27 D. H. HAYES, F. HAYES AND M. F. GUERIN, .[. ]VIol. Biol., 18 (1966) 499. 28 S. PENMAN, J. Mol. Biol., 17 (1966) 117 . 29 E. F. ZIMMERMAN AND B. W. HOLLER, J. ]Viol. Biol., 23 (1967) 149. 3 ° M. H. VAUGHAN, J. I~. WARNER AND J. E. DARNELL, .[. 2VIol. Biol., 25 (I967) 23531 K. F. JERVELL, C. R. DINIZ AND G. C. MUELLER, J. Biol. Chem., 231 (1958 ) 94532 D. D. HAGERMAN, Endocrinology, 76 (1965) 553. 33 J. GORSKI AND G. C. MUELLER, Arch. Biochem. Biophys., lO 3 (1963) 21. 34 K. SCHERRER AND J. E. DARNELL, Biochem. Biophys. Res. Commun., 7 (1962) 486. 35 T. UCHIYAMA, N. FAUSTO AND J. L. VAN LANCKER, J. Biol. Chem., 241 (1966) 991. 36 S. CHAUDHURI AND l. L1EBERMAN, J. Mol, Biol., 33 (1968) 323 . 37 K. SCHERRER, L. MARCAUD, F. ZAJDELA, [. LONDON AND F. GROS, ProC. Natl. Acad. Sci. U.S., 56 (1966) 157I. 38 S. PENMAN, I. SMITH AND E. HOLTZMAN, Science, 154 (1966) 786. 39 S. PENMAN, C. VESCO AND M. PENMAN, J. ~'F[ol. Biol., 34 (1968) 494 ° D. P. LEADER AND J. M. BARRY, Nature, 215 (1967) 1374. 41 C. D. JACKSON AND B. H. SELLS, Biochim. Biophys. Acla, 155 (1968) 417 . 42 T. H. HAMILTON, C. C. WTIDNELL AND J. R. TATA, J. Biol. Chem., 243 (1968) 408. 43 J. GORSKI AND N. J. NELSON, Arch. Biochem. Biophys., Ii O (1965) 284.
Biochim. Biophys. Acta, 195 (1969) 328-339
328 BBA 96338
E X T R A C T I O N OF RNA FROM RAT U T E R U S PETERANNE
B. JOEL"
AND
DWAIN
D. HAGERMAN*"
Fearing Research Laboratory at the Boston Hospital for Women and the Department o/ Biological Chemistry and o[ Obstetrics and Gynecology, Harvard Medical School, Boston, Mass. (U.S.A.) (Received J u l y Ioth, 1969)
SUMMARY
Methods for the extraction of RNA from the immature rat uterus have been critically evaluated. Primary emphasis was placed upon minimizing degradation of RNA. The procedure of choice consists of extraction with phenol and sodium dodecyl sulfate at 5°o in the presence of bentonite, followed by deoxyribonuclease treatment of the extracted material. The product contains an average of 55 % of the total RNA and > 26 % of the rapidly-labeled RNA (2o-min pulse of V5-aHluridine) of the uterus. (The recoveries are based on values obtained by hot HC104 extraction of RNA.) Almost all of the rapidly-labeled RNA in the product is of high molecular weight, with sedimentation coefficients in the range 30-50 S on sucrose gradients, which suggests that degradation of the material has been minimized. Estradiol given intraperitoneally to immature rats 2, 4, or 12 tl before sacrifice did not affect the pattern of distribution of this high-molecular-weight, rapidlylabeled RNA (io-, 2o-, or 4o-min pulse of [5-aH]uridine) on sucrose gradients. Estradiol given intraperitoneally 20 rain before sacrifice to rats ovariectomized at 28 days and used for experimentation 18 33 days after ovariectomy also did not affect the pattern of distribution of rapidly-labeled RNA (Io-min pulse of [5-3H]uridine) on sucrose gradients.
I NTRODUCTION
Several studies 1-7 have indicated that estradiol increases the synthesis of RNA as one of the hormone's early effects upon the metabolism of the rat uterus. Furthermore, some investigations s 1~ have suggested that there may be qualitative differences in the RNA synthesized in the uterus under estradiol stimulation as compared with controls. Methods previously used to extract the total uterine RNA have generally yielded a contaminated and/or degraded RNA product as judged by either the nature of the procedure or by sucrose gradients of the product. These methods are not acceptable if one is looking for qualitative differences in the new RNA. We sought to develop a procedure for the extraction of RNA from the rat uterus which would yield an undegraded and uncontaminated product coupled with a reasonable recovery of all classes of RNA. * 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 C h e m i s t r y , L a w r e n c e U n i v e r s i t y , Appleton, W i s c o n s i n ** P r e s e n t address: D e p a r t m e n t of B i o c h e m i s t r y a n d D e p a r t m e n t of O b s t e t r i c s a n d G y n e cology, U n i v e r s i t y of Colorado School of Medicine, Denver, Colorado.
Biochim. Biophys. Aeta, 195 (1969) 328-339
UTERINE RNA
329
MATERIALS AND METHODS
Immature Wistar strain rats (Charles River Breeding Co., Boston, Mass.), 22-26 days of age, were used in all of the experiments unless indicated otherwise. Estradioltreated rats received 5 #g of I7/~-estradiol in 0.5 ml of o.154 M NaC1 containing 1 % ethanol injected intraperitoneally. Control animals received the same solution without estradiol. Standard procedure/or R N A isolation Each animal received 50 t*C of [5-3H~uridine IO, 20 or 4 ° rain prior to sacrifice. The rat was decapitated; the uterus was trimmed free of adipose tissue, was removed, and was placed in a tube suspended in a --75 ° ethanol bath. Each uterus was frozen within 2 rain after the rat was decapitated. Five to eight frozen uteri were placed in a conical ground glass Potter-Elvehjem type homogenizer containing 2 ml of 0.05 M sodium acetate buffer (pH 5.2) with I m g bentonite per ml, and 2 nfl of 88 % phenol. The uteri were homogenized about 30 sec with a 5-1o ° water bath around the tube. The homogenate was combined with an additional 1.6 ml of 0.05 M sodium acetate buffer (pH 5.2) with I m g bentonite per ml, 2.0 ml of 88 % phenol, and 0. 4 ml of IO % sodium dodecyl sulfate in 0.05 M sodium acetate buffer (pH 5.2). The mixture was then blended in a Lourdes homogenizer for 3o sec at 60 V operated at room temperature. The resulting homogenate was heated for 3 rain in a bath at 52 ° (final solution temperatule of 5 o°) while being mixed with a footed stirring rod rapidly raised and lowered through the mixture. The solution was cooled in an ice bath for 2 rain and centrifuged for 6 min at 20 ooo × g (4°). The phenol layer was discarded, leaving the interphase behind, and the extraction at 5 °° was repeated with 3.2 ml of 88 % phenol. The aqueous layer was removed and set aside at 4 ° after the second extraction. 4 mg of bentonite were added to this aqueous layer. To the remaining phenol layer and interphase were added 2.0 ml of 0.05 M sodium acetate buffer (pH 5.2), containing 2 mg bentonite, for a third partition. The aqueous layer from this third extraction was combined with the first aqueous layer, and the combination was centrifuged (15 rain at 20 ooo ×g, 4 °) to remove most of the bentonite. RNA (and DNA) were precipitated from the resulting supernatant fluid by the addition of NaC1 to o.15 M followed by 2 vol. of ethanol and storage overnight at --20 °. This procedure for the extraction of RNA follows that of WARNER et al. 14 with some modifications. The precipitate was collected by centrifugation and was washed twice with cold 95 % ethanol. The washed precipitate was treated with o.15 mg pancreatic deoxyribonuclease (electrophoretically pure, Worthington Biochemical Corp., Freehold, N.J.) in I.O ml of o.oi M Tris buffer (pH 7.5) containing o.ooi M MgCI2 with a 5-rain incubation at o ° followed by IO rain at 25 ° (ref. 15). (A larger amount of deoxyribonuclease was required to digest the DNA than was used by DIGIROLAMOet al. is, presumably as a result of residual bentonite in the extract.) The cooled solution was made to I mg/ml with bentonite and to 1 % sodium dodecyl sulfate, and was then extracted with 0.5 vol. of 88 % phenol for IO rain at IO°. After centrifugation as before, the aqueous phase was saved and the phenol and interphase were reextracted with 0.5 ml of Tris buffer (pH 7-5) containing o.ooi M MgC12. The combined aqueous phases were centrifuged as before to remove bentonite. The RNA was then preBiochim. Biophys. Acta, 195 (1969) 328-339
330
P . B . JOEL, D. D. HAGERMAN
cipitated from the supernatant fluid with NaC1 and ethanol for 1. 5 h at --20 °. The precipitate was collected by centrifugation, dissolved in 0.5 ml of o.ooi M MgC12 and was reprecipitated with 2 M potassium acetate, 25 % ethanol and chilling as described by DIGIROLAMOet al. 1~. The reprecipitation was performed 3 times. The final RNA precipitate was washed twice with cold 95 % ethanol.
Sucrose gradients lO-5O% sucrose gradients in o.oi M sodimn acetate buffer (pH 5) containing o.I M NaC1 were used unless otherwise indicated. The initial sucrose solutions were made with o.5 mg bentonite per 1111and were centrifuged before the gradients were prepared. RNA was dissolved in o.oi M sodium acetate buffer (pH 5) containing o.i M NaC1 and o.I ml of the solution was layered on the top of 4.9-ml gradients. The gradients were centrifuged at 4 ° for 165 rain at 65 ooo rev/min in the SW65 rotor of a Model L2-65 ultracentrifuge, Beckman Instruments, Palo Alto, Calif. The gradients were fractionated into about 35 sanlples by the collection of 6-drop samples from the bottom of the tube through a siphon. The absorbance at 260 nm was determined on each fraction in mierocuvettes. When necessary, a portion was diluted with water for the absorbance reading. For determination of radioactivity, o.I ml of each fraction was mixed in a vial with I.I ml of 0.3 M "Nuclear-Chicago solubilizer" in toluene (Nuclear Chicago Corp., Des Plaines, Ill.). 15 ml of scintillator solution (4 g 2,5diphenyloxazole and o.I g 1,4-bis-E2-(5-phenyloxazolyl)~henzene per 1 of toluene) were added and the vials were counted in a Nuclear-Chicago scintillation spectrometer equipped with an external standardization device. In all of the figures, the solid lines represent the absorbance at 260 nm and the dotted lines the disint,/min in o.I ml of the fractions.
Analyses For DNA and RNA analyses, samples were extracted with 0. 5 M HC1Q for 15 rain at 7 o°. DNA was analyzed on a portion of the HCI04 extract by the procedure of BURTO~16. Calf thymus DNA (Calbioehem, Los Angeles, Calif.) was used as the standard, assuming the absorbance at 260 nm of a I mg/ml solution to be 20. RNA was analyzed on a portion of the HC104 extract by a modification of the procedure of Dische and Schwarz ~7. The concentrations in the final mixture of reagent and sample were: orcinol, 2.67 mg/ml; ferric alum, 0.59 mg/ml; HC1, 4 M. The mixtures were heated 15 rain at Ioo °, cooled and read at 660 nm. Yeast RNA (Nutritional Biochemieals Co,, Cleveland, Ohio) was used as the standard.
Materials Labeled V5-~H~uridine (8.o C/mmole) was purchased from Schwartz Bio-Research, Orangeburg, N.Y. Bentonite was purified according to the procedure of BROWNHILL et al. is. Sodium dodeeyl sulfate was purified according to the procedure of CRESTFIELD et al. ~9. Phenol (Mallinckrodt 0025, liquified) was taken from a newly opened bottle for each experiment or was redistilled before use. RESULTS
Comparison o] [our extraction procedures A method was sought for extraction of RNA from rat uterus which would give reasonable recovely of all classes of RNA with minimum degradation. The uteri were Biochim. Biophys. Acta, 195 (1969) 328-339
UTERINE RNA
331
labeled in vivo with a 2o-min pulse of [5-~H]uridine in order to ascertain the extent to which rapidly-labeled RNA was extracted. Rapidly-labeled RNA is the most difficult class to extract and is also the most susceptible tc degradation. Due to the small size of the immature rat uterus, it was necessary to combine several uteri for a single extraction. In order to avoid degradation of RNA in uteri between excision and homogenization, uteri were frozen at --75 ° immediately upon excision. The rat uterus has unusually potent nucleases (unpublished observations; ref. 13). Therefore no attempt was made to fractionate the cell into nuclei and cytoplasm; instead, homogenization of the uteri was carried out in the presence of phenol and bentonite, both of which inhibit ribonuclease ls,2°-~2. The large amount of connective tissue in the rat uterus makes homogenization and extraction of nucleic acids difficult. (KIRBY 23 has shown that collagen absorbs DNA under certain conditions.) The combination of homogenization first with a Potter-Elvejhem homogenizer, followed by homogenization in the Lourdes homogenizer was the most effective method found for disrupting cells and connective tissue. For the extraction of RNA four methods using phenol from the laboratories of WARNER et al. 14, KIRBY 24, DIGIROLAMO et al. 15, and ATTARDI et al. 25 were compared for their applicability to the rat uterus. The four methods differ chiefly in the temperature at which the extraction is carried out. The final temperature of the mixture in the procedure of WARNER et al. is 5 o°, whereas the procedure of KIRBY uses 20 °, and the procedures of DIGIROLAMO et al. and ATTERDI et al. extract at 4 °. The proce dure of WARNER et al. uses sodium dodecyl sulfate in conjunction with phenol to release RNA at a p H of 2. 5. The procedure of KIRBY relies on 4-aminosalicylate to release DNA as well as RNA and uses a phenol-clesol mixture, containing 8-hydroxyquinoline, as deproteinizing agent. The procedures of DIGIROLAMOet al. and ATTARDI et al. are very similar, both using sodium dodecyl sulfate and naphthalene disulfate with phenol containing 8-hydroxyquinoline. They differ in the concentration of sodium dodecyl sulfate used and in the ionic and osmotic conditions of the aqueous phase. Bentonite was added to all the extraction media except in the procedure of KIRBY.
All four procedures extracted a large portion of the uterine DNA. In the procedure of KIRBY, DNA was removed by extraction with 3 M sodium acetate. The DNA in the extraction products from the other three procedures was hydrolyzed by deoxyribonuclease. The oligodeoxyribonucleotides were then removed with 2 M potassium acetate and 25 % ethanol. The modified Darnell extraction procedure, including removal of DNA b y deoxyribonuclease, is referred to and described in ~ATERIALS AND METHODS as t h e " s t a n d a r d p r o c e d u r e " .
The yield of total RNA and rapidly-labeled RNA in the final product using each of the four methods is presented in Table I. The results given in Table I under the heading HC104 indicate values obtained when nucleic acids are extracted with hot HCIO 4 as the basis of determining approximate recoveries by the other methods. Both the recovery of total RNA and rapidly-labeled RNA were by far superior in the product extracted b y the adopted standard procedure. The iecoveries averaged 55 % for total RNA and ~ 26 % for rapidly-labeled RNA for the standard prccedure as compared to hot HC104 extraction. Analysis of the final products from the four procedures by sucrose gradient centrifugation is shown in Fig. I. Only in the product extracted by the adopted Biochim. Biophys. Acta, 195 (1969) 328-339
332
P. B. JOEL, D. D. HAGERMAN
TABLE I
COMPARISON OF FOUR METHODS FOR THE EXTRACTIONOF R N A
FROM THE
IMMATURERAT UTERUS
Descriptions of the four m e t h o d s used for extraction are given in the legend to Fig. i. The last c o l u m n (HC104) indicates the values o b t a i n e d w h e n the frozen uteri were homogenized in 0. 5 M HC104, the precipitates w a s h e d 3 times w i t h cold 0. 5 M HC104, and R N A and D N A extracted w i t h 0.5 M HCIO~ at 7 °o for 15 miD. The values for the HC104 extraction are averages of eight uteri f r o m r a t s 22 d a y s of age.
Quantity isolated per uterus ,4 *
B
C
D
HCIO~
6o-~ io 6 4 ~ 12 i I 4oo~43oo
23** 57 96o
29 73 25oo
21 79 28oo
lO9 t53 44 5 ° 0
R N A (ktg) D N A (/,g)*** Total disint./min in R N A
• The values for Method A are the m e a n s of 6-8 separate e x p e r i m e n t s i S . D , • * The 3 M p o t a s s i u m acetate used in the m e t h o d of KIRBY r e m o v e s t R N A . "** ~'rior to r e m o v a l b y deoxyribonuclease or 3 M s o d i u m acetate.
~.5
t
2B,
-! 5oc
500 1 3O0
5~ ? 0 0 05 Ec oo
Z 100 z
300 i I0
2I0
i
110
3I0
210
3t0
D
1.5
E
~
-
~
"~ 300100"~ T~
-j ~-
(?.5
I0
2O
10
30 ER#,CTION
20
30
NUMBER
Fig. i. Sucrose g r a d i e n t analysis of the R N A extracted f r o m r a t u t e r u s b y four methods. The m e t h o d s used for e x t r a c t i o n and purification of R N A were as follows. A. WARNER el al. a4 as modified and described in detail in MATERIALS AND METHODS. ]3. KIRBY, Method 2 f r o m ref. 24. C. DIGIROLAMO et al. 1~, the conditions used for the D N A h y d r o l y s i s varied f r o m the published procedure in t h a t the deoxyribonuclease c o n c e n t r a t i o n w a s 25/~g/ml and the i n c u b a t i o n was for 5 miD at 25 °. D. ATTARDI et al. 25. I n all of the m e t h o d s , i m m a t u r e r a t s each received 5 ° #C of [5-DH~uridine 20 miD prior to sacrifice. Frozen uteri were homogenized in the combined a q u e o u s and phenol p h a s e s described for each m e t h o d . I n A, B and C, seven uteri were extracted w i t h an initial a q u e o u s plus phenol v o l u m e of 8.0 ml; in D, five uteri in an initial v o l u m e of 8.0 ml. I n A, C and D the sucrose gradients were p r e p a r e d as described in MATERIALS AND METHODS; in B the purified R N A was dissolved in o.i M s o d i u m acetate buffer (pH 5.2) and the gradients were p r e p a r e d in o.i M s o d i u m acetate buffer (pH 5.2). standard mentation
procedure
did the major
p o r t i o n of t h e l a b e l e d R N A
coefficients equal to or greater than
sediment
with sedi-
28 S.
EMect o/variations in the standard procedure upon the extraction o/ R N A Several conditions determine
whether
of t h e s t a n d a r d
procedure
were varied systematically
these conditions were critical and whether
Biochim. Biophys. Acta, 195 (1969) 328-339
to
the procedure might
UTERINE R N A
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l
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Fig. 2. Sucrose gradient analysis of the effect of variations in the standard procedure for extraction of RNA from rat uterus. Immature rats received 5o/~C of F5-3H]uridine 20 mid prior to sacrifice. In Expt. I each group contained six uteri; in Expt. II, five uteri. RNA was extracted according to the standard procedure (see MATERIALS AND METHODS) with individual variations as indicated below. In each group the final RNA product was dissolved in o.15 ml, and o. I ml was layered on the sucrose gradient. Expt. L A. Control; no variation. B. 1 % sodium dodecyl sulfate was present in the 2 ml of 0.05 M sodium acetate buffer added for the third extraction at 5o°. C. The final temperature of the extraction mixture at the end of the 3-mid heating was 6o ° rather than 5 o°. D. The uteri were cut into 2-ram slices and placed in o.154 M NaC1 at 4 ° rather than being frozen. Expt. II. A. Control; no variation. B. The initial homogenization of the uteri in the glass conical homogenizer was carried out in 2 ml of 0.05 M sodium acetate buffer (pN 5.2) with i mg bentonite per ml (no phenol present). Phenol was added prior to the subsequent homogenization in the Lourdes homogenizer.
Expt. I A RNA per uterus (/,g) Total disint./min in RNA per uterus Recovery of disint./min from gradient (%) be of to in
Expt. I I B
C
D
A
B
55
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56
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53
54
12 500
12 800
13 200
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i m p r o v e d (Fig. 2). T h e f o u r a l t e r a t i o n s , o m i s s i o n of f r e e z i n g of t h e u t e r i , o m i s s i o n p h e n o l f r o m t h e f i r s t h o m o g e n i z a t i o n , a d d i t i o n of m o r e s o d i u m d o d e c y l s u l f a t e t h e t h i r d e x t r a c t i o n a t 5 °0 , a n d i n c r e a s e of t h e f i n a l t e m p e r a t u r e of t h e m i x t u r e t h e e x t r a c t i o n s t e p s t o 60 °, h a d l i t t l e o r n o e f f e c t o n t h e t o t a l q u a n t i t y of R N A
e x t r a c t e d (Fig. 2). W h e n t h e u t e r i w e r e k e p t in s a l i n e r a t h e r t h a n b e i n g f r o z e n b e f o r e h o m o g e n i z a t i o n , c o n s i d e r a b l y less of t h e r a p i d l y - l a b e l e d R N A w a s e x t r a c t e d (Fig. 2, E x p t . I D). S i n c e t h e m a i o r p o r t i o n of t h e r a p i d l y - l a b e l e d R N A s e d i m e n t e d f a s t e r t h a n t h e 28-S R N A , t h e d e c r e a s e d a m o u n t of r a p i d l y - l a b e l e d R N A in t h e p r o d u c t is p r o b a b l y
Biochim. Biophys. Acta, 195 (1969) 328-330
334
P . B . JOEL, D. D. HAGERMAN
not due to degradation while the uteri were in saline. Freezing and thawing may facilitate release of rapidly-labeled RNA, or the uteri may be disrupted more effectively when they are frozen at the initiation of the homogenization. When the phenol was omitted from the first homogenization step, the total amount of rapidly-labeled RNA extracted increased (Fig. 2, Expt. II B). This finding may reflect the fact that the uteri are more easily homogenized when phenol is absent and may therefore be more thoroughly disrupted. A greater portion of the rapidly-labeled RNA extracted was of a size less than the 28-S RNA. Either the increased amount of rapidly-labeled RNA which was extracted actually has a lower molecular weight or, more likely, some degradation occurred when phenol was absent from the homogenization medium. Adding sodium dodecyl sulfate to the third extraction at 5 °° did not affect the amount of rapidly-labeled RNA extracted (Fig. 2, Expt. I C). The recovery of labeled RNA from the sucrose gradient was, however, greater with the RNA isolated in the presence of extra sodium dodecyl sulfate than with the control RNA. The lower recovery of labeled RNA flom the sucrose gradient in the control plus the shape of the curve of the control labeled RNA suggests that some material may have sedimented to the bottom of the tube (v.i.). Increasing the final temperature of the mixture to 6o ° in the extraction steps did not affect the amount of rapidly-labeled RNA extracted (Fig. 2, Expt. I B). The recovery of the RNA extracted at 6o ° (both labeled and unlabeled) from the sucrose gradient was very poor. The shape of the curves suggest that RNA had sedimented to the bottom of the tube. WAGNER et al. 26 have reported that extraction of RNA by the procedure of WARNER et al. at high temperatures (65 °) may result in noncovalently bonded aggregates of the two species of rRNA. The shape of tile absorbance curve with a large amount of absorbance at positions greater than 28 S for the material extracted at 6o ° suggests that a similar type of aggregation may have occurred here.
E//ect o/ varying conditions in the sucrose gradient analysis upon the high-molecularweight labeled R N A The possibility exists that the high-molecular-weight rapidly-labeled RNA (about 30-50 S) seen in the sucrose gradients represents aggregates of RNA. WAGNER et al. 26 reported aggregation of rRNA when the RNA was extracted at temperatures higher than 55 ° or when the RNA in the sodimn dodecyl sulfate buffer solution was more concentrated than 0. 5 mg/ml. According to their results this phenomenon would not be expected to occur to any great extent at the 5°° extraction temperature and RNA concentration of 0.2 mg/ml used in the present experiments. Although there is an occasional shoulder or trailing on the heavy side of the 28-S absorbance peak, the curves generally suggest little or no significant aggregation of rRNA. Under appropriate conditions rapidly-labeled RNA will associate with rRNA 27. This association apparently requires high concentrations of NaC1 (0.6 M) or the presence of Mg 2+ (o.oi M). The complex between rapidly-labeled RNA and rRNA disscciates only partially in o.I M NaC1 but separates completely in o.oi M Tris buffer (pH 7.0). The complex does not form at pH > 9-5 since the - N H - C O groups are dissociated and unable to form hydrogen bonds. Biochim. t~iophys. ,4cta, 195 (1969) 328-339
UTERINE RNA
335
40 ~A t ~ O 0
+ZB
500
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Fig. 3. Effect of variations in the sucrose gradient media on the s e d i m e n t a t i o n p a t t e r n of rapidlylabeled uterine RNA. I m m a t u r e r a t s received 5 o,uC of ~5-3HJuridine 20 rain (A and B) or 4 ° min (C) prior to sacrifice. Uterine RNA was extracted b y the s t a n d a r d procedure, i o - 5 o % sucrose gradients were p r e p a r e d in the media indicated below. The purified R N A was dissolved in the same m e d i u m for layering on the gradient. These R N A p r e p a r a t i o n s were also centrifuged on sucrose gradients prepared in o.oi M s o d i u m acetate buffer (pH 5.2) containing o.i M NaC1 and gave p a t t e r n s of d i s t r i b u t i o n of labeled R N A essentially the same as t h a t s h o w n in Fig. IA. A. 0.02 M g l y c i n e - N a O H buffer (pH 9-7) containing o.i M NaC1. B. 0.02 M glycine N a O H buffer (pH 9.7). C. 0.005 M Tris buffer (pH 7.2) containing o.oot M EDTA.
To determine whether association of rapidly-labeled RNA and rRNA was responsible for the rapidly-labeled RNA sedimenting between 3o and 5° S, RNA isolated by the standard procedure was centrifuged through sucrose gradients prepared in glycine buffer at pH 9.7 with or without o.I M NaC1 (Figs. 3A and 3 B) and in o.oo 5 M Tris buffer (pH 7.2) containing o.ooi M EDTA (Fig. 3C) to remove any residual Mg2÷. Increasing the pH to 9.7 did not decrease the portion of the rapidlylabeled RNA sedimenting between 3o and 5o S. The heavy side of the 28-S RNA absorbance curve was somewhat steeper at pH 9.7 and the rapidly-labeled RNA was slightly more concentrated toward 3o S. Therefore association of rapidly-labeled RNA with rRNA may contribute slightly but not to any great extent to the patterns seen in o.oi M sodium acetate buffer (pH 5) containing o.I M NaC1. In the gradients prepared at low ionic strengths in the presence of EDTA there was a shift of the peak of rapidly-labeled RNA toward 24-3o S. The shift in these latter gradients may reflect the fact that lowering the ionic strength reduces the sedimentation coefficients of a single stranded RNA.
E//ect o/the administration o~ estradiol and varying pulse times on the sucrose gradient sedimentation characteristics o/ rapidly-labeled uterine R N A The adopted standard procedure for extraction of uterine RNA was used to investigate whether the rapidly-labeled RNA made under estradiol-stimulation might contain a greater predominance of precursor rRNA of 45 and 32 S (refs. 28-30) than in the control. Estradiol-I7/~ was administered to immature rats 2 h prior to sacrifice. The F5-aHluridine (5o/~C) was injected IO, 20 or 40 rain prior to sacrifice. RNA was extracted by the standard procedure and analyzed on sucrose gradients. The labeled RNA from the control uteri gave the same pattern of distribution on sucrose gradients regardless whether the pulse time had been IO, 20 or 4 ° rain. The labeled RNA from the estradiol-treated animals gave the same pattern of distribution as the control RNA at each pulse time. No specific increase in radioactivity in the region of 45 and 32 S could be distinguished. Since all of these patterns were similar to that shown in Fig. IA, they are not included. In additional experiments estradiol-I7/~ was administered to immature rats at 4 or 12 h prior to sacrifice and [5-3HJuridine injected 20 rain prior to sacrifice. Biochim. Biophys. Acts, I95 (1969) 328-339
336
P.B. JOEL, D. D. HAGERMAN
Again the uterine rapidly-labeled RNA from estradiol-treated rats gave the same pattern of distribution on sucrose gradients as that from the controls. In these experiments on the effects of estradiol, an average of 6 8 ± 1 9 ~g (S.E., 9 groups) of RNA per uterus were isolated. This represents an average recovery of 62 % compared to RNA levels determined by hot HC104 extraction (Table I). (The group receiving estradiol 12 h prior to sacrifice is not included in the average since JERVELL et al. 31 have shown that there is a 30 O//oincrease in total uterine RNA by 12 h after administration of estradiol.) In the experiment in which estradiol was administered 2 h prior to sacrifice and I5-3Hluridine injected 20 rain prior to sacrifice, the specific activity of the isolated RNA was 85 % greater than that of the control. The same percent increase (89 %) was found when the total RNA was extracted by hot HC10 4 in a comparable experiment. Thus although the recovery of RNA is not quantitative, the increase in the amount of I5-aHluridine incorporated into RNA due to estradiol administration is evident to the same extent in the fraction of RNA isolated as in the total RNA. Although the uteri after 4 h of estradiol treatment were distended and obviously stimulated by the estradiol, the specific activity of the RNA was 30 % lower than the control level. The same decrease occurred when RNA was extracted by hot HC10 4. This finding is not interpreted as a decrease in RNA synthesis but rather as a consequence of a large increase in the size of the uridine nucleotide pool which is caused by estradio131. The E5-3Hluridine incorporated into F5-3HJUMP is diluted to a much greater extent by unlabeled UMP in the case of the estradiol-treated uteri than in the control. Effects of estradiol on the turnover rate 32 and specific activity of uterine nucleoside triphosphates 31,3a and possible effects on transport processes make changes in the specific activity of RNA uninterpretable without concomitant measurement of the specific activity of the immediate precursor (UTP in this case). E[lect o/estradiol on the sucrose density sedimentation characteristics oI rapidly-labeled uterine R N A [rom ovariectomized rats Rats were ovariectomized at 28 days of age and were used for experimentation 18-33 days after ovariectomy (I9O-25o-g rats). Ovariectomized rats received IO/~g of estradiol in 0.2 ml propanediol intraperitoneally 20 nfin prior to sacrifice and 50 /,C of E5-~Hjuridine in 0.2 ml of o.154 M NaC1 intraperitoneally io rain prior to sacrifice as described by HAMILTON et al. 4. RNA was extracted from the total uterus by the standard procedure and examined on a sucrose gradient. RNA isolated from uteri of estradiol-treated rats had the same pattern of isotope and mass distribution on the sucrose gradient as did the RNA from control animals. Both patterns resembled the pattern of RNA from immature rats. Four such experiments were carried out and the average total disint./min in RNA per uterus extracted from the estradioltreated rats (396O:klO5 o (S.E.)) in these four experiments was not significantly different from that of the controls (478o~:27oo). An average of 48:ki6 #g RNA per uterus were isolated.
DISCUSSION Of the four procedures for the extraction of RNA initially tested with rat uterus, the procedure developed in the laboratory of WARNER et al. 14 clearly gave the Biochim. Biophys. Mcta, 195 (1969) 328-339
UTERINE RNA
337
highest recovery both of total RNA and of rapidly-labeled RNA. Most likely it is the higher temperature (5°0 ) of extraction used in this procedure which is responsible for the higher extraction efficiency. However, unlike H e L a cells from which little if any DNA is extracted by the procedure of SCHERRER AND DARNELL34, a considerable portion of the uterine DNA is extracted and must be removed from the extract. Therefore, the final procedure found most suitable for extraction of RNA from rat uterus combines the extraction technique developed in the laboratory of WARNER et al. 14 with the removal of DNA from the product by procedures used in the laboratory of DIGIROLAMO et al. 15. The modifications introduced (freezing the tissue after excision, omitting fractionation of the cells, using phenol in the homogenization medium and keeping bentonite present whenever feasible) were for the purpose of minimizing degradation of RNA. The rapidly-labeled RNA extracted from uteri b y the procedure developed was predominantly of high molecular weight (i.e., with sedimentation coefficients in the range of 30-50 S). The fact that the sedimentation coefficients of the rapidly-labeled RNA changed very little when the sucrose gradients were carried out at p H 9.7 (at similar ionic strength) suggests that the high-molecular-weight rapidly-labeled RNA is not the result of aggregation of individual RNA molecules by hydrogen bonding. The possibility of residual DNA or protein causing aggregation of smaller molecules of rapidly-labeled RNA to give the material sedimenting between 30 and 50 S cannot be ruled out but seems unlikely for several reasons: the DNA digestion is extensive; the extracted RNA is treated a second time with phenol after the deoxyribonuclease treatment for removal of protein; and the general pattern of the labeled RNA on sucrose gradients is very reproducible and similar to that seen with other tissues when care is taken to avoid RNA degradation 25,34,s5. The existence of rapidly-labeled rRNA precursors of 32 and 45 S is well substantiated 2s-a°. CHAUDHURIAND LIEBERMAN~6 have reported the labeling in rat liver of 45-, 32- and I8-S RNA within 8 rain after intravenous injection of F14C~orotate. Rapidly-labeled RNA of 30-80 S differing from rRNA in base ratios has been observed in other tissues 14,25,3v-39. The primary disadvantage of the standard procedure is the fact that recovery of total RNA averaged 55 % while that of the rapidly-labeled RNA was probably lower. Comparison of the amount of radioactive label in the RNA extracted by the standard procedure with the amount of label extracted by hot HC1Q suggests an average recovery of only 26 % for rapidly-labeled RNA. However, it is quite likely that the hot HC104 extract contains some short (incomplete) labeled oligonucleotides and possibly some contaminating E5-3H~uridine and acid-soluble [5-3Hluridine nucleotides. In contrast, the RNA isolated by the standard procedure contains very little labeled material with a sedimentation rate less than 4 S. Therefore the average recovery of RNA ~ 4 S with the standard procedure is probably significantly higher than 26 %. The large amount of connective tissue in the uterus makes it difficult to homogenize the tissue completely and to extract RNA by any method which does not degrade the RNA. This is presumably the major factor in the low recoveries. The major advantage of the standard procedure is the fact that the rapidlylabeled RNA isolated is of high molecular weight indicating that RNA degradation has been minimized. In addition, DNA and protein are specifically removed from the product. For studies of possible qualitative changes in the nature of RNA produced under hormonal stimulation, it is essential to avoid degradation of RNA and to reBiochim. Biophys. dcta, I95 (I969) 328-339
338
P.B.
J O E L , D. D. HAGERMAN
move contaminants which may be bound to RNA and influence its behavior. (The work of LEADERAND BERRY4° and JACKSONAND SELLS41 suggests that the differences in countercurrent distribution profiles of rapidly-labeled liver RNA from hormonetreated and control rats observed by KIDSON AND KIRBY8 may have been introduced during preparation of the RNA.) Although recovery of RNA by the standard procedure is not quantitative, the relative specific activities of uterine RNA from control and estradiol-treated rats have been the same whether isolated by the standard procedure or hot HC1Q. Hence the standard procedure does seem to isolate a similar sample of RNA from uteri of control and estradiol-treated ra~s. Although the standard procedure would not be applicable in studies requiring rigorous quantitative recovery of RNA, it would be applicable in studies of possible qualitative changes in RNA due to estradiol. There has been speculation 42 that estradiol may specifically increase the synthesis of rRNA as one of the earliest effects on the uterus. Although no specific increases in RNA with the sedimentation values of precursor rRNA were found 2o rain, or 2, 4, or 12 h after estradiol administration, the limited resolving power of tile sucrose gradient technique makes further study of this question necessary. In 1965 GORSKI AND NELSON4a performed experiments similar to some reported here. The rapidly-labeled RNA which they isolated, however, sedimented predominantly between 5 and 2o S in contrast to the 3o-5o-S rapidly-labeled RNA iso-. lated by the procedure in this paper. HAMILTON"and co-workers4-v reported that the rate of incorporation of i5-3H] uridine into rapidly-labeled RNA in the ovariectomized rat uterus was accelerated 5-6-fold within 20 nlin after estradiol was injected intraperitoneally. Judging from tile DNA concentrations and weights reported for the uteri of their ovariectomized rats 5, HAMILTON and co-workers used rats ovariectomized after they had reached maturity. In the similar experiments reported in the present paper, the rats used were ovariectomized while immature (28 days). Whether the difference in age at which the rats were ovariectomized can account for the failure to repeat the observation of HAMILTONand co-workers remains to be investigated. ACKNOWLEDGMENTS
We thank Mrs. Sandra Cressman for capable technical assistance. This investigation was supported in part by National Institutes of Health Research Grant No. AM-II846 and its predecessors, National Institute of Arthritis and Metabolic Diseases. REFERENCES i 2 3 4 .5 6 7 8 9 IO
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UTERINE R N A
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Biochim. Biophys. Acta, 195 (1969) 328-339